Co-reporter:Qun Zhao, Fei Fang, Yichu Shan, Zhigang Sui, Baofeng Zhao, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry May 16, 2017 Volume 89(Issue 10) pp:5179-5179
Publication Date(Web):April 22, 2017
DOI:10.1021/acs.analchem.6b04232
Although great achievement has been made in the mapping of human proteome, the efficiency of sample preparation still needs to be improved, especially for membrane proteins. Herein, we presented a novel method to deepen proteome coverage by the sequential extraction of proteins using urea and 1-dodecyl-3- methylimidazolium chloride (C12Im-Cl). With such a strategy, the commonly lost hydrophobic proteins by 8 M urea extraction could be further recovered by C12Im-Cl, as well as the suppression effect of high abundance soluble proteins could be decreased. Followed by the in situ sample preparation and separation with different stationary phases, more than 9810 gene products could be identified, covering 8 orders of magnitude in abundance, which was, to the best of our knowledge, the largest data set of HeLa cell proteome. Compared with previous work, not only the number of proteins identified was obviously increased, but also the analysis time was shortened to a few days. Therefore, we expect that such a strategy has great potential applications to achieve unprecedented coverage for proteome analysis.
Co-reporter:Huiming Yuan, Shen Zhang, Baofeng Zhao, Yejing Weng, Xudong Zhu, Senwu Li, Lihua Zhang, and Yukui Zhang
Analytical Chemistry June 20, 2017 Volume 89(Issue 12) pp:6324-6324
Publication Date(Web):May 18, 2017
DOI:10.1021/acs.analchem.7b00682
Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography–electrospray ionization-tandem mass spectrometry (nanoHPLC–ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.
Co-reporter:Kaiguang Yang, Jianhui Liu, Jingdi Sun, Yuan Zhou, ... Yukui Zhang
Science Bulletin 2017 Volume 62, Issue 18(Volume 62, Issue 18) pp:
Publication Date(Web):30 September 2017
DOI:10.1016/j.scib.2017.08.026
The complications of hemodialysis accompanied the hemodialysis and threaten the patients’ life. Besides the loss of nutrient substance, such as amino acid and vitamin, we found new clues that the adsorbed proteins on common-used polysulfone-based dialysis membrane might be the reason according to the qualitative proteomic study by ionic liquid assisted sample preparation method. Our results indicated that the adsorbed proteins on the membrane were related with complement activation, blood coagulation, and leukocyte-related biological process. The quantitative proteome further demonstrated some significant changes of signal proteins in the post-dialysis plasma after the hemodialysis, such as beta-2-microglobulin and platelet factor-4, which would further verify these new clues.New clues were found that the adsorbed proteins on dialysis membrane might be the reason of complications of hemodialysis according to the novel proteomic techniques, ionic liquid assisted sample preparation method (ILSP method) and pseudo-isobaric dimethyl labeling method (pIDL method).Download high-res image (56KB)Download full-size image
Co-reporter:Shu-Rong Zhang, Yi-Chu Shan, Hao Jiang, Jian-Hui Liu, Yuan Zhou, Li-Hua Zhang, Yu-Kui Zhang
Journal of Proteomics 2017 Volume 163(Volume 163) pp:
Publication Date(Web):23 June 2017
DOI:10.1016/j.jprot.2017.05.010
•Control the reliability of the peptide identification algorithm at design stage.•Null-Test: a general evaluation strategy for the identification algorithm.•Null-Test can check the potential bugs, faultiness, errors etc.•None of the five famous software could pass the most rigorous Null-Test.•Fuzzy logics based method has the possibility to pass the Null-Test.The present research proposed general evaluation strategy named Null-Test for peptide identification algorithm in Shotgun proteomics. The Null-Test method based on random matching can be utilized to check whether the algorithm has a tendency to make a mistake or has potential bugs, faultiness, errors etc., and to validate the reliability of the identification algorithm. Unfortunately, none of the five famous identification software could pass the most stringent Null-Test. PatternLab had good performance in both Null-Test and routine search by making a good control on the overfitting with sound design. The fuzzy logics based method presented as another candidate strategy could pass the Null-Test and has competitive efficiency in peptide identification. Filtering the results by appropriate FDR would increase the number of discoveries in an experiment, at the cost of losing control of Type I errors. Thus, it is necessary to utilize some more stringent criteria when someone wants to design or analyze an algorithm/software. The more stringent criteria will facilitate the discovery of latent bugs, faultiness, errors etc. in the algorithm/software. It would be recommended to utilize independent search combining random database with statistics theorem to estimate the accurate FDR of the identified results.Biological significanceIn the past decades, considerable effort has been devoted to developing a sensitive algorithm for peptide identification in Shotgun proteomics. However, little attention has been paid to controlling the reliability of the identification algorithm at the design stage. The Null-Test based on random matching can be utilized to check whether the algorithm has a tendency to make a mistake or has potential bugs, faultiness, errors etc. However, it turns out that none of the five famous identification software could pass the most stringent Null-Test in the present study, which should be taken into account seriously. Accordingly, a candidate strategy based on fuzzy logics has been demonstrated the possibility that an identification algorithm can pass the Null-Test. PatternLab shows that earlier control on overfitting is valuable for designing an efficient algorithm.Download high-res image (225KB)Download full-size image
Co-reporter:Bo Jiang;Qiong Wu;Lihua Zhang;Yukui Zhang
Nanoscale (2009-Present) 2017 vol. 9(Issue 4) pp:1607-1615
Publication Date(Web):2017/01/26
DOI:10.1039/C6NR09260H
Although selective enrichment of glycopeptides from complex biological samples is indispensable for mass spectrometry (MS)-based glycoproteomics, it still remains a great challenge due to the low abundance of glycoproteins and suppression of non-glycopeptides. In this study, silver nanoparticle-functionalized magnetic graphene oxide nanocomposites (GO/Fe3O4/PEI/Ag) were synthesized. Silver nanoparticles were generated in situ on the surface of magnetic graphene oxide using polyethylenimine as a reducing and stabilizing agent. The resulting material was used as an adsorbent for selective enrichment of glycopeptides. GO/Fe3O4/PEI/Ag nanocomposites offered excellent enrichment ability, which was attributed to the synergistic effect of polyethylenimine and silver nanoparticles. The nanocomposites showed superior specificity for glycopeptides even when non-glycopeptides were 100 times more concentrated than glycopeptides. The nanocomposites displayed advantages including rapid adsorption (1 min), low detection limit (25 fmol), repeatability (6 times), and high recovery (77.8%). Using these nanocomposites, 91 different glycoproteins and 136 N-linked glycopeptides were identified from among 20 μg tryptic human serum proteins and this demonstrated the superior performance of the nanocomposites for glycopeptides enrichment.
Co-reporter:Lukuan Liu, Kaiguang Yang, Xudong Zhu, Yu Liang, Yuanbo Chen, Fei Fang, Qun Zhao, Lihua Zhang, Yukui Zhang
Talanta 2017 Volume 175(Volume 175) pp:
Publication Date(Web):1 December 2017
DOI:10.1016/j.talanta.2017.07.041
•An aptamer-immobilized open tubular capillary column were developed by ATRP and biotin-avidin system.•The immobilized aptamers provided the open tubular column with functions capable of capturing CTCs specifically.•Owing to the compatibility of the column, the captured cells could be analyzed by LC-MS for proteome analysis.Circulating tumor cells hold the key to predicting the prognosis and discovering the therapeutic targets. Herein, we proposed a strategy to develop an aptamer-immobilized open tubular capillary column by which SMMC-7721 human hepatoma cells (SMMC-7721 cells) could be captured with an over 70% of capture efficiency and a 3.0 ± 0.2 of enrichment factor. Owing to the compatibility of the column, the captured cells by the column could be analyzed by LC-MS from protein level and 5 unique proteins of SMMC-7721 cells were identified which could be used as markers to identify SMMC-7721 cells when Jurkat T-leukemia cells (Jurkat cells) were employed as interfering cells. As the key component, the aptamer-immobilized column had the potential to be integrated into the platform for separating, enriching and characterizing rare cells simultaneously.As the key component, a strategy to develop an aptamer-immobilized column was proposed, by which SMMC-7721 cells could be captured with an over 70% of capture efficiency and a 3.0 ± 0.2 of enrichment factor. The captured cells could be analyzed by LC-MS for proteome analysis.Download high-res image (152KB)Download full-size image
Co-reporter:Senwu Li, Kaiguang Yang, Baofeng Zhao, Xiao Li, Lukuan Liu, Yuanbo Chen, Lihua Zhang and Yukui Zhang
Journal of Materials Chemistry A 2016 vol. 4(Issue 15) pp:2739-2739
Publication Date(Web):30 Mar 2016
DOI:10.1039/C6TB90044E
Correction for ‘Epitope imprinting enhanced IMAC (EI-IMAC) for highly selective purification of His-tagged protein’ by Senwu Li et al., J. Mater. Chem. B, 2016, 4, 1960–1967.
Co-reporter:Senwu Li, Kaiguang Yang, Baofeng Zhao, Xiao Li, Lukuan Liu, Yuanbo Chen, Lihua Zhang and Yukui Zhang
Journal of Materials Chemistry A 2016 vol. 4(Issue 11) pp:1960-1967
Publication Date(Web):17 Feb 2016
DOI:10.1039/C5TB02505B
Recombinant protein technology occupies an important position in fields including biopharmaceutics, proteomics, structural and functional biology. However, the purification of His-tagged protein, the majority portion of recombinant protein, is seriously hindered by impurities. These impurities, including host proteins with inherent cysteine and histidine-rich regions or metal centers, are usually beyond the purification ability of commonly used IMAC materials. To remove this barrier, a novel purification material was developed through enhancing the selectivity of IMAC by means of surface epitope imprinting using His-tag, the common terminal of His-tagged protein, as the template. Characterizations including TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy, measurement of DLS size and zeta potential were carried out to prove the fabrication of the imprinted shell. Results exhibited a high imprinting factor of 7.1. Besides, the adsorption kinetics were not affected by the surface imprinted shell and could reach adsorption equilibrium within 15 min. Compared with the substrate IMAC, the novel epitope imprinting enhanced IMAC (EI-IMAC) showed an obvious improvement (5% increase of purity) in the selectivity of His-tagged recombinant protein from crude cell lysis.
Co-reporter:Bo Jiang, Qi Wu, Nan Deng, Yuanbo Chen, Lihua Zhang, Zhen Liang and Yukui Zhang
Nanoscale 2016 vol. 8(Issue 9) pp:4894-4897
Publication Date(Web):02 Feb 2016
DOI:10.1039/C5NR08126B
GO/Fe3O4/Au/PEG nanocomposites were synthesized via bonding gold nanoparticles on magnetic graphene oxide using polyethylenimine as the reducing and immobilizing reagent, followed by thiol-terminal polyethylene glycol immobilization. With the use of this nanocomposite, 255 glycopeptides, mapped to 127 different glycoproteins, were identified from human serum, demonstrating its great potential for glycosylation analysis.
Co-reporter:Senwu Li, Kaiguang Yang, Nan Deng, Yi Min, Lukuan Liu, Lihua Zhang, and Yukui Zhang
ACS Applied Materials & Interfaces 2016 Volume 8(Issue 9) pp:5747
Publication Date(Web):February 24, 2016
DOI:10.1021/acsami.5b11415
Among various artificial antibodies, epitope imprinted polymer has been paid increasingly attention. To modulate the “adsorption and release” behavior by environment stimuli, N-isopropylacrylamide, was adopted to fabricate the thermoresponsive epitope imprinted sites. The prepared imprinted materials could adsorb 46.6 mg/g of target protein with the imprinting factor of 4.0. The template utilization efficiency could reach as high as 8.21%. More importantly, in the real sample, the materials could controllably capture the target protein from the human plasma at 45 °C and release it at 4 °C, which demonstrated the “on-demand” application potentials of such materials in the biomolecule recognition field.Keywords: controllable release; epitope; molecularly imprinting; protein; stimuli-response
Co-reporter:Jianxi Liu, Kaiguang Yang, Wenya Shao, Yanyan Qu, Senwu Li, Qi Wu, Lihua Zhang, and Yukui Zhang
ACS Applied Materials & Interfaces 2016 Volume 8(Issue 15) pp:9552
Publication Date(Web):April 6, 2016
DOI:10.1021/acsami.6b01829
A novel organic–inorganic hybrid particle with high hydrophilicity three-dimensional boronic acid functional polymer branches was facilely synthesized through thiol–ene surface-initiated click reaction, by which the target glycoprotein could be captured selectively in the 5000-fold disrupting protein. This highest selectivity ever reported demonstrated that this boronic acid functionalized particle exhibited great potential in the recognition of cis-diol-containing biomolecules, including the glycoproteins.Keywords: boronic acid; glycoprotein; hybrid particle; polymer branches; thiol−ene click reaction
Co-reporter:Jianxi Liu, Kaiguang Yang, Wenya Shao, Senwu Li, Qi Wu, Shen Zhang, Yanyan Qu, Lihua Zhang, and Yukui Zhang
ACS Applied Materials & Interfaces 2016 Volume 8(Issue 34) pp:22018
Publication Date(Web):August 8, 2016
DOI:10.1021/acsami.6b06343
Because of the low abundance of glycopeptide in natural biological samples, methods for efficient and selective enrichment of glycopeptides play a significant role in mass spectrometry (MS)-based glycoproteomics. In this study, a novel kind of zwitterionic hydrophilic interaction chromatography polymer particles, namely, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@l-Cys (poly(MBAAm-co-MAA)@l-Cys), for the enrichment of glycopeptides was synthesized by a facile and efficient approach that combined distillation precipitation polymerization (DPP) and “thiol–ene” click reaction. In the DPP approach, residual vinyl groups explored outside the core with high density, then the functional ligand cysteine was immobilized onto the surface of core particles by highly efficient thiol–ene click reaction. Taking advantage of the unique structure of poly(MBAAm-co-MAA)@l-Cys, the resulting particles possess remarkable enrichment selectivity for glycopeptides from the tryptic digested human immunoglobulin G. The polymer particles were successfully employed for the analysis of human plasma, and 208 unique glycopeptides corresponding to 121 glycoproteins were reliably identified in triple independent nano-LC-MS/MS runs. The selectivity toward glycopeptides of these particles poly(MBAAm-co-MAA)@l-Cys is ∼2 times than that of the commercial beads. These results demonstrated that these particles had great potential for large-scale glycoproteomics research. Moreover, the strategy with the combination of DPP and thiol–ene click chemistry might be a facile method to produce functional polymer particles for bioenrichment application.Keywords: click chemistry; core−shell structures; glycopeptides; nanoparticles; separation
Co-reporter:Ci Wu, Yu Liang, Kaiguang Yang, Yi Min, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2016 Volume 88(Issue 3) pp:1521
Publication Date(Web):January 11, 2016
DOI:10.1021/acs.analchem.5b04641
A novel clickable periodic mesoporous organosilica monolith with the surface area up to 1707 m2 g–1 was in situ synthesized in the capillary by the one-step condensation of the organobridged-bonded alkoxysilane precursor bis(triethoxysilyl)ethylene. With Si–C bonds in the skeleton, the monolith possesses excellent chemical and mechanical stability. With vinyl groups highly loaded and homogeneously distributed throughout the structure, the monolith can be readily functionalized with functional groups by effective thiol–ene “click” chemistry reaction. Herein, with “click” modification of C18, the obtained monolith was successfully applied for capillary liquid chromatographic separation of small molecules and proteins. The column efficiency could reach 148 000 N/m, higher than most reported hybrid monoliths. Moreover, intact proteins could be separated well with good reproducibility, even after the monolithic column was exposed by basic mobile phase (pH 10.0) overnight, demonstrating the great promising of such monolith for capillary chromatographic separation.
Co-reporter:Yejing Weng, Zhigang Sui, Yichu Shan, Hao Jiang, Yuan Zhou, Xudong Zhu, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2016 Volume 88(Issue 9) pp:4971
Publication Date(Web):April 4, 2016
DOI:10.1021/acs.analchem.6b00910
Secreted proteins play key roles during cellular communication, proliferation, and migration. The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been performed under serum-free conditions. However, these conditions might alter the status of the cells. Herein, we describe an efficient strategy that avoids the disturbance of serum by combining metabolic labeling, protein “equalization,” protein fractionation, and filter-aided sample preparation, called MLEFF, enabling the identification of 534 secreted proteins from HeLa conditioned media, including 31 cytokines, and growth factors. This MLEFF strategy was also successfully applied during a comparative secretome analysis of two human hepatocellular carcinoma cell lines with differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumor invasion and metastasis.
Co-reporter:Fei Fang, Qun Zhao, Zhigang Sui, Yu Liang, Hao Jiang, Kaiguang Yang, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2016 Volume 88(Issue 10) pp:5065
Publication Date(Web):April 18, 2016
DOI:10.1021/acs.analchem.6b01082
Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling.
Co-reporter:Lingfan Chen, Yichu Shan, Yejing Weng, Zhigang Sui, Xiaodan Zhang, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2016 Volume 88(Issue 17) pp:8390
Publication Date(Web):August 17, 2016
DOI:10.1021/acs.analchem.6b02453
The analysis of protein N-termini is of great importance for understanding the protein function and elucidating the proteolytic processing. Herein, we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted N-termini enrichment (HYTANE) to achieve a global N-terminome analysis. The HYTANE strategy showed a high efficiency in hydrophobic tagging and C18 material-assisted depletion using bovine serum albumin (BSA) as the sample. This strategy was applied to N-termini profiling from S. cerevisiae cell lysates and enabled the identification of 1096 protein N-termini, representing the largest N-terminome data set of S. cerevisiae. The identified N-terminal peptides accounted for 99% of all identified peptides, and no deficiency in acidic, histidine (His)-containing, and His-free N-terminal peptides was observed. The presented HYTANE strategy is therefore a highly selective, efficient, and unbiased strategy for the large scale N-terminome analysis. Furthermore, using the HYTANE strategy, we identified 329 cleavage sites and 291 substrates of caspases in Jurkat cells, demonstrating the great promise of HYTANE strategy for protease research. Data are available via ProteomeXchange with identifier PXD004690.
Co-reporter:Kaiguang Yang, Senwu Li, Jianxi Liu, Lukuan Liu, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2016 Volume 88(Issue 11) pp:5621
Publication Date(Web):May 17, 2016
DOI:10.1021/acs.analchem.6b01247
To achieve the simultaneous capture of various target proteins, the multiepitope templates imprinted particles were developed by phase inversion-based poly(ether sulfone) (PES) self-assembly. Herein, with the top three high-abundance proteins in the human plasma, serum albumin, immunoglobulin G, and transferrin, as the target proteins, their N-terminal peptides were synthesized as the epitope templates. After the preorganization of three epitopes and PES in dimethylacetamide, the multiepitope templates imprinted particles were formed in water through self-assembly, by which the simultaneous recognition of three target proteins in human plasma was achieved with high selectivity. Furthermore, the binding kinetics study proved that the adsorption mechanism in this imprinting system toward three epitope templates was the same as that on the single-epitope imprinting polymer. These results demonstrate that our proposed multiepitope templates imprinting strategy might open a new era of artificial antibodies to achieve the recognition of various targets simultaneously.
Co-reporter:Bo Jiang, Yanyan Qu, Lihua Zhang, Zhen Liang, Yukui Zhang
Analytica Chimica Acta 2016 Volume 912() pp:41-48
Publication Date(Web):17 March 2016
DOI:10.1016/j.aca.2016.01.018
•Hydrophilicity GO/PEI/Au composites was prepared for immobilizing 4-MPB.•The immobilized amount of 4-MPB was calculated as 14.3% wt.•The GO/PEI/Au/4-MPB composites showed superior selective for glycopeptide.Selective enrichment and isolation of glycopeptides from complex biological samples was indispensable for mass spectrometry (MS)-based glycoproteomics, however, it remained a great challenge due to the low abundance of glycoproteins and the ion suppression of non-glycopeptides. In this work, 4-mercaptophenylboronic acid functionalized graphene oxide composites were synthesized via loading gold nanoparticles on polyethylenimine modified graphene oxide surface, followed by 4-mercaptophenylboronic acid immobilization by the formation of Au–S bonding (denoted as GO/PEI/Au/4-MPB composites). The composites showed highly specific and efficient capture of glycopeptides due to their excellent hydrophilicity and abundant boronic acid groups. The composites could selectively capture the glycopeptides from the mixture of glycopeptides and nonglycopeptides, even when the amounts of non-glycopeptides were 100 times more than glycopeptides. Compared with commercial meta-amino phenylboronic acid agarose, the composites showed better selectivity when the sample was decreased to 10 ng. These results clearly verified that the GO/PEI/Au/4-MPB composites might be a promising material for glycoproteomics analysis.GO/PEI/Au/4-MPB composites were synthesized, and exhibited high selectivity to capture glycopeptides.Figure optionsDownload full-size imageDownload as PowerPoint slide
Co-reporter:Qun Zhao, Fei Fang, Ci Wu, Qi Wu, Yu Liang, Zhen Liang, Lihua Zhang, Yukui Zhang
Analytica Chimica Acta 2016 Volume 912() pp:58-64
Publication Date(Web):17 March 2016
DOI:10.1016/j.aca.2016.01.049
•An integrated sample preparation method was developed for fast protein analysis.•All the sample treatment processes were performed in situ with high sample recovery.•Proteins were digested by high concentration trypsin with the aid of microwave.•More proteins and peptides were identified while throughput was improved by 14 times.•At least seven times improvement was achieved for protein analysis at nanogram level.An integrated sample preparation method, termed “imFASP”, which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50–500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples.
Co-reporter:Fei Fang, Qun Zhao, Xiao Li, Zhen Liang, Lihua Zhang, Yukui Zhang
Analytica Chimica Acta 2016 Volume 945() pp:39-46
Publication Date(Web):16 November 2016
DOI:10.1016/j.aca.2016.09.032
•A sample preparation method was developed for in-depth membrane proteome profiling.•Our strategy utilized the differential dissolving capability of extraction solvents.•A most comprehensive membrane proteome dataset of HeLa was achieved by our study.•Referred to neXtProt database, 358 missing proteins were discovered in our dataset.•110 of the identified 358 missing proteins were annotated to be membrane proteins.Profiling membrane proteins would facilitate revealing disease mechanism and discovering new drug targets as they play essential roles in cellular signaling, substrate transport, and cell adhesion. However, the analysis of membrane proteins still remains a challenge due to their high hydrophobicity, as well as the suppression effect of high abundant soluble proteins. In this work, to achieve a membrane proteome profiling, a sample preparation strategy based on sequential extraction at the protein level assisted by a range of extraction reagents with different dissolving capabilities, followed by nano-RPLC-ESI-MS/MS analysis was developed and applied for HeLa cell line analysis. It was found that with progressively harsher extraction reagents (i.e., 2 M NaCl, 4 M urea, 0.1 M Na2CO3, and 10% 1-dodecyl-3- methyl-imidazolium chloride (C12ImCl) performed, much more high hydrophobic proteins and low abundant proteins were identified. With our developed strategy, 5553 of the identified proteins (4419 gene products) were annotated to be membrane proteins and 2573 proteins (2183 gene products) have at least one transmembrane domain, to our best knowledge, which is the most comprehensive membrane proteome dataset for HeLa cell line. Notably, 110 of the identified membrane proteins were discovered in the “missing proteins” list referred to those in the neXtProt database. All above results indicated that our strategy has great potential to tackle the difficult but relevant task of identifying and profiling membrane proteins.
Co-reporter:Shen Zhang, Lingfan Chen, Yichu Shan, Zhigang Sui, Qi Wu, Lihua Zhang, Zhen Liang and Yukui Zhang
Analyst 2016 vol. 141(Issue 16) pp:4912-4918
Publication Date(Web):09 Jun 2016
DOI:10.1039/C6AN00388E
The pseudo isobaric peptide termini labeling (IPTL) method is a remarkable strategy in quantitative proteomics, and has been efficiently applied in biological studies due to its high quantitative accuracy. However, irreproducible precursor ion selection caused by data dependent acquisition and the chromatographic shift caused by isotope effects limit the wide application of this method. Herein, we expand the use of pseudo IPTL to SWATH MS application and develop a novel quantitative strategy, termed SWATH-pseudo-IPTL, by which the relative quantification could be achieved by comparing the “complete” extracted ion chromatogram (XIC) intensity of MS/MS scan instead of a single intensity measurement in DDA-pseudo-IPTL which only reflected the peptide abundances at that given time. The quantitative analysis of various proportions of mixed HeLa samples revealed the strong accuracy and precision of our SWATH-pseudo-IPTL method, both of which were better than that of the DDA-pseudo-IPTL strategy. SWATH-pseudo-IPTL was also applied to the quantitative profiling of the proteome from human hepatocellular carcinoma cell lines with high and low metastatic potential, and most of the differentially expressed proteins were related to tumorigenesis and tumor metastasis, demonstrating the feasibility of this methodology for biological applications.
Co-reporter:Yejing Weng, Zhigang Sui, Yichu Shan, Yechen Hu, Yuanbo Chen, Lihua Zhang and Yukui Zhang
Analyst 2016 vol. 141(Issue 15) pp:4640-4646
Publication Date(Web):04 May 2016
DOI:10.1039/C6AN00892E
Exosomes are secreted nanovesicles shed by almost all kinds of cells. Recently, increased interest has been focused on these extracellular vesicles as natural carriers transporting biological contents for intercellular communication. However, current isolation techniques, such as ultracentrifugation, are not convenient and often require specialized equipment. Herein, we describe a polyethylene glycol (PEG)-based approach, which could permit facile, low-cost and effective isolation of exosomes from cell culture supernatant. High-resolution electron microscopes clearly visualized the size and morphology of isolated exosome aggregates, implying the mechanism of PEG-based precipitation. Combined with tandem mass spectrometry analysis, 6299 protein groups encoded by 5120 genes were successfully characterized from HeLa cell culture supernatant, including numerous exosome proteins which could overlap 97% of the Top 100 exosome marker proteins recorded in the ExoCarta database, as well as a series of low-abundance cytokines and biomarkers. Furthermore, we found a higher ratio of neo-cleavage sites in proteins identified from exosomes compared with cellular proteins, revealing the potential roles of exosomes in accumulation and transportation of protein degradation intermediates.
Co-reporter:Nan Deng, Bo Jiang, Yuanbo Chen, Zhen Liang, Lihua Zhang, Yu Liang, Kaiguang Yang, Yukui Zhang
Journal of Chromatography A 2016 Volume 1427() pp:16-21
Publication Date(Web):4 January 2016
DOI:10.1016/j.chroma.2015.12.018
•Apt/Au/PEI/GO nanocomposites were prepared by a facile method.•Au/PEI/GO nanocomposites were used as a aptamer immobilization substrate.•The immobilizing amount of aptamer was high to 36.1 nmol/mg.•Apt/Au/PEI/GO nanocomposites showed high capture efficiency and high recovery.The specific recognition toward target proteins from complex biological samples has great potential in clinical diagnostics and therapeutics, receiving more and more attention. Herein, we achieved the specific detection of human α-thrombin from human serum by aptamer-conjugated gold functionalized graphene oxide nanocomposites (denoted as Apt/Au/PEI/GO nanocomposites). Gold functionalized graphene oxide nanocomposites were synthesized by in situ growth of Au nanoparticles on graphene oxide surface using polyethylenimine as reducing and stabilizing reagents, and then it was used as support for aptamer immobilization through forming an Au–S bonding. The obtained Apt/Au/PEI/GO nanocomposites inherited not only the large surface area which made the immobilizing amount of aptamer up to 36.1 nmol/mg, but also the excellent hydrophilicity which showed remarkable selectivity for human α-thrombin specific recognition, even with the interference of 3000 fold human serum proteins. Furthermore, with its superior properties, Apt/Au/PEI/GO nanocomposites showed advantages of high capture efficiency (>86%) and excellent recognition repeatability. Finally, the Apt/Au/PEI/GO nanocomposites were successfully applied for human α-thrombin specific recognition in human serum, verifying its great potential in clinical applications.
Co-reporter:Lingfan Chen;Yichu Shan;Yejing Weng
Analytical and Bioanalytical Chemistry 2016 Volume 408( Issue 14) pp:3867-3874
Publication Date(Web):2016 May
DOI:10.1007/s00216-016-9476-1
The analysis of protein C-termini is of great importance, because it not only provides valuable information about protein function, but also facilitates the elucidation of proteolytic processing. However, even with the recent methods for the global profiling of protein C-termini, the identification of C-termini is still far behind that of N-termini due to the lack of basic residue and low reactive carboxyl group. Therefore, an unbiased and complementary method for C-termini profiling is imperative. In this work, we developed a negative enrichment strategy to achieve the in-depth analysis of C-terminome. Proteins were firstly amidated to block carboxyl groups, followed by lysyl endoproteinase (LysC) digestion to generate C-terminal peptides with α-amines and internal peptides bearing both α- and ε-amines. After the α-amines were blocked by site-selective dimethylation or succinylation, the remaining ε-amines on internal peptides were labeled with phosphate groups. Finally, internal peptides were depleted by TiO2, leaving exclusively the fraction of C-terminal peptides for LC-MS/MS analysis. With Escherichia coli (E. coli) digests as the sample, the efficiency of amidation, dimethylation/succinylation, phosphate labeling and TiO2 depletion was proved high. With the combination of dimethyl and succinic blocking strategy, our method enabled the identification of 477 unique C-terminal peptides in E. coli. In comparison with the C-terminal amine-based isotope labeling of substrates (C-TAILS) method, 83 C-termini were identified by both methods, whereas 369 C-termini were unique to C-TAILS and 394 to our dataset. The method proposed is therefore efficient and possibly promotes the comprehensive profiling of C-termini.
Co-reporter:Hao Jiang, Huiming Yuan, Yanyan Qu, Yu Liang, Bo Jiang, Qi Wu, Nan Deng, Zhen Liang, Lihua Zhang, Yukui Zhang
Talanta 2016 Volume 146() pp:225-230
Publication Date(Web):1 January 2016
DOI:10.1016/j.talanta.2015.08.037
•An amide functionalized hydrophilic monolithic capillary column was synthesized.•The column was of stable structure and nice permeability.•High selectivity and good specificity for glycopeptide enrichment were achieved.•We successfully apply it for the N-glycosylation sites profiling of real samples.In this study, a novel kind of amide functionalized hydrophilic monolith was synthesized by the in situ photo-polymerization of N-vinyl-2-pyrrolidinone (NVP), acrylamide (AM), and N, N’-methylenebisacrylamide (MBA) in a UV transparent capillary, and successfully applied for hydrophilic interaction chromatography (HILIC) based enrichment of N-linked glycopeptides. With 2 μg of the tryptic digests of IgG as the sample, after enrichment, 18 glycopeptides could be identified by MALDI-TOF/TOF MS analysis. Furthermore, with the mixture of BSA and IgG digests ( 10,000:1, m/m) as the sample, 6 N-linked glycopeptides were unambiguously identified after enrichment, indicating the high selectivity and good specificity of such material. Moreover, such a monolithic capillary column was also applied for the N-glycosylation sites profiling of 6 μg protein digests from HeLa cells and 1 μL human serum. In total, 530 and 262 unique N-glycosylated peptides were identified, respectively, corresponding to 282 and 124 N-glycoproteins, demonstrating its great potential for the large scale glycoproteomics analysis.
Co-reporter:Yuanbo Chen, Nan Deng, Ci Wu, Yu Liang, Bo Jiang, Kaiguang Yang, Zhen Liang, Lihua Zhang, Yukui Zhang
Talanta 2016 Volume 154() pp:555-559
Publication Date(Web):1 July 2016
DOI:10.1016/j.talanta.2016.02.054
•Aptamer functionalized monolith with AuNPs modification was prepared.•The monolithic column was homogeneous and with stable structure.•High selectivity and high recovery for target protein were achieved.•The α-thrombin in human plasma was detected with high sensitivity.Low abundant proteins of body fluids participate nearly all physiological processes and indicate various kinds of diseases. The development of specific enrichment techniques is the key to identify and quantify the low abundant proteins. Herein, a novel kind of aptamer functionalized hydrophilic polymer monolith was developed for the specific enrichment and detection of human α-thrombin from the human plasma. Human α-thrombin aptamer, with thiol group modified at the 5′ terminal, was immobilized on the gold nanoparticles (AuNPs) modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate) monolithic column, with the binding capacity of 277.1 μmol/L. Due to the hydrophilic poly(ethylene glycol) diacrylate) as the cross-linking monomer, the detection recovery of the aptamer-functionalized hydrophilic polymer monolithic column could reach to 92.6±5.2% (n=3) and the dynamic range could reach 0.5–300 ng/μL (S/N>10) with on-line UV detection. Meanwhile, the column could run over 100 times, because the poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate) stability structure and the AuNPs improved the stability of the matrix material. Furthermore, this column could even capture the target α-thrombin, which was spiked in 1000 folds of original human plasma. All these results demonstrated the great potential of the prepared aptamer functionalized hydrophilic polymer monolith for the recognition of the trace proteins in the biological samples.
Co-reporter:Xudong Zhu, Yu Liang, Yejing Weng, Yuanbo Chen, Hao Jiang, Lihua Zhang, Zhen Liang, and Yukui Zhang
Analytical Chemistry 2016 Volume 88(Issue 23) pp:
Publication Date(Web):November 9, 2016
DOI:10.1021/acs.analchem.6b03422
To improve the stability and sensitivity of nanoelectrospray for liquid chromatography-mass spectroscopy (LC-MS) analysis, we present a new method to fabricate gold-coated emitters. Via gravity-assisted etching self-termination, the emitter with a tapered outer surface and a straight inner surface is prepared with good reproducibility, without the need of fluid introduced to protect internal surface during etching. Followed by electroless deposition, the emitter is further coated with gold film homogeneously, by which the relative standard deviation (RSD) value of total ion current in 160 h is <5%, showing good stability. Compared to that obtained by a commercial emitter, the identified protein number from 2 μg HeLa cell digests is increased over 10%, contributed by the stable electrospray and improved signal intensity of peptides. Furthermore, the integrated gold-coated emitter is prepared at the end of the ultranarrow-bore packed column (inner diameter of 25 μm), and 218 proteins are identified from 2 ng HeLa cell digests. All of these results demonstrate the great promise of such emitters for use in ultrasensitive proteome analysis.
Co-reporter:Wenya Shao, Jianxi Liu, Kaiguang Yang, Yu Liang, Yejing Weng, Senwu Li, Zhen Liang, Lihua Zhang, Yukui Zhang
Talanta 2016 Volume 158() pp:361-367
Publication Date(Web):1 September 2016
DOI:10.1016/j.talanta.2016.05.034
•Branched copolymer modified HILIC materials were obtained by thiol-ene click reaction.•Hydrophilicity was improved benefitting from the branched copolymer.•The separations of polar compounds, sugars and peptides were successfully performed.•High selectivity of glycopeptide capture was achieved by the assisted hydrogen-bond interaction.Hydrophilic interaction chromatography (HILIC) has attracted increasing attention in recent years due to its efficient application in the separation of polar compounds and the enrichment of glycopeptides. However, HILIC materials are still of weak hydrophilicity and thereby present weak retention and selectivity. In this work, branched copolymer modified hydrophilic material Sil@Poly(THMA-co-MBAAm), with high hydrophilicity and unique “claw-like” polyhydric groups, were prepared by “grafting from” thiol-ene click reaction. Due to the abundant functional groups provided by branched copolymer, the material showed excellent retention for nucleosides, necleobases, acidic compounds, sugars and peptides. Furthermore, Sil@Poly(THMA-co-MBAAm) was also applied for the N-glycosylation sites profiling towards the digests of the mouse brain, and 1997N-glycosylated peptides were identified, corresponding to 686 glycoprotein groups. Due to the assisted hydrogen-bond interaction, the selectivity for glycopeptide enrichment in the real sample reached 94.6%, which was the highest as far as we know. All these results indicated that such hydrogen-bond interaction assisted branched copolymer HILIC material possessed great potential for the separation and large scale glycoproteomics analysis.
Co-reporter:Zhigang Sui, Yejing Weng, Qun Zhao, Nan Deng, Fei Fang, Xudong Zhu, Yichu Shan, Lihua Zhang, Yukui Zhang
Talanta 2016 Volume 161() pp:541-546
Publication Date(Web):1 December 2016
DOI:10.1016/j.talanta.2016.08.083
•Direct aggregation of [C12-mim]Cl with proteoglycans on the surface of cartilage.•Exclusion of proteoglycans and collagens during the protein extraction.•Selective extraction of cellular proteins from cartilage.•Facile preparation procedure and short processing time.•Dramatic improvement in identification of proteins, especially membrane proteins.The cartilage zone of the velvet antler is richly vascularized, this being a major difference to the classical cartilage, in which there are no blood vessels. Angiogenesis and rapid growth of vasculature in velvet antler cartilage (VAC) make it an ideal model for discovering the novel angiogenic regulatory factors. However, the proteomic analysis of VAC is challenging due to the serious interference of proteoglycans (PGs) and collagens. To achieve a comprehensive proteome characterization of VAC, herein, we developed an ionic liquid-based method using 1-dodecyl-3-methylimidazolium chloride ([C12-mim]Cl) for selective extraction of cellular proteins from VAC. Compared with the previous cetylpyridinium chloride (CPC)-based method, the developed [C12-mim]Cl-based method takes much less processing time, shows facile preparation procedure and good compatibility towards downstream proteomic analysis, leading to the identification of more protein groups (1543 vs 753), membrane proteins (663 vs 279) and transmembrane proteins (217 vs 58).
Co-reporter:Lukuan Liu;Kaiguang Yang;Lihua Zhang;Yukui Zhang
Science Bulletin 2016 Volume 61( Issue 24) pp:1890-1891
Publication Date(Web):2016 December
DOI:10.1007/s11434-016-1207-7
Co-reporter:Yanyan Qu, Jianxi Liu, Kaiguang Yang, Qi Wu, Yichu Shan, Lihua Zhang, Zhen Liang and Yukui Zhang
Journal of Materials Chemistry A 2015 vol. 3(Issue 19) pp:3927-3930
Publication Date(Web):15 Apr 2015
DOI:10.1039/C5TB00156K
Biocompatible boronate core–shell polymeric particles were grown in a unique polymerization system via a one-pot strategy making full use of the residual soluble boronate oligomer to in situ build the core–shell structure. The obtained submicron particles were shown to exhibit excellent recognition affinity toward glycoproteins with high binding capacity and specificity.
Co-reporter:Jianxi Liu, Kaiguang Yang, Yanyan Qu, Senwu Li, Qi Wu, Zhen Liang, Lihua Zhang and Yukui Zhang
Chemical Communications 2015 vol. 51(Issue 18) pp:3896-3898
Publication Date(Web):30 Jan 2015
DOI:10.1039/C4CC10004B
Stimuli-responsive core–shell nanoparticles, with 3-acrylamidophenyl boronic acid (APBA) as the functional group, were synthesized via combined distillation precipitation polymerization and RAFT media precipitation polymerization. The well-defined boronic acid-polymer branch on the nanoparticle surface displayed high binding capacity and good selectivity towards cis-diol-containing molecules.
Co-reporter:Senwu Li, Kaiguang Yang, Jianxi Liu, Bo Jiang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2015 Volume 87(Issue 9) pp:4617
Publication Date(Web):April 17, 2015
DOI:10.1021/ac5047246
The specific recognition of biomolecules by artificial antibodies has inspired fascination among chemists and biologists. Herein, we propose a new method to prepare epitope-oriented surface-imprinted nanoparticles with high template utilization efficiency. Using a His-tag as the anchor to facilitate the epitope immobilization/removal and the self-polymerization of dopamine to control the imprinted shell thickness, the prepared epitope-imprinted nanoparticles show specific recognition of the target protein. Moreover, with improved hydrophilicity of the His-tag-anchored epitope, this method opens up a universal route for imprinting epitopes with various polarities.
Co-reporter:Shen Zhang, Qi Wu, Yichu Shan, Yuan Zhou, Lihua Zhang, Yukui Zhang
Journal of Proteomics 2015 Volume 114() pp:152-160
Publication Date(Web):30 January 2015
DOI:10.1016/j.jprot.2014.11.014
•Combine the advantages of quantitative strategies based on MS and MS/MS spectrum•Easy manipulation of the total labeling process•Any samples including cells, tissues and body fluids can be quantified.•Accurate quantification of Hca-F and Hca-P samples was performed.Isotopic labeling and isobaric labeling are two kinds of the typical quantification method that have been widely used in proteomics analysis. Herein, a novel quantitative strategy, partially isobaric peptide termini labeling (PITL), was developed to overcome the drawbacks of each above-mentioned labeling strategy, by simultaneously collecting the quantitative information from both MS and MS/MS spectrum. PITL is based on the site-selective N-terminus dimethylation labeling of peptide α-N-termini and the free ε-amino group of lysines, resulting in the partially isobaric labeling of peptides. The relative quantification can then be achieved by comparing the intensities of precursor ions in MS spectra and a, b and y ions in MS/MS spectra. The quantitative analysis of differently labeled yeast digests pooled with various ratios indicated the good quantitative accuracy, reproducibility, coverage and wide dynamic range of PITL strategy. Furthermore, PITL was applied to the quantitative proteome analysis of two mouse hepatocarcinoma ascites syngeneic cell lines with low and high lymph node metastasis rates (Hca-F and Hca-P). Given its low cost, simple operation and good accuracy, PITL might have great potential in the quantitative proteome analysis of biological samples.Biological significanceThe partially isobaric peptide termini labeling (PITL) method enabled to simultaneously obtain the quantitative information from MS and MS/MS spectrum, which combined the advantages of these two strategies. Relative quantification could be achieved by comparing the intensities of parent ions in MS spectra and a, b, y ions in the MS/MS spectra. The quantitative analysis for differently labeled yeast digests mixed at various ratios indicated the good accuracy, reproducibility, quantitative coverage and wide dynamic range of the PITL strategy. Finally, we found 84 differentially expressed proteins in mouse hepatocarcinoma ascites syngeneic cell lines with low and high lymph node metastasis rates with PITL strategy and 77 proteins of them were consistently quantified in our previous studies.
Co-reporter:Shen Zhang, Huiming Yuan, Baofeng Zhao, Yuan Zhou, Hao Jiang, Lihua Zhang, Zhen Liang and Yukui Zhang
Analyst 2015 vol. 140(Issue 15) pp:5227-5234
Publication Date(Web):27 May 2015
DOI:10.1039/C5AN00887E
A novel automated integrated platform for quantitative proteome analysis was established with a combination of online digestion of proteins and in situ18O labeling by an immobilized enzyme reactor (IMER); digests were captured and desalted by a C18 trap column, and peptides were analyzed by nanoRPLC-ESI-MS/MS. Bovine serum albumin (BSA) was used to evaluate the performance of the developed platform. Compared with traditional offline methods, not only the digestion and labeling time was shortened from 36 h to just 1 h, but also the labeling efficiency was improved from 95% to 99%. Furthermore, the back-exchange from 18O to 16O could also be efficiently avoided by the use of IMER. The platform was further evaluated by the quantitative analysis of 100 ng 18O and 16O online labeled yeast sample with a mixing ratio of 1:1, and the results showed significantly improved sensitivity and reproducibility, as well as improved quantitative accuracy than offline method. With these advantages, the integrated platform was finally applied to the quantitative profiling of 100 ng proteins extracted from two mouse hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastases rates, and ten differentially expressed proteins were successfully found, most of which were related to tumorigenesis and tumor metastasis. All these results demonstrate that the developed integrated platform can provide a new way for high efficiency 18O labeling and the quantitative analysis of trace amounts of sample with high accuracy and high reproducibility.
Co-reporter:Yi Min, Zhigang Sui, Zhen Liang, Lihua Zhang, Yukui Zhang
Journal of Pharmaceutical and Biomedical Analysis 2015 Volume 114() pp:247-253
Publication Date(Web):10 October 2015
DOI:10.1016/j.jpba.2015.05.035
•Teicoplanin bonded sub-2 μm superficially porous particles were synthesized.•The particles were of narrow particle size distribution and large surface area.•The enantioseparation of native amino acids was achieved with high resolution and short analysis time.Superficially porous particles (SPPs) demonstrate superior efficiency than totally porous particles in chiral separations. In order to obtain high efficiency and fast separation, sub-2 μm SPPs with high surface area are synthesized, and with teicoplanin bonded, such materials are successfully applied into the rapid enantioseparation of native amino acids. In brief, 1.27 ± 0.06 μm nonporous silica particles are prepared by a modified seeded growth method, followed by mesoporous shell fabrication via one-pot templated dissolution and redeposition strategy, and pore size expansion via acid-refluxing. The diameter of the formed SPPs is 1.49 ± 0.04 μm, with the shell thickness as 206 nm. Nitrogen physisorption experiments show that the Brunauer–Emmett–Teller (BET) specific surface area is 213.6 m2/g and pore size is 9 nm. After teicoplanin derivatization with bonding capacity as 83.3 μmol/g, the prepared chiral stationary phase is packed into a stainless steel tube with the geometry of 50 mm × 2.1 mm i.d.. In less than 6.4 min, six native amino acids (norleucine, alanine, valine, methionine, leucine, norvaline) are enantioseparated with resolution factors ranging from 1.9 to 5.0. Besides, the resolution for chiral separation is improved with ethanol-water instead of methanol-water as the mobile phase. Moreover, the low temperature gives higher resolution, but longer retention time and higher backpressure. Finally, the effect of flow rate on enantiomeric separation is studied and fast chiral separation within 1 min is obtained with flow rate of 0.4 mL/min. All these results show that the synthesized teicoplanin bonded sub-2 μm SPPs have great potential to achieve the enantioseparation of native amino acids with high resolution and rapid speed.
Co-reporter:Yi Min, Kaiguang Yang, Zhen Liang, Lihua Zhang and Yukui Zhang
RSC Advances 2015 vol. 5(Issue 33) pp:26269-26272
Publication Date(Web):06 Mar 2015
DOI:10.1039/C4RA16890A
Dandelion-like core–shell silica microspheres with hierarchical pores are synthesized by condensing a dissolved silica species on the surface of homologous nonporous silica particles in a microemulsion system formed by the complexes of N,N-dimethyldecylamine and hexadecyltrimethylammonium chloride. The microspheres have good monodispersity and center radial pores.
Co-reporter:Si-Min Xia, Hui-Ming Yuan, Zheng Liang, Li-Hua Zhang, Yu-Kui Zhang
Chinese Chemical Letters 2015 Volume 26(Issue 9) pp:1068-1072
Publication Date(Web):September 2015
DOI:10.1016/j.cclet.2015.05.042
In this work, a novel kind of particulate capillary precolumns with double-end polymer monolithic frits has been developed. Firstly, the polymer monolithic frit at one end was prepared via photo-initiated polymerization of a mixture of lauryl methacrylate and ethyleneglycol dimethacrylate with 1-propanol and 1,4-butanediol as porogens and 2,2-dimethoxy-2-phenylacetophenone as a photo-initiator in UV transparent coating capillary (100 μm i.d.). Subsequently, C18 particles (5 μm, 100 Å) were packed into the capillary, and sealed with the polymer monolithic frit at another end. To prevent the reaction of monomers and C18 particles, the packed C18 particles were masked during UV exposure. The loading capacity of such a precolumn was determined to be about 9 μg by frontal analysis with a synthetic peptide APGDRIYVHPF as a model sample. Furthermore, two parallel precolumns were incorporated into a two-dimensional nano-liquid chromatography (2D nano-LC) system with dual capillary trap columns for peptide trapping and concentration. Compared to 2D nano-LC system with a single trap column, such two dimensional separations could be operated simultaneously to improve the analysis throughput. All these results demonstrated that such capillary precolumns with double frits would be promising for high-throughput proteome analysis.2D-nano-SCX-dual capillary trap columns-RPLC–MS/MS platform constructed with double-end polymer monolithic frits capillary precolumns.
Co-reporter:Zhi HUANG, Nan DENG, Guo-Quan YAN, Ming-Xia GAO, Zhen LIANG, Li-Hua ZHANG, Xiang-Min ZHANG, Yu-Kui ZHANG
Chinese Journal of Analytical Chemistry 2015 Volume 43(Issue 10) pp:1472-1478
Publication Date(Web):October 2015
DOI:10.1016/S1872-2040(15)60865-9
The human plasma proteome has the characteristics of complexity in component and large dynamic range of protein concentrations. Herein, an array-based online two dimensional liquid chromatography system combined with protein equalizer technology was developed for the large-scale depletion of high abundance proteins and enrichment of low abundance proteins in human plasma. The array-based online two dimensional liquid chromatography system could be used to separate the plasma at the intact protein level with good reproducibility and high throughput. The total separation time was only 4 h and the fast location of high abundance proteins was also achieved. After the high abundance protein fractions was treated by ampholine@PM polymer microsphere, the number of identified low abundance proteins increased ten-fold, which significantly decreased the loss of low abundance proteins in high abundance protein fractions. The techniques combined were then applied to perform the proteomic analysis of human plasma sample. The total number of identified proteins was 1474 and the dynamic range of protein concentration was 7. In this work, 252 proteins were identified in high abundance protein fractions, among which 61 proteins belonged to high abundance proteins. These results demonstrated that an array-based online two dimensional liquid chromatography system combined with protein equalizer technology could efficiently achieve the large-scale depletion of high abundance proteins and the enrichment of low abundance proteins in human plasma, with a remarkable improvement in protein identification and a great prospect in the proteomic research of other complex samples.An array-based online two dimensional liquid chromatography system combined with protein equalizer technology was developed. This strategy could achieve fast location and large-scale depletion of high abundance proteins, as well as the enrichment of low abundance proteins in human plasma.
Co-reporter:Simin Xia, Huiming Yuan, Yuanbo Chen, Zheng Liang, Lihua Zhang, Yukui Zhang
Talanta 2015 Volume 141() pp:235-238
Publication Date(Web):15 August 2015
DOI:10.1016/j.talanta.2015.04.011
• A novel integrated device for SDS-assisted proteome analysis was developed.• The integrated device was constructed by an HFMI and an IMER.• Online SDS removal and protein digestion was achieved by the integrated device.• The device was applied for the analysis of SDS extracted proteins from rat brain.In this work, a novel integrated sample preparation device for SDS-assisted proteome analysis was developed, by which proteins dissolved in 4% (w/v) SDS were first diluted by 50% methanol, and then SDS was online removed by a hollow fiber membrane interface (HFMI) with 50 mM ammonium bicarbonate (pH 8.0) as an exchange buffer, finally digested by an immobilized enzyme reactor (IMER). To evaluate the performance of such an integrated device, bovine serum albumin dissolved in 4% (w/v) SDS as a model sample was analyzed; it could be found that similar to that obtained by direct analysis of BSA digests without SDS (the sequence coverage of 60.3±1.0%, n=3), with HFMI as an interface for SDS removal, BSA was identified with the sequence coverage of 61.0±1.0% (n=3). However, without SDS removal by HFMI, BSA could not be digested by the IMER and none peptides could be detected. In addition, such an integrated sample preparation device was also applied for the analysis of SDS extracted proteins from rat brain, compared to those obtained by filter-aided sample preparation (FASP), not only the identified protein group and unique peptide number were increased by 12% and 39% respectively, but also the sample pretreatment time was shortened from 24 h to 4 h. All these results demonstrated that such an integrated sample preparation device would provide an alternative tool for SDS assisted proteome analysis.
Co-reporter:Qinran Li, Kaiguang Yang, Yu Liang, Bo Jiang, Jianxi Liu, Lihua Zhang, Zhen Liang, and Yukui Zhang
ACS Applied Materials & Interfaces 2014 Volume 6(Issue 24) pp:21954
Publication Date(Web):December 1, 2014
DOI:10.1021/am5072783
A novel kind of lysozyme (Lys) surface imprinted core–shell particles was synthesized by reversible addition–fragmentation chain transfer (RAFT) strategy. With controllable polymer shell chain length, such particles showed obviously improved selectivity for protein recognition. After the RAFT initial agent and template protein was absorbed on silica particles, the prepolymerization solution, with methacrylic acid and 2-hydroxyethyl methacrylate as the monomers, and N,N′-methylenebis(acrylamide) as the cross-linker, was mixed with the silica particles, and the polymerization was performed at 40 °C in aqueous phase through the oxidation–reduction initiation. Ater polymerization, with the template protein removal and destroying dithioester groups with hexylamine, the surface Lyz imprinted particles were obtained with controllable polymer chain length. The binding capacity of the Lys imprinted particles could reach 5.6 mg protein/g material, with the imprinting factor (IF) as 3.7, whereas the IF of the control material prepared without RAFT strategy was only 1.6. The absorption equilibrium could be achieved within 60 min. Moreover, Lys could be selectively recognized by the imprinted particles from both a four-proteins mixture and egg white sample. All these results demonstrated that these particles prepared by RAFT strategy are promising to achieve the protein recognition with high selectivity.Keywords: core−shell particles; lysozyme; molecularly imprinting; protein recognition; RAFT
Co-reporter:Jianxi Liu, Yanyan Qu, Kaiguang Yang, Qi Wu, Yichu Shan, Lihua Zhang, Zhen Liang, and Yukui Zhang
ACS Applied Materials & Interfaces 2014 Volume 6(Issue 3) pp:2059
Publication Date(Web):January 14, 2014
DOI:10.1021/am405144x
The development of a highly specific recognition system for glycoprotein capture from complex biological samples is a prerequisite to the success of mass spectra-based glycoproteomics analysis. To achieve this purpose, a one-pot precipitation polymerization (PP) strategy with a novel solvent system composed of water/ethanol (4:1, v/v) is described for preparing boronate-affinity polymeric micro/nano particles using 4-vinylphenylboronic acid (VPBA) as the functional monomer and N,N′-methylenebis(acrylamide) (MBA) alone or together with divinylbenzene (DVB) as the cross-linker(s). The proposed polymerization strategy not only affords monodisperse polymeric submicrometer particles with a smooth surface and controllable size, ranging from 300 to 700 nm, but also increases the loading capacity of boronic acid, which could hardly be achieved by other polymerization methods, thus leading to the specific recognition of glycoproteins. The effects of solvent systems and monomers on the morphology and boronate-affinity capacity of prepared materials were further investigated based on the Flory–Huggins model. It was proved that the reaction rate of various monomers during particle formation might be the key factor affecting the affinity capacity for glycoproteins. Our results demonstrated that under the theoretical guidance of the Flory–Huggins model the PP strategy with a selected monomer and solvent system might provide a good approach to prepare submicrometer polymer particles with plenty of boronic acid groups on the surface to achieve a highly selective enrichment of glycoproteins.Keywords: boronate affinity; Flory−Huggins model; glycoprotein response; one pot; polymerization;
Co-reporter:Kaiguang Yang, Jianxi Liu, Senwu Li, Qinran Li, Qi Wu, Yuan Zhou, Qun Zhao, Nan Deng, Zhen Liang, Lihua Zhang and Yukui Zhang
Chemical Communications 2014 vol. 50(Issue 67) pp:9521-9524
Publication Date(Web):30 Jun 2014
DOI:10.1039/C4CC03428G
Polymer self-assembly was developed as an epitope imprinting strategy involving facile processes and high recognition site density. As a model, transferrin epitope imprinted polyethersulfone (PES) beads were successfully fabricated using this technique. The imprinted beads demonstrated excellent selectivity toward the transferrin epitope and transferrin even in the real sample.
Co-reporter:Qun Zhao, Fei Fang, Yu Liang, Huiming Yuan, Kaiguang Yang, Qi Wu, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2014 Volume 86(Issue 15) pp:7544
Publication Date(Web):June 18, 2014
DOI:10.1021/ac5013267
Due to their extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenge. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference with MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs. Compared with other commonly used additives, such as sodium dodecyl sulfate (SDS), Rapigest, and methanol, C12Im-Cl showed the best performance. In addition, with a strong cation exchange trap column, it could be easily removed after trypsin digestion, which not only was beneficial to avoid protein precipitation during digestion but also had no adverse effect on LC-MS-based separation and detection. Such a C12Im-Cl-assisted sample preparation method was further applied to the membrane proteome analysis of rat brain. Compared with the SDS-assisted method, 1.4 and 3.5 times improvement on the identified IMP and hydrophobic peptide number were achieved (251 vs 178, and 982 vs 279). All these results demonstrated that the C12Im-Cl-assisted sample preparation method is of great promise to promote the large-scale membrane proteome profiling.
Co-reporter:Nan Deng, Guijie Zhu, Yuanbo Chen, Qi Wu, Zhen Liang, Zhigang Sui, Lihua Zhang, Kaiguang Yang, Yukui Zhang
Analytica Chimica Acta 2014 Volume 826() pp:43-50
Publication Date(Web):15 May 2014
DOI:10.1016/j.aca.2014.04.004
•Dynamic range reduction in abundance of plasma by ampholine immobilized microspheres fractionation.•High efficiency and good complementary of three-step elution and on-particle digestion.•Promotion the deep coverage study of human plasma proteome.A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2 M NaCl, 100 mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6–97.2%, and the identified low abundance protein (<100 ng mL−1) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.
Co-reporter:Yejing Weng, Yanyan Qu, Hao Jiang, Qi Wu, Lihua Zhang, Huiming Yuan, Yuan Zhou, Xiaodan Zhang, Yukui Zhang
Analytica Chimica Acta 2014 Volume 833() pp:1-8
Publication Date(Web):23 June 2014
DOI:10.1016/j.aca.2014.04.037
•An integrated platform for quantitative N-glycoproteome analysis was established.•On-line enrichment, deglycosylation and labeling could be achieved within 160 min.•A N2-assisted interface was applied to improve the compatibility of the platform.•The platform exhibited improved quantification accuracy, precision and throughput.Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N2-assisted HILIC–RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD = 6.2%, n = 3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD = 11.6%, n = 3). Additionally, the total on-line sample preparation time was greatly shortened to 160 min, much faster than that of offline approach (24 h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log 2 ratios (H/L) of quantified glycopeptides ranged from −1 to 1, demonstrating high accuracy of the developed sample preparation strategy. By triplicated analysis of glycopeptides and non-glycopeptides of Hca-F and Hca-P lysates, 43 up-regulated and 30 down-regulated (Hca-F/P) N-glycosylation sites, and 11 significantly changed N-glycoproteins were successfully quantified, and most of them were related to tumorigenesis and tumor metastasis. All these results demonstrate the developed integrated N-glycoprotein pretreatment platform is of great power for the accurate, precise and high-throughput analysis of N-glycoproteomes.
Co-reporter:Yi Min, Bo Jiang, Ci Wu, Simin Xia, Xiaodan Zhang, Zhen Liang, Lihua Zhang, Yukui Zhang
Journal of Chromatography A 2014 Volume 1356() pp:148-156
Publication Date(Web):22 August 2014
DOI:10.1016/j.chroma.2014.06.049
•Sub-2 μm superficially porous packing materials were synthesized.•Pore diameter could be well tailored.•Radially oriented pores were obtained.•Column efficiency over 200,000 plates per m was demonstrated.•Rapid separation of peptides and proteins was achieved.In this work, 1.9 μm reversed-phase packing materials with superficially porous structure were prepared to achieve the rapid and high efficient separation of peptides and proteins. The silica particles were synthesized via three steps, nonporous silica particle preparation by a modified seeded growth method, mesoporous shell formation by a one pot templated dissolution and redeposition strategy, and pore size expansion via acid-refluxing. By such a method, 1.9 μm superficially porous materials with 0.18 μm shell thickness and tailored pore diameter (10 nm, 15 nm) were obtained. After pore enlargement, the formerly dense arrays of mesoporous structure changed, the radially oriented pores dominated the superficially porous structure. The chromatographic performance of such particles was investigated after C18 derivatization. For packing materials with 1.9 μm diameter and 10 nm pore size, the column efficiency could reach 211,300 plates per m for naphthalene. To achieve the high resolution separation of peptides and proteins, particles with pore diameter of 15 nm were tailored, by which the baseline separation of 5 peptides and 5 intact proteins could be respectively achieved within 1 min, demonstrating the superiority in the high efficiency and high throughput analysis of biomolecules. Furthermore, BSA digests were well separated with peak capacity of 120 in 30 min on a 15 cm-long column. Finally, we compared our columns with a 1.7 μm Kinetex C18 column under the same conditions, our particles with 10 nm pore size demonstrated similar performance for separation of the large intact proteins. Moreover, the particles with 15 nm pore size showed more symmetrical peaks for the separation of large proteins (BSA, OVA and IgG) and provided rapid separation of protein extracts from Escherichia coli in 5 min. All these results indicated that the synthesized 1.9 μm superficially porous silica packing materials would be promising in the ultra-fast and high-resolution separation of biomolecules.
Co-reporter:Huiming Yuan, Lihua Zhang, Yukui Zhang
Journal of Chromatography A 2014 Volume 1371() pp:48-57
Publication Date(Web):5 December 2014
DOI:10.1016/j.chroma.2014.10.067
•A novel organic–silica monolith based IMER was prepared.•The monolithic support was prepared by a single step “one-pot” strategy.•Polyethylenimine was bound onto the surface to increase active reaction sites.•The carry-over of protein/peptides on the IMER was not greatly decreased.•The IMER was further online integrated with 2D-nanoHPLC–MS/MS system.In this work, a novel kind of organic–silica hybrid monolith based immobilized enzymatic reactor (IMER) was developed. The monolithic support was prepared by a single step “one-pot” strategy via the polycondensation of tetramethoxysilane and vinyltrimethoxysilane and in situ copolymerization of methacrylic acid and vinyl group on the precondensed siloxanes with ammonium persulfate as the thermal initiator. Subsequently, the monolith was activated by N-(3-dimethylaminopropyl) – N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), followed by the modification of branched polyethylenimine (PEI) to improve the hydrophilicity. Finally, after activated by EDC and NHS, trypsin was covalently immobilized onto the monolithic support. The performance of such a microreactor was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin, followed by MALDI-TOF-MS analysis. Compared to those obtained by traditional in-solution digestion, not only higher sequence coverages for BSA (74 ± 1.4% vs. 59.5 ± 2.7%, n = 6) and myoglobin (93 ± 3% vs. 81 ± 4.5%, n = 6) were obtained, but also the digestion time was shortened from 24 h to 2.5 min, demonstrating the high digestion efficiency of such an IMER. The carry-over of these two proteins on the IMER was investigated, and peptides from BSA could not be found in mass spectrum of myoglobin digests, attributed to the good hydrophilicity of our developed monolithic support. Moreover, the dynamic concentration range for protein digestion was proved to be four orders of magnitude, and the IMER could endure at least 7-day consecutive usage. Furthermore, such an IMER was coupled with nano-RPLC–ESI/MS/MS for the analysis of extracted proteins from Escherichia coli. Compared to formerly reported silica hybrid monolith based IMER and the traditional in-solution counterpart, by our developed IMER, although the identified protein number was similar, the identified distinct peptide number was improved by 7% and 25% respectively, beneficial to improve the reliability of protein identification. The IMER was further online integrated with two-dimensional nano-HPLC–MS/MS system for the analysis of protein extracts from hepatocellular carcinoma (HCC) cells with low metastasis rate, and more than 3000 protein groups were identified, with only 46 proteins identified from the residues of the IMER. All these results demonstrated that such a hybrid monolith based IMER would be of great promise in the high throughput and high confidence proteome analysis.
Co-reporter:Yuan Zhou, Yichu Shan, Lihua Zhang, Yukui Zhang
Journal of Chromatography A 2014 Volume 1365() pp:1-11
Publication Date(Web):24 October 2014
DOI:10.1016/j.chroma.2014.08.098
•Isotope labeling based method is most widely applied for proteome quantification.•Merits and drawbacks of various kinds of labeling methods were described.•The development of isotope labeling methods in the future was prospected.The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected.
Co-reporter:Xiaoqiang Qiao, Rui Wang, Guangyue Li, Hongyuan Yan, Yuan Zhou, Lihua Zhang and Yukui Zhang
Analyst 2014 vol. 139(Issue 4) pp:705-708
Publication Date(Web):05 Dec 2013
DOI:10.1039/C3AN01907A
New types of imidazolium-based iodoacetamide tags were designed, synthesized and further exploited for cysteinyl-peptide analysis with superior labeling efficiency, high stability, improved ionization efficiency, and increased charge states by mass spectrometry. For the first time, the effects of these kinds of tags on the mass spectrometry performance of the derivatized peptides were investigated, which is of great importance to help us design more efficient tags for the analysis of peptides or proteins, especially for those with low abundance.
Co-reporter:Qi Wu, Yichu Shan, Yanyan Qu, Hao Jiang, Huiming Yuan, Jianxi Liu, Shen Zhang, Zhen Liang, Lihua Zhang and Yukui Zhang
Analyst 2014 vol. 139(Issue 1) pp:138-146
Publication Date(Web):17 Oct 2013
DOI:10.1039/C3AN01738A
Proteome scale absolute quantification is fundamental for the quantitative understanding of an organism. The unsatisfactory accuracy for protein abundance estimation of current algorithms has been partially improved by the Absolute Protein EXpression profiling (APEX) algorithm, which implements the prior expectations of peptides' appearances in the calculation of protein abundances. However, the abundance feature (AF) in APEX is the spectral count (SC); an AF suffers from a narrow dynamic range, thus, unsatisfactory accuracy. Therefore, we adopted another tandem mass spectrometric (MS/MS) level AF called Summed MS/MS Total ion current (SMT), which cumulates the MS/MS fragment intensities rather than simply counting the MS/MS spectra, to surmount this particular deficiency. The combination of APEX and SMT (abbreviated as APEX-SMT) is capable of improving the accuracy of absolute quantification by reducing the average relative deviation by ∼55–85% compared to that of APEX, through a series of tests on the Universal Proteomics Standard sample with a dynamic range of 5 orders of magnitude (UPS2). The algorithm could also be used for relative quantification. When applied to the relative quantification of a publicly available benchmark dataset, APEX-SMT could provide comparable accuracy to APEX. All these results suggest that APEX-SMT is a promising alternative to APEX for proteome quantification.
Co-reporter:Zhigang Sui, Lihua Zhang, Yushu Huo, Yukui Zhang
Journal of Pharmaceutical and Biomedical Analysis 2014 Volume 87() pp:229-240
Publication Date(Web):18 January 2014
DOI:10.1016/j.jpba.2013.07.044
Velvet antler is one of the most important animal medicines, and has been used with a variety of functions, such as anti-fatigue, tissue repair and health promotion. In the past few years, the investigation on chemical compositions, bioactive components, and pharmacological effects has been performed, which demonstrates that velvet antlers could be used as an important health-promoting tonic with great nutritional and medicinal values. This review focuses on the recent advance in studying the bioactive components of velvet antlers.
Co-reporter:Xiaoqiang Qiao, Xinying Qin, Dandan She, Rui Wang, Xiaodan Zhang, Lihua Zhang, Yukui Zhang
Talanta 2014 Volume 126() pp:91-102
Publication Date(Web):1 August 2014
DOI:10.1016/j.talanta.2014.03.012
•The recent reported tags for high efficiency analysis of peptides by mass spectrometry are reviewed.•These tags are categorized based on their target reactive groups on peptides.•Tags for analysis of post-translational modifications have been included.•A brief introduction of their applications for peptide analysis is presented.Chemical derivatization is a very promising technique for improving analysis of peptides by mass spectrometry (MS). Thereinto, development of novel tags compatible with MS and/or MS/MS has always been the focus point of study. In this review, the recent reported tags for derivatization of thiol groups of cysteine, carboxyl groups, and amino groups on peptides as well as peptides with post-translational modifications (PTMs) are summarized. Moreover, the tags used for derivatization of glycans or oligosaccharides released from glycoproteins are also reviewed.
Co-reporter:Bo Jiang, Kaiguang Yang, Lihua Zhang, Zhen Liang, Xiaojun Peng, Yukui Zhang
Talanta 2014 Volume 122() pp:278-284
Publication Date(Web):May 2014
DOI:10.1016/j.talanta.2014.01.056
•Dendrimer grafted GO was prepared as substrate for covalently bonding of trypsin.•Trypsin-linked dGO showed no interference on MALDI-TOF MS signal.•Trypsin-linked dGO showed advantages of high efficiency and long-term stability.•Trypsin-linked dGO was successfully applied for on-plate digestion of trace samples.In this study, dendrimer grafted graphene oxide nanosheets (dGO) were prepared by covalent reaction. The successful synthesis of dGO was confirmed by Fourier-transform infrared spectra, Raman spectra, Thermo gravimetric analysis and Zeta potential. Taking advantages of large surface area, excellent biocompatibility and abundant functional groups, dGO provided an ideal substrate for trypsin immobilization. Trypsin-linked dGO was synthesized through covalent bonding using glutaraldehyde as coupling agents. The amount of trypsin immobilized on dGO nanosheets was calculated to be about 649±20 mg/g. The activity of immobilized trypsin could be maintained for over 10 days at 4 °C. On-plate proteolysis could be performed without removing trypsin-linked dGO, because dGO did not interfere with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry analysis. By such an immobilized enzymatic reactor, standard proteins could be efficiently digested within 15 min, with sequence coverages comparable or better than those obtained by conventional over-night in-solution digestion. Furthermore, trypsin-linked dGO showed high sensitivity when applied to trace samples analysis. All these results demonstrated that the developed dGO based enzymatic reactor might provide a promising tool for high throughput proteome identification.Trypsin-linked dGO was prepared and used as IMER for on-plate digestion. High efficient and rapid on-plate proteolysis was obtained by trypsin-linked dGO.
Co-reporter:ZiQi Yan;Yuan Zhou;YiChu Shan;Qi Wu;Shen Zhang;Zhen Liang
Science China Chemistry 2014 Volume 57( Issue 5) pp:718-722
Publication Date(Web):2014 May
DOI:10.1007/s11426-014-5093-z
Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods. Herein, we used a new strategy: data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode, to study the differentially expressed proteins in mouse liver cancer metastasis cells. A total of 1528 protein groups were identified, among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins (86 proteins up-regulated and 163 down-regulated). This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.
Co-reporter:Ci Wu;Dr. Yu Liang;Qun Zhao;Yanyan Qu;Shen Zhang;Qi Wu;Dr. Zhen Liang;Dr. Lihua Zhang; Yukui Zhang
Chemistry - A European Journal 2014 Volume 20( Issue 28) pp:8737-8743
Publication Date(Web):
DOI:10.1002/chem.201402787
Abstract
As low abundance is the great obstacle for glycoprotein analysis, the development of materials with high efficiency and selectivity for glycoprotein enrichment is a prerequisite in glycoproteome research. Herein, we report a new kind of hydrophilic boronate affinity monolith by attaching 4-mercaptophenylboronic acid (MPBA) with 2-mercaptoethylamine (MPA) on the gold nanoparticle-modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate)) monolith for glycoprotein enrichment. With poly(ethylene glycol) diacrylate as the cross-linker and the further modification of gold nanoparticles, the matrix has advantages of good hydrophilicity and enhanced surface area, which are beneficial to improve the enrichment selectivity and efficiency for glycoproteins. The attachment of MPBA and MPA provide intramolecular BN coordination, which could further enhance the specificity of glycoprotein capture. Such a boronate affinity monolith was applied to enrich horseradish peroxidase (HRP) from the mixture of HRP and bovine serum albumin (BSA), and high selectivity was obtained even at a mass ratio of 1:1000. In addition, the binding capacity of ovalbumin on such monolith reached 390 μg g−1. Furthermore, the average recovery of HRP on the prepared affinity monoliths was (84.8±1.9) %, obtained in three times enrichment with the same column. Finally, the boronate affinity monolith was successfully applied for the human-plasma glycoproteome analysis. As a result, 160 glycoproteins were credibly identified from 9 μg of human plasma, demonstrating the great potential of such a monolith for large-scale glycoproteome research.
Co-reporter:Qun Zhao, Yu Liang, Huiming Yuan, Zhigang Sui, Qi Wu, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2013 Volume 85(Issue 18) pp:8507
Publication Date(Web):August 19, 2013
DOI:10.1021/ac402076u
Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an “SCX-SAX” biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 μg of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional in-solution method were 10 and 1, respectively. All these results demonstrated that such an “SCX-SAX” based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.
Co-reporter:Yuan Zhou, Yichu Shan, Qi Wu, Shen Zhang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2013 Volume 85(Issue 22) pp:10658
Publication Date(Web):November 1, 2013
DOI:10.1021/ac402834w
Discovering differentially expressed proteins in various biological samples requires proteome quantification methods with accuracy, precision, and wide dynamic range. This study describes a mass defect-based pseudo-isobaric dimethyl labeling (pIDL) method based on the subtle mass defect differences between 12C/13C and 1H/2H. Lys-C protein digests were labeled with CD2O/13CD2O and reduced with NaCNBD3/NaCNBH3 as heavy and light isotopologues, respectively. The fragment ion pairs with mass differences of 5.84 mDa were resolved by high-resolution tandem mass spectrometry (MS/MS) and used for quantification. The pIDL method described here resulted in highly accurate and precise quantification results with approximately 100-fold dynamic range. Furthermore, the pIDL method was extended to 4-plex proteome quantification and applied to the quantitative analysis of proteomes from Hca-P and Hca-F, two mouse hepatocarcinoma ascites syngeneic cell lines with low and high lymph node metastasis rates.
Co-reporter:Yichu Shan;Lihua Zhang;Yukui Zhang
Analytical and Bioanalytical Chemistry 2013 Volume 405( Issue 21) pp:6611-6612
Publication Date(Web):2013 August
DOI:10.1007/s00216-013-7116-6
Co-reporter:Qinran Li;Kaiguang Yang;Jinxiang Liu;Lihua Zhang;Zhen Liang
Microchimica Acta 2013 Volume 180( Issue 15-16) pp:1379-1386
Publication Date(Web):2013 November
DOI:10.1007/s00604-013-0994-7
A novel kind of transferrin imprinted polymer particles was synthesized by a hierarchical strategy. First, transferrin was immobilized on silica beads by non-covalent absorption. Then, a pre-polymerization mixture, composed of 1,4-bis(acryloyl)piperazine, methacrylamide, methacrylic acid, ammonium sulfate and polyoxyethylene sorbitan monolaurate, was irrigated into the pores of silica particles, and polymerized at 25 °C. Finally, the silica matrix was etched with ammonium hydrogen fluoride, not only to remove the template protein, but also to expose protein recognition sites on the surface of the imprinted polymer. The binding capacity of the transferrin-imprinted particles is 6.3 mg of protein per gram of material, and the time required to reach adsorption equilibrium was less than 10 min. The imprinting factor of transferrin is ca. 3.3 in the presence of ribonuclease B, cytochrome c and β-lactoglobulin. The results indicate that these imprinted polymer particles can recognize transferrin with good selectivity, high binding capacity and fast mass transfer. They may be applied as an artificial antibody to remove the high abundance proteins in plasma.
Co-reporter:Zhigang Sui, Huiming Yuan, Zhen Liang, Qun Zhao, Qi Wu, Simin Xia, Lihua Zhang, Yushu Huo, Yukui Zhang
Talanta 2013 Volume 107() pp:189-194
Publication Date(Web):30 March 2013
DOI:10.1016/j.talanta.2013.01.015
The exceptional growth rate of velvet antler makes it a valuable model for studying the development of tissues, such as blood vessels, cartilage and bone. Meanwhile, investigating the activities of extracted proteins from velvet antlers promisingly leads to the discovery of new active factors which regulate the development of above-mentioned tissue types. In this study, a novel sequential protein extraction method was developed for proteome profiling and bioactivity study of velvet antlers. Herein, four antler growing tips were pooled to create a proportional pooled sample, and three aliquots of which were extracted in parallel using the developed extraction method. For each sample, proteins were extracted sequentially by saline solvent (0.15 M sodium chloride, pH 7.0), mild acid buffer (0.15 M acetate buffer, pH 4.0) and mild alkaline buffer (0.15 M glycine-sodium hydroxide buffer, pH 10.0) with good bio-compatibility to prevent proteins denaturation. Then STD lysis buffer, containing 4% SDS, 0.1 M Tris–HCl and 0.1 M DTT, was used to extract hydrophobic proteins. The tryptic digest of each fraction was analyzed by nanoRPLC-ESI-MS/MS in triplicates, with false discovery rate for peptide identification adjusted to 1% to create filtered protein group list. In total, 1423 protein groups were identified, which expanded up to 3 times of the previous published dataset. The relative standard deviation of identified peptide and protein group number for all analyses indicated the good reproducibility of the developed sequential protein extraction method. Additionally, proteins extracted by acid buffer and alkaline buffer showed obvious promoting effect on the proliferation of human umbilical vein endothelial cells. All these results demonstrate that the developed sequential extraction method is efficient for the comprehensive proteome analysis and activity investigation of velvet antlers.Highlights► The complexity of sample was efficiently decreased during protein extraction. ► This strategy has good reproducibility of peptide and protein group identification. ► Remarkable improvement in protein identification was achieved by this strategy. ► Fractions obtained by biocompatible solvents showed activities in different manner.
Co-reporter:Yu Liang;Lihua Zhang;Yukui Zhang
Analytical and Bioanalytical Chemistry 2013 Volume 405( Issue 7) pp:2095-2106
Publication Date(Web):2013 March
DOI:10.1007/s00216-012-6570-x
Capillary liquid chromatography (cLC) has great potential for protein and peptide separation, with advantages of high efficiency, high resolution, low sample consumption, and high sensitivity when coupled with mass spectrometry. In recent years, monoliths have been widely used as the stationary phases for capillary columns, owing to easy preparation, high permeability, fast mass transfer, and low backpressure. This review summarizes recent advances (2007–2012) in monolithic columns for protein and peptide separation by cLC. After a brief introduction on the preparation of monolithic capillary columns, the emphasis of this review is focused on the recent application of such columns for protein and peptide separation by cLC. Furthermore, the challenges and potential hot points of monolithic capillary columns in the future are discussed.
Co-reporter:XiaoQiang Qiao;Yuan Zhou;ChunYan Hou;XiaoDan Zhang
Science China Life Sciences 2013 Volume 56( Issue 3) pp:240-245
Publication Date(Web):2013 March
DOI:10.1007/s11427-013-4446-8
The cationic reagent 1-(3-aminopropyl)-3-butylimidazolium bromide (BAPI) was exploited for the derivatization of carboxyl groups on peptides. Nearly 100% derivatization efficiency was achieved with the synthetic peptide RVYVHPI (RI-7). Furthermore, the peptide derivative was stable in a 0.1% TFA/water solution or a 0.1% (v/v) TFA/acetonitrile/water solution for at least one week. The effect of BAPI derivatization on the ionization of the peptide RI-7 was further investigated, and the detection sensitivity was improved >42-fold via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), thus outperforming the commercial piperazine derivatization approach. Moreover, the charge states of the peptide were largely increased via BAPI derivatization by electrospray ionization (ESI) MS. The results indicate the potential merits of BAPI derivatization for high sensitivity peptide analysis by MS.
Co-reporter:Liyuan Zhang, Qun Zhao, Zhen Liang, Kaiguang Yang, Liangliang Sun, Lihua Zhang and Yukui Zhang
Chemical Communications 2012 vol. 48(Issue 50) pp:6274-6276
Publication Date(Web):01 May 2012
DOI:10.1039/C2CC31641B
A new type of IMAC material, with ATP as the chelating ligand, was synthesized and applied to capture phosphopeptides. For the first time, the approach for phosphopeptide enrichment could provide selectivity under 5000-fold dilution by nonphosphopeptides, and sensitivity of on-target enrichment at 3 amol.
Co-reporter:Huiming Yuan, Yuan Zhou, Simin Xia, Lihua Zhang, Xiaodan Zhang, Qi Wu, Zhen Liang, and Yukui Zhang
Analytical Chemistry 2012 Volume 84(Issue 11) pp:5124
Publication Date(Web):April 23, 2012
DOI:10.1021/ac3006796
An online integrated platform for proteome profiling was established, with the combination of protein separation by microreversed phase liquid chromatography (μRPLC), online acetonitrile (ACN) removal, and pH adjustment by a hollow fiber membrane interface (HFMI), online digestion by an immobilized enzymatic microreactor (IMER), as well as peptide separation and proteins identification by μRPLC or nano-RPLC-electrospray ionization tandem mass spectrometry (μRPLC-ESI-MS/MS). To evaluate the performance of such a platform, a three-protein mixture with mass ranging from 5 to 500 ng was analyzed automatically. Compared to the offline counterpart, although similar protein sequence coverages were obtained by the integrated platform, the signal intensity of total ion chromatogram was improved by almost 4 times. In addition, such an integrated platform was further applied for the analysis of extracted proteins from rat brain. Compared to the results obtained by offline counterpart and traditional MudPIT approach under similar conditions, by the integrated platform, the identified protein group number was comparable, but the analysis time was shortened to less than half of that taken by the traditional approaches. All these results demonstrated that our developed integrated platform might offer a promising tool for high-throughput and large-scale profiling of proteomes.
Co-reporter:Guijie Zhu, Peng Zhao, Nan Deng, Dingyin Tao, Liangliang Sun, Zhen Liang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2012 Volume 84(Issue 18) pp:7633
Publication Date(Web):August 21, 2012
DOI:10.1021/ac3017746
Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC–ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.
Co-reporter:Nan Deng, Zhen Liang, Yu Liang, Zhigang Sui, Liyuan Zhang, Qi Wu, Kaiguang Yang, Lihua Zhang, and Yukui Zhang
Analytical Chemistry 2012 Volume 84(Issue 23) pp:10186
Publication Date(Web):November 9, 2012
DOI:10.1021/ac302779u
A novel kind of aptamer modified organic–inorganic hybrid silica monolithic capillary column has been developed, via the covalent bonding of 5′-NH2-modified aptamer for human α-thrombin on hybrid silica monolith, prepared by sol–gel method, with tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors. Due to the large specific surface area of the hybrid matrix, the average coverage density of aptamer reached 568 pmol/μL, and the thrombin binding capacity was 1.15 μg/μL, 14 times higher than that of aptamer modified open tubular capillaries. By such an affinity capillary column, the limit of detection of thrombin was decreased to 3.4 nM with a UV detector. Furthermore, even when thrombin was mixed with 1000 times more concentrated human serum, it could be selectively enriched and detected with the signal-to-noise ratio as ca.10. These results indicate that the developed preparation strategy for aptamer based hybrid silica monolithic capillary column might provide an effective method to achieve highly selective recognition of trace targets.
Co-reporter:Liyuan Zhang, Zhen Liang, Kaiguang Yang, Simin Xia, Qi Wu, Lihua Zhang, Yukui Zhang
Analytica Chimica Acta 2012 Volume 729() pp:26-35
Publication Date(Web):4 June 2012
DOI:10.1016/j.aca.2012.04.005
The enrichment of low abundance phosphopeptides before MS analysis is a critical step for in-depth phosphoproteome research. In this study, mesoporous titanium dioxide (TiO2) aerogel was prepared by precipitation and supercritical drying. The specific surface area up to 490.7 m2 g−1 is achieved by TiO2 aerogel, much higher than those obtained by commercial TiO2 nanoparticles and by the latest reported mesoporous TiO2 spheres. Due to the large specific surface area and the mesoporous structure of the aerogel, the binding capacity for phosphopeptides is six times higher than that of conventional TiO2 microparticles (173 vs 28 μmol g−1). Because of the good compatibility of enrichment procedure with MALDI-TOF-MS and the large binding capacity of TiO2 aerogel, a detection limit as low as 30 amol for analyzing phosphopeptides in β-casein digest was achieved. TiO2 aerogel was further applied to enrich phosphopeptides from rat liver mitochondria, and 266 unique phosphopeptides with 340 phosphorylation sites, corresponding to 216 phosphoprotein groups, were identified by triplicate nanoRPLC-ESI-MS/MS runs, with false-positive rate less than 1% at the peptide level. These results demonstrate that TiO2 aerogel is a kind of promising material for sample pretreatment in the large-scale phosphoproteome study.Graphical abstractHighlights► The specific surface area of TiO2 aerogel is 10 times larger than TiO2 nanoparticle. ► The limit of detection for phosphopeptides was down to 30 amol by TiO2 aerogel. ► The loading capacity for phosphopeptides was 6-fold larger than TiO2 microparticle.
Co-reporter:Bo Jiang, Kaiguang Yang, Qun Zhao, Qi Wu, Zhen Liang, Lihua Zhang, Xiaojun Peng, Yukui Zhang
Journal of Chromatography A 2012 Volume 1254() pp:8-13
Publication Date(Web):7 September 2012
DOI:10.1016/j.chroma.2012.07.030
In this paper, magnetic Fe3O4 nanoparticles modified graphene oxide nanocomposites (GO–CO–NH–Fe3O4) were prepared by covalent bonding, via the reaction between the amino groups of fuctionalized Fe3O4 and the carboxylic groups of GO, confirmed by Fourier-transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. With GO–CO–NH–Fe3O4 as a novel substrate, trypsin was immobilized via π–π stacking and hydrogen bonding interaction, and the binding capacity of trypsin reached as high as 0.275 mg/mg. Since GO–CO–NH–Fe3O4 worked as not only support for enzyme immobilization, but also as an excellent microwave irradiation absorber, the digestion efficiency could be further improved with microwave assistance. By such an immobilized enzymatic reactor (IMER), standard proteins could be efficiently digested within 15 s, with sequence coverages comparable or better than those obtained by conventional in-solution digestion (12 h). Since trypsin was immobilized under mild conditions, the enzymatic activity of IMER preserved at least for a month. In addition, due to the good hydrophilicity of GO, no peptide residue was observed in the sequent digestion of bovine serum albumin and myoglobin. To further confirm the efficiency of such an IMER for proteome analysis, it was applied to digest proteins extracted from rat liver, followed by nanoRPLC–ESI-MS/MS analysis. With only 5 min microwave-assisted digestion, in 3 parallel runs, totally 456 protein groups were identified, comparable to that obtained by 12 h in-solution digestion, indicating the great potential of IMERs with GO–CO–NH–Fe3O4 as the support for high throughput proteome study.Highlights► GO–CO–NH–Fe3O4 nanocomposites were prepared by a facile covalent bonding. ► GO–CO–NH–Fe3O4 nanocomposites were used as trypsin immobilization substrate. ► Such an IMER showed advantages of high efficiency and low residue. ► Such an IMER was successfully applied to the digestion of rat liver samples.
Co-reporter:Hao Jiang, Huiming Yuan, Yu Liang, Simin Xia, Qun Zhao, Qi Wu, Lihua Zhang, Zhen Liang, Yukui Zhang
Journal of Chromatography A 2012 Volume 1246() pp:111-116
Publication Date(Web):13 July 2012
DOI:10.1016/j.chroma.2012.03.014
In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix. With NVP modification, the hydrophilicity of IMER was greatly improved, resulting in not only the sequence coverage of myoglobin increased from 63% to 73%, but also no residual peptides from BSA observed in myoglobin digests. Although the sequence coverages of proteins obtained by the IMER were comparable to those obtained by in-solution digestion, the digestion time was shortened from 24 h to 1 min. By such an IMER, a protein mixture, containing BSA, myoglobin, and cytochrome c (100, 1 and 0.01 μg/mL, respectively), was digested, and all proteins were unambiguously identified with improved sequence coverages than that achieved by in-solution digestion. Furthermore, the hydrophilic IMER was also off-line coupled to nano-RPLC–ESI-MS/MS for the analysis of proteins extracted from yeast. After 1.5 min digestion, 271 protein groups with at least 2 distinct peptides were identified, much more than those obtained by 24 h in-solution digestion (192 protein groups), indicating the great potential of such an IMER for proteome analysis.Highlights► A novel kind of immobilized enzyme reactor with low peptide residue was prepared. ► NVP modified poly acrylic ester microspheres were used for trypsin immobilization. ► Trypsin was modified by EDC and NHS before immobilization. ► Such an IMER shows advantages of high efficiency, low residue and easy preparation. ► Such an IMER was successfully applied to the digestion of yeast lysates.
Co-reporter:Qi Wu, Qun Zhao, Zhen Liang, Yanyan Qu, Lihua Zhang and Yukui Zhang
Analyst 2012 vol. 137(Issue 13) pp:3146-3153
Publication Date(Web):19 Apr 2012
DOI:10.1039/C2AN35173K
Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies. Herein, we implemented two formats of MS/MS fragment ion intensity, Spectral Index (SI) and Summed MS/MS TIC (SMT), to alleviate this particular deficiency arising from SC. Both were in forms of replacing SC in the Normalized Spectral Abundance Factor (NSAF) formula, resulting in two algorithms, abbreviated as NSI and NSMT, respectively. The necessity of the normalization process was validated using a publicly available dataset. Furthermore, when applied to another well characterized benchmark dataset, both NSI and NSMT showed improved overall accuracy over NSAF for the relative quantification of proteomes. Hereinto, NSI enabled the sensitive detection of differentially expressed proteins, while NSMT ensured accurate calculation for protein abundance fold change. Therefore, the selective use of both algorithms might facilitate the screening and quantification of potential biomarkers on the proteome scale.
Co-reporter:Simin Xia;Dingyin Tao;Huiming Yuan;Yuan Zhou;Zhen Liang;Lihua Zhang;Yukui Zhang
Journal of Separation Science 2012 Volume 35( Issue 14) pp:1764-1770
Publication Date(Web):
DOI:10.1002/jssc.201200052
An integrated multidimensional nano-flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successfully applied for proteome analysis. By this platform, proteins were first separated by a weak anion and weak cation mixed-bed microcolumn under a series of salt steps, online digested by a trypsin immobilized enzymatic reactor, digests trapped and desalted by a C18 precolumn, separated by nano-reversed phase liquid chromatography, and finally identified by electrospray ionization-MS/MS. To evaluate the performance of such a platform, Escherichia coli whole cell lysate proteins were analyzed. Compared with the results obtained by shotgun approach, the identified protein number was increased by 6%, with the consumed time decreased from 38 to 14 h. We also compared with integrate platform based on micro-HPLC, and the required sample amount was decreased to 8 μg. These results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.
Co-reporter:Yanyan Qu;Jianxi Liu;Dr. Kaiguang Yang;Dr. Zhen Liang;Dr. Lihua Zhang; Yukui Zhang
Chemistry - A European Journal 2012 Volume 18( Issue 29) pp:9056-9062
Publication Date(Web):
DOI:10.1002/chem.201103514
Abstract
The boronic acid-functionalized core–shell polymer nanoparticles, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@4-vinylphenylboronic acid (poly(MBA-co-MAA)@VPBA), were successfully synthesized for enriching glycosylated peptides. Such nanoparticles were composed of a hydrophilic polymer core prepared by distillation precipitation polymerization (DPP) and a boronic acid-functionalized shell designed for capturing glycopeptides. Owing to the relatively large amount of residual vinyl groups introduced by DPP on the core surface, the VPBA monomer was coated with high efficiency, working as the shell. Moreover, the overall polymerization route, especially the use of DPP, made the synthesis of nanoparticles facile and time-saving. With the poly(MBA-co-MAA)@VPBA nanoparticles, 18 glycopeptides from horseradish peroxidase (HRP) digest were captured and identified by MALDI-TOF mass spectrometric analysis, relative to eight glycopeptides enriched by using commercially available meta-aminophenylboronic acid agarose under the same conditions. When the concentration of the HRP digest was decreased to as low as 5 nmol, glycopeptides could still be selectively isolated by the prepared nanoparticles. Our results demonstrated that the synthetic poly(MBA-co-MAA)@VPBA nanoparticles might be a promising selective enrichment material for glycoproteome analysis.
Co-reporter:Qi Wu, Huiming Yuan, Lihua Zhang, Yukui Zhang
Analytica Chimica Acta 2012 731() pp: 1-10
Publication Date(Web):
DOI:10.1016/j.aca.2012.04.010
Co-reporter:Qun Zhao, Liangliang Sun, Yu Liang, Qi Wu, Huiming Yuan, Zhen Liang, Lihua Zhang, Yukui Zhang
Talanta 2012 Volume 88() pp:567-572
Publication Date(Web):15 January 2012
DOI:10.1016/j.talanta.2011.11.035
Analysis of integral membrane proteins (IMPs) presents great challenges due to their hydrophobic nature. Recently, much attention has been paid to improve the solubilization of IMPs. However, besides that, the separation of hydrophobic peptides with high recovery is also a dominating factor, but with rare report. Here, the prefractionation of the digests by reverse phase trap column during desalting was presented to efficiently decrease the complexity of samples, with the identified hydrophobic peptides and IMPs increased by more than 43%. Furthermore, the effect of C18 and C8 stationary phases on the separation of membrane protein digests was studied. A total of 301 proteins (536 peptides) with C18 stationary phase and 398 proteins (703 peptides) with C8 stationary phase were identified by μRPLC–ESI-MS/MS using an LCQ instrument in duplicate runs, with false discovery rate (FDR) less than 5% at peptide level. In addition, with C8 stationary phase, the number of identified hydrophobic peptides and IMPs was obviously improved by 29% and 20%, respectively, compared with that identified by C18 stationary phase, indicating that the polarity of stationary phase has evident effect on the analysis of membrane protein digests. All these results show that the prefractionation by reverse phase trap column during desalting and the separation by C8 stationary phases could facilitate the efficient identification of IMPs.Highlights► Prefractionation of sample digests was successfully achieved during desalting. ► Prefractionation during desalting could efficiently simplify the sample digests. ► C8 column was found superior to C18 column for hydrophobic peptide separation. ► C8 column separation with prefractionation could favour membrane protein analysis.
Co-reporter:Kaiguang Yang;Lihua Zhang;Zhen Liang
Analytical and Bioanalytical Chemistry 2012 Volume 403( Issue 8) pp:2173-2183
Publication Date(Web):2012 June
DOI:10.1007/s00216-012-5840-y
Protein imprinting is a promising tool for generating artificial biomimetic receptors with antibody-like specific recognition sites. Recently, protein-imprinted materials, as potential antibody substitutes, have attracted much attention in many fields, for example chemical sensors, chromatographic stationary phases, and artificial enzymes, owing to their long-term storage stability, potential re-usability, resistance to harsh environment, and low cost. In this critical review, we focus our discussion on the rational preparation of protein-imprinted materials in terms of choice of template, functional monomer, crosslinker, and polymerization format. In addition, several highlighted applications of protein-imprinted materials are emphasized, not only in well-known fields but also in some unique fields, for example proteomics and tissue engineering. Finally, we propose challenges arising from the intrinsic properties of protein imprinting, for example obtaining the template, heterogeneous binding, and extrinsic competition, for example immobilized aptamers.
Co-reporter:Jinxiang Liu, Kaiguang Yang, Qiliang Deng, Qinran Li, Lihua Zhang, Zhen Liang and Yukui Zhang
Chemical Communications 2011 vol. 47(Issue 13) pp:3969-3971
Publication Date(Web):24 Feb 2011
DOI:10.1039/C0CC05317A
A new approach, combining metal-coordination with molecular imprinting technology, was developed to prepare protein-affinity materials, which showed higher specific recognition ability towards the target protein than those prepared using either metal-coordination or molecular imprinting technology.
Co-reporter:Yanyan Qu, Simin Xia, Huiming Yuan, Qi Wu, Man Li, Lijuan Zou, Lihua Zhang, Zhen Liang, and Yukui Zhang
Analytical Chemistry 2011 Volume 83(Issue 19) pp:7457
Publication Date(Web):August 16, 2011
DOI:10.1021/ac201665e
An integrated sample pretreatment system, composed of a click maltose hydrophilic interaction chromatography (HILIC) column, a strong cation exchange (SCX) precolumn, and a PNGase F immobilized enzymatic reactor (IMER), was established for the simultaneous glycopeptide enrichment, sample buffer exchange, and online deglycosylation, by which the sample pretreatment for glycoproteome could be performed online automatically, beneficial to improve the efficiency and sensitivity of the N-linked glycosylation site identification. With such a system, the deglycosylated glycopeptide from the digests of avidin with the coexistence of 50 times (mass ratio) BSA could be selectively detected, and the detection limit as low as 5 fmol was achieved. Moreover, the sample pretreatment time was significantly shortened to ∼1 h. Such a system was further successfully applied for analyzing the digest of the soluble fraction extracted from rat brain. A total of 120 unique glycoprotein groups and 196 N-linked glycosylation sites were identified by nanoreversed phase liquid chromatography–electrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS), with the injected digests amount as 6 μg. All these results demonstrate that the integrated system is of great promise for N-linked glycosylation site profiling and could be further online coupled with nanoHPLC-ESI-MS/MS to achieve high-throughput glycoproteome analysis.
Co-reporter:Dingyin Tao, Xiaoqiang Qiao, Liangliang Sun, Chunyan Hou, Liang Gao, Lihua Zhang, Yichu Shan, Zhen Liang, and Yukui Zhang
Journal of Proteome Research 2011 Volume 10(Issue 2) pp:732-738
Publication Date(Web):2017-2-22
DOI:10.1021/pr100893j
Two dimensional high performance liquid chromatography−electrospray ionization−tandem mass spectrometry (2D-HPLC−ESI−MS/MS) is one of the most powerful techniques for high resolution, efficiency, and throughput separation and identification of proteomes. For a bottom-up strategy-based proteome analysis, usually multistep salt elution was needed in the first dimension separation by SCX, to simplify the peptides for the further second dimensional separation by RPLC. Here, by using a 30 cm-long serially coupled long column (SCLC) in the second dimension, we reduced the salt steps of SCX from 13 to 5 to shorten the total analysis time. Compared to the commonly applied 2D-HPLC with over 10-step salt elution in SCX and microRPLC with a short column (SC), named as SC-2D, the peak capacity of 2D-HPLC with a SCLC column, named as SCLC-2D, was increased 3.3-folds while the analysis time was increased by only 1.17-folds. Therefore, the time-based protein identification efficiency was ∼55 protein groups/h, nearly 2-fold of that for SC-2D (∼28 protein groups/h). With the further combination of assisted solubilization by ionic liquids and SCLC-2D, 608 integral membrane proteins (IMPs) (27.66% of the total 2198 proteins, FDR < 1%) were identified from rat brain, more than those obtaind by the traditional urea method (252 unique IMPs, occupying 17.03% of total 1480 proteins). All of these results demonstrate the promise of the developed technique for large-scale proteome analysis.
Co-reporter:Yu Liang, Dingyin Tao, Junfeng Ma, Liangliang Sun, Zhen Liang, Lihua Zhang, Yukui Zhang
Journal of Chromatography A 2011 Volume 1218(Issue 20) pp:2898-2905
Publication Date(Web):20 May 2011
DOI:10.1016/j.chroma.2011.02.073
A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12 s to 71 s. With μRPLC–ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12 h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.
Co-reporter:Dingyin Tao;Liangliang Sun;Guijie Zhu;Yu Liang;Zhen Liang;Lihua Zhang;Yukui Zhang
Journal of Separation Science 2011 Volume 34( Issue 1) pp:83-89
Publication Date(Web):
DOI:10.1002/jssc.201000632
Abstract
To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.
Co-reporter:Tingting Wang;Agnes Fekete;Andras Gaspar;Junfeng Ma;Zhen Liang;Huiming Yuan;Lihua Zhang;Philippe Schmitt-Kopplin;Yukui Zhang
Journal of Separation Science 2011 Volume 34( Issue 4) pp:422-427
Publication Date(Web):
DOI:10.1002/jssc.201000720
Abstract
A novel method for the separation and detection of low molecular weight (LMW) acids was developed using monolithic immobilized pH gradient-based capillary isoelectric focusing coupled with mass spectrometry. Two main parameters, focusing conditions and delivery buffer conditions, which might affect separation efficiency, were optimized with the focusing time of 7 min at 350 V/cm and the delivery buffer of 50% (v/v) acetonitrile in 10 mmol/L ammonium formate (pH 3.0). Under these conditions, the linear correlation between the volume of delivery solvent and the pKa of the model components was observed. In addition, the separation mechanism of LMW acids was proposed as well. We suppose that this method may provide a useful tool for the characterization of LMW components (e.g. natural organic matter of different origins).
Co-reporter:Liyuan Zhang;Hui Wang;Zhen Liang;Kaiguang Yang;Lihua Zhang;Yukui Zhang
Journal of Separation Science 2011 Volume 34( Issue 16-17) pp:2122-2130
Publication Date(Web):
DOI:10.1002/jssc.201100169
Abstract
The selective enrichment of phosphopeptides with good reproducibility is vital for the in-depth study of the phosphoproteome. Herein, we presented a novel method to prepare monolithic Ti4+ or Zr4+ immobilized metal affinity chromatography (IMAC) columns. Since succinimide was of high reaction activity with nitrilotriacetic acid (NTA) than traditional epoxy group, through the reaction of succinimide group on the monolithic matrix with nitrilotriacetic acid, the preparation of monolithic IMAC columns became facile. By the developed new method, not only the time required for Ti4+ or Zr4+ immobilization could be shortened from 10 to 1 h, but also the RSD of obtained phosphopeptide peak area was below 13%, even with IMAC columns prepared in different batches (n=3). With such monolithic Ti4+- and Zr4+-IMAC columns, ten and seven phosphopeptides were effectively identified from the digests of a mixture of β-casein and BSA, even with the molar ratio as low as 1:100, respectively. The phosphopeptide recovery was over 73%, and the loading capacity was over 0.8 μmol/mL. Compared with commercial IMAC beads, the monolithic IMAC columns provide outstanding reproducibility, good selectivity, large loading capacity, and high recovery, which demonstrates that such monolithic IMAC columns might facilitate the in-depth analysis of phosphoproteomes.
Co-reporter:Hui Wang;Jicheng Duan;Hongjiu Xu;Liang Zhao;Yu Liang;Yichu Shan;Lihua Zhang;Zhen Liang;Yukui Zhang
Journal of Separation Science 2011 Volume 34( Issue 16-17) pp:2113-2121
Publication Date(Web):
DOI:10.1002/jssc.201100168
Abstract
To meet the demands of protein phosphorylation study, immobilized zirconium ion affinity chromatography (Zr4+-IMAC) monolith was prepared by combining UV-initiated polymerization of monolithic support and subsequent photografting in both capillary columns and microchannels. Hydrophilic poly(2-hydroxyethyl methacrylate (HEMA)-co-ethylene dimethacrylate (EDMA)) monolithic support was prepared under UV irradiation at the wavelength of 365 nm with monomer HEMA, crosslinker EDMA and 2,2-dimethoxy-2-phenylacetophenone as photoinitiator in 1-decanol solution, which provides good biocompatibility and permeability for biomolecule analysis. To introduce chelating ligands, such as phosphate groups, on the pore surface of monolith for metal ion immobilization, photografting of ethylene glycol methacrylate phosphate with benzophenone as the photoinitiator was performed at 254 nm for 300 s. The grafting process and metal ion immobilization can be monitored by measuring the electroosmotic flow produced by the modified monolith, providing a quantitative evaluation of post-modification. This new method for the preparation of Zr4+-IMAC monolith simplifies the optimization of monolith preparation and avoids the time-consuming chemical modification process. Additionally, advantages include facile preparation in microdevices, easy regenerability and good reproducibility. After optimization, the microchip-based Zr4+-IMAC monolith was used for phosphopeptide analysis and showed good selectivity in phosphopeptide enrichment with matrix-assisted laser desorption ionization mass spectrometry detection.
Co-reporter:Qi Liang Deng, Yan Li Li, Li Hua Zhang, Yu Kui Zhang
Chinese Chemical Letters 2011 Volume 22(Issue 11) pp:1351-1354
Publication Date(Web):November 2011
DOI:10.1016/j.cclet.2011.05.044
Synthetic materials that can specifically recognize proteins will find wide application in many fields. In this report, bovine serum albumin was chosen as the template protein. Acrylamide and N,N′-methylenebisacrylamide were employed as the functional and cross-linker monomers, respectively. Molecularly imprinted macroporous monolithic materials that can preferentially bind the template protein in an aqueous environment were prepared by combination of molecular imprinting technique and freezing/thawing preparation method. The resulted imprinted macroporous monolithic columns were evaluated by utilizing as stationary phase in high performance liquid chromatography and solid-phase extraction materials. The experimental results indicated that the imprinted macroporous monolithic column exhibited good recognition for template protein, as compared with the control protein (hemoglobin), whereas the non-imprinted polymer (prepared under the same conditions except without addition template protein) had no selective properties.
Co-reporter:Xiaoqiang Qiao, Liangliang Sun, Li Wang, Yu Liang, Lihua Zhang, Yichu Shan, Xiaojun Peng, Zhen Liang, Yukui Zhang
Journal of Chromatography B 2011 Volume 879(17–18) pp:1439-1443
Publication Date(Web):15 May 2011
DOI:10.1016/j.jchromb.2010.11.002
A novel device, composed of a syringe pump for sample loading, a hydrophilic hollow fiber membrane interface for protein concentration and small molecules removal, and a centrifugation tube for buffer exchange, was designed for protein preconcentration and in situ fluorescence derivatization. With the outlet of the interface blocked, denatured proteins were continually introduced. Restricted by the membrane with the molecular weight cutoff (MWCO) of 3000 Da, proteins were concentrated within the membrane. However, denaturant and other small molecules, which might affect the further fluorescence derivatization, were driven out of the membrane. Then, the membrane with proteins restricted inside was directly put into the fluorescence derivatization buffer. Here, the water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide of 3H-indolium,1-(5-carboxypentyl)-2-[3-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-,monopotassium salt (sw-cy3-NHS), synthesized in our lab, was used for protein labeling. By such a method, the detection sensitivity of bovine serum albumin (BSA) was improved by nearly 200 folds, compared to that obtained by direct in-solution derivatization. Through the derivatization of a fraction of E. coli protein separated by reversed phase HPLC, proteins with low concentration were efficiently labeled, which indicated the potential merit of the developed method for the high sensitive detection of low abundance proteins.
Co-reporter:Tingting Wang, Junfeng Ma, Shuaibin Wu, Liangliang Sun, Huiming Yuan, Lihua Zhang, Zhen Liang, Yukui Zhang
Journal of Chromatography B 2011 Volume 879(11–12) pp:804-810
Publication Date(Web):1 April 2011
DOI:10.1016/j.jchromb.2011.02.020
An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ∼200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.
Co-reporter:Shuaibin Wu, Liangliang Sun, Junfeng Ma, Kaiguang Yang, Zhen Liang, Lihua Zhang, Yukui Zhang
Talanta 2011 Volume 83(Issue 5) pp:1748-1753
Publication Date(Web):15 February 2011
DOI:10.1016/j.talanta.2010.12.011
A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10−13 m2). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography–electrospray ionization-mass spectrometry (nanoRPLC–ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.
Co-reporter:Liyuan Zhang;Jin Xu;Liangliang Sun;Junfeng Ma
Analytical and Bioanalytical Chemistry 2011 Volume 399( Issue 10) pp:3399-3405
Publication Date(Web):2011 April
DOI:10.1007/s00216-011-4657-4
In this study, zirconium oxide (ZrO2) aerogel was synthesized via a green sol–gel approach, with zirconium oxychloride, instead of the commonly used alkoxide with high toxicity, as the precursor. With such material, phosphopeptides from the digests of 4 pmol of β-casein with the coexistence of 100 times (mol ratio) BSA could be selectively captured, and identified by MALDI-TOF MS. Due to the large surface area (416.0 m2 g−1) and the mesoporous structure (the average pore size of 10.2 nm) of ZrO2 aerogel, a 20-fold higher loading capacity for phosphopeptide, YKVPQLEIVPN[pS]AEER (MW 1952.12), was obtained compared to that of commercial ZrO2 microspheres (341.5 vs. 17.87 mg g−1). The metal oxide aerogel was further applied in the enrichment of phosphopeptides from 100 ng nonfat milk, and 17 phosphopeptides were positively identified, with a 1.5-fold improvement in phosphopeptide detection compared with previously reported results. These results demonstrate that ZrO2 aerogel can be a powerful enrichment material for phosphoproteome study.
Co-reporter:Liangliang Sun;Dingyin Tao;Bin Han;Junfeng Ma
Analytical and Bioanalytical Chemistry 2011 Volume 399( Issue 10) pp:3387-3397
Publication Date(Web):2011 April
DOI:10.1007/s00216-010-4381-5
The solubility and digestion efficiency are two crucial factors that might affect the identification of integral membrane proteins (IMPs). In this work, 1% (v/v) ionic liquid (IL), 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIM BF4), added in NH4HCO3 buffer (pH 8.3), was applied as a sample preparation buffer for IMPs analysis. Compared to the commonly used sodium dodecyl sulfate and methanol methods, the number of identified IMPs from rat brain by microcolumn reversed phase liquid chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was improved by over three times, which might be due to the fact that BMIM BF4 offered high solubilizing ability for IMPs and good compatibility for tryptic digestion. Furthermore, compared to Rapigest and urea methods, with BMIM BF4 method, the number of identified IMPs from rat brain could be improved 25% and 80%, respectively, which might be contributed to the good solubilizing ability and high thermal stability of such IL. With the sample treated by BMIM BF4 method, by 2D-nanoSCX-RPLC-ESI-MS/MS, 1,450 non-redundant proteins and 7,978 unique peptides were identified from rat brain, and 418 proteins contained at least one predicted transmembrane domain, with false discovery rates of less than 1% for peptide identification, and at least two identified unique peptides per protein. All these results demonstrate that the BMIM BF4 method is of high potential for the large-scale identification of IMPs.
Co-reporter:Lihua Zhang;Qiankun Zhuang;Yukui Zhang
Analytical and Bioanalytical Chemistry 2011 Volume 399( Issue 10) pp:3305-3306
Publication Date(Web):2011 April
DOI:10.1007/s00216-011-4731-y
Co-reporter:ShuaiBin Wu;JunFeng Ma;KaiGuang Yang;JinXiang Liu
Science China Life Sciences 2011 Volume 54( Issue 1) pp:54-59
Publication Date(Web):2011 January
DOI:10.1007/s11427-010-4108-z
In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.
Co-reporter:Liangliang Sun, Junfeng Ma, Xiaoqiang Qiao, Yu Liang, Guijie Zhu, Yichu Shan, Zhen Liang, Lihua Zhang and Yukui Zhang
Analytical Chemistry 2010 Volume 82(Issue 6) pp:2574
Publication Date(Web):February 12, 2010
DOI:10.1021/ac902835p
An integrated sample treatment device, composed of a membrane interface and a monolithic hybrid silica based immobilized enzymatic reactor (IMER), was developed for the simultaneous sample buffer exchange, protein enrichment, and online digestion, by which for the sample buffer, the acetonitrile content was reduced to ∼1/10 of the initial one, and the pH value was adjusted from ∼3.0 to ∼8.0, compatible for online trypsin digestion. Furthermore, the signal intensity of myoglobin digests was improved by over 10 times. Such an integrated device was successfully applied to the online treatment of three protein eluates obtained by reverse-phase liquid chromatography (RPLC) separation, followed by further protein digest analysis with microreverse-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (μRPLC-ESI-MS/MS). The experimental results showed that the performance of such an integrated sample treatment device was comparable to that of the traditional offline sample treatment method, including lyophilization and in-solution digestion. However, the consumed time was reduced to 1/192. All these results demonstrate that such an integrated sample treatment device could be further online coupled with protein separation, peptide separation, and identification, to achieve high-throughput proteome analysis.
Co-reporter:Junfeng Ma, Chunyan Hou, Liangliang Sun, Dingyin Tao, Yanyan Zhang, Yichu Shan, Zhen Liang, Lihua Zhang, Ling Yang, and Yukui Zhang
Analytical Chemistry 2010 Volume 82(Issue 23) pp:9622
Publication Date(Web):November 3, 2010
DOI:10.1021/ac1023099
In this study, a facile system for membrane proteome profiling was established, in which membrane proteins were solubilized by formic acid, online digested by a pepsin-based immobilized enzyme reactor (pepsin-IMER), and analyzed by strong cation exchange and microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (SCX-μRPLC-ESI-MS/MS). Under optimized conditions, such a system showed excellent compatibility between all crucial steps and was successfully applied for analyzing integral membrane proteins extracted from rat liver microsomes. Out of the 235 unique proteins positively identified, 39% (91/235) were annotated as membrane proteins with one or more transmembrane domains (TMDs). It is anticipated that the efficient sample treatment and the relevant online analytical system might provide a promising tool for automated and comprehensive profiling of membrane proteomes.
Co-reporter:Chunyan Hou, Junfeng Ma, Dingyin Tao, Yichu Shan, Zhen Liang, Lihua Zhang and Yukui Zhang
Journal of Proteome Research 2010 Volume 9(Issue 8) pp:4093-4101
Publication Date(Web):2017-2-22
DOI:10.1021/pr100294z
A novel kind of immobilized metal affinity chromatography (IMAC) column based on organic−inorganic hybrid silica monolith has been developed. The monolithic support was prepared in a 250 μm i.d. capillary by the sol−gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, amine groups were functionalized by glutaraldehyde, and then activated with (aminomethyl) phosphonic acid, followed by Ti4+ chelation. By such a hybrid silica monolithic Ti4+-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of α-casein and BSA with the molar ratio as low as 1:200, illustrating its superior selectivity. With a synthetic phosphorylated peptide, YKVPQLEIVPNSpAEER, as the sample, the loading capacity and recovery of the Ti4+-IMAC monolithic column were measured to be 1.4 μmol/mL and 69%, respectively. Such an IMAC monolithic column was further applied to enrich phosphopeptides from rat liver mitochondria. In total, 224 unique phosphopeptides, corresponding to 148 phosphoprotein groups, were identified by duplicate nanoRPLC-LTQ MS/MS/MS runs with a false-positive rate of less than 1% at the peptide level. These results demonstrate that the hybrid silica monolith based Ti4+-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.
Co-reporter:Pingli Wang;Lihua Zhang;Yichu Shan;Yongzheng Cong;Yu Liang;Bin Han;Zhen Liang;Yukui Zhang
Journal of Separation Science 2010 Volume 33( Issue 13) pp:2039-2044
Publication Date(Web):
DOI:10.1002/jssc.201000162
Abstract
A one-step etching method was developed to fabricate glass free-flow electrophoresis microchips with a rectangle separation microchamber (42 mm-long, 23 mm-wide and 28 μm-deep), in which two glass bridges (0.5 mm-wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free-flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and β-lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na2SO4 added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free-flow electrophoresis microchip fabricated by one-step etching method is suitable for the continuous separation of proteins, which might become an effective pre-fractionation method for proteome study.
Co-reporter:Jinxiang Liu;Qiliang Deng;Kaiguang Yang;Lihua Zhang;Zhen Liang;Yukui Zhang
Journal of Separation Science 2010 Volume 33( Issue 17-18) pp:2757-2761
Publication Date(Web):
DOI:10.1002/jssc.201000350
Abstract
Macroporous cytochrome c (cyc)-imprinted monolithic polyarylamide columns were prepared, and applied for the template protein recognition by HPLC. With cyc (18.8 mg) as template, the imprinted monolithic materials were in situ polymerized in an HPLC column tube, with methacrylamide (450 mg), methacrylic acid (15.8 mg), piperazine diacrylamide (720 mg) and ammonium sulfate (390 mg) dissolved in 5 mL of phosphate buffer (pH 7.4), initiated by ammonium persulfate and TEMED. After the reaction, cyc was removed with acetic acid (10%, v/v) containing 10% w/v SDS. The non-imprinted monolithic column was prepared under the same procedure except without cyc. Retention of cyc and its competitive protein, lysozyme (lys), on molecular-imprinting polymer (MIP) and non-imprinted polymer columns was studied. When the pH value of mobile phase was 4.0, on MIP column, the retention factors of cyc and lys were 2.0 and 1.3, respectively. However, those on non-imprinted polymer column were very similar, both as 1.1. Even in competitive environment, a mixture of cyc and lys could be separated on MIP column under gradient elution, with resolution as 1.2. These results indicate that protein-imprinted monolithic polymer columns could offer obvious affinity and specific recognition to the template protein.
Co-reporter:Tingting Wang;Junfeng Ma;Guijie Zhu;Yichu Shan;Zhen Liang;Lihua Zhang;Yukui Zhang
Journal of Separation Science 2010 Volume 33( Issue 20) pp:3194-3200
Publication Date(Web):
DOI:10.1002/jssc.201000324
Abstract
An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M-IPG), on-line digestion by trypsin-based immobilized enzyme microreactor (trypsin-IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M-IPG CIEF column and trypsin-IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M-IPG CIEF, followed by on-line digestion by trypsin-IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE-based on-line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.
Co-reporter:Chunyan Hou;Huiming Yuan;Xiaoqiang Qiao;Jinxiang Liu;Yichu Shan;Lihua Zhang;Zhen Liang;Yukui Zhang
Journal of Separation Science 2010 Volume 33( Issue 21) pp:3299-3303
Publication Date(Web):
DOI:10.1002/jssc.201000440
Abstract
To separate proteins with a wide distribution of pIs under the conditions compatible to online tryptic digestion (with preferable pH=8.0), weak anion and cation exchange chromatography (WAX/WCX) mixed-bed microcolumn has been developed. With a mixture of five proteins with pIs ranging from 4.2 to 11.4, the effect of WAX/WCX ratio on the separation performance was investigated, and an optimum packing ratio of 1:1 w/w was obtained. Moreover, the undesirable hydrophobic interaction between the proteins and the stationary phase was suppressed with 10% ACN v/v added in the mobile phases. Under the optimized conditions compatible to tryptic digestion, basic and acidic proteins were resolved simultaneously, with RSDs of relative retention time on six columns less than 6%, indicating the good resolution and packing reproducibility. Furthermore, one RPLC fraction of proteins extracted from rat middle brain and the whole protein mixture extracted from rat liver were analyzed, respectively. The results demonstrated better separation performance on WAX/WCX microcolumns than that on both weak anion exchange chromatography and weak cation exchange chromatography at pH ∼8. We anticipate that WAX/WCX microcolumns are promising for the integration of protein separation and tryptic digestion aiming at high-throughput proteome study.
Co-reporter:Liang Gao;Jie Zhang;Weibing Zhang;Yichu Shan;Zhen Liang;Lihua Zhang;Yushu Huo;Yukui Zhang
Journal of Separation Science 2010 Volume 33( Issue 23-24) pp:3817-3821
Publication Date(Web):
DOI:10.1002/jssc.201000453
Abstract
In this study, a comprehensive 2-D chromatography was constructed, consisting of normal phase LC (NPLC) with a CN column as the first dimension, and supercritical fluid chromatography (SFC), with a Merck Chromolith Flash C18 column as the second dimension, which were connected by a 10-port, dual-position valve controlled automatically by a self-designed software. Such platform was applied into the analysis of the fruiting bodies of Ganoderma lucidum, a traditional Chinese medicine, and within 2 h analysis, the obtained peak capacity of the 2-D-NPLC-SFC system was about 350, obviously higher than that of each dimension. These results demonstrate that 2-D-NPLC-SFC is not only of good orthogonality, but also of high throughput for the analysis of complex samples.
Co-reporter:Li-Yuan ZHANG, Hui WANG, Zhen LIANG, Yi-Chu SHAN, Li-Hua ZHANG, Yu-Kui ZHANG
Chinese Journal of Analytical Chemistry 2010 Volume 38(Issue 5) pp:659-662
Publication Date(Web):May 2010
DOI:10.1016/S1872-2040(09)60042-6
A novel method to prepare Fe3+ immobilized metal affinity chromatography monolithic column was developed, which included in situ polymerization of N-acryloxysuccinimide and ethylene dimethacrylate, nitrilotriacetic acid activation, and Fe3+ immobilization. The monolithic column showed high selectivity, high binding capacity, good recovery, and good reproducibility for the enrichment of phosphopeptides. Such a column was successfully applied for the enrichment of phosphopeptides from the nonfat milk protein digests. All these results show the promising potential of monolithic column for phosphoproteome analysis.
Co-reporter:Liang Gao, Dingyin Tao, Yichu Shan, Zhen Liang, Lihua Zhang, Yushu Huo, Yukui Zhang
Journal of Chromatography B 2010 Volume 878(Issue 32) pp:3370-3374
Publication Date(Web):15 December 2010
DOI:10.1016/j.jchromb.2010.10.022
Deer antlers mature rapidly in 60 days, and subsequently shed in 5 days with rapid ossification. During this procedure, the function of deer antlers changes significantly. Therefore, the profiling of antler proteome is helpful to discover important growing and shedding regulation proteins, which might be of great significance for studying development and regeneration. In this study, a parallel protein extraction strategy was developed to extract proteins from antlers of red deer with five different lysis solutions, followed by shotgun proteomic analysis by microflow reversed-phase liquid chromatography/electrospray ionization/tandem mass spectrometry (μRPLC–ESI-MS/MS) with a 30 cm-long serially coupled microcolumn. Our experimental results showed that the identified proteins extracted by five kinds of lysis solution were complementary to each other. In total, 416 unique proteins were identified, with relative molecular masses from 2000 to 600,000, and isoelectric points from 3.84 to 11.57. All these results demonstrate that the combination of parallel protein extraction strategy and μRPLC–ESI-MS/MS analysis with serially coupled long microcolumns might be of great significance for comprehensive proteomic research of deer antler.
Co-reporter:Junfeng Ma, Jinxiang Liu, Liangliang Sun, Liang Gao, Zhen Liang, Lihua Zhang and Yukui Zhang
Analytical Chemistry 2009 Volume 81(Issue 15) pp:6534
Publication Date(Web):July 7, 2009
DOI:10.1021/ac900971w
A facile integrated platform for proteome profiling was established, in which native proteins were online denatured and reduced within a heater, digested with an immobilized trypsin microreactor, and analyzed by microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (μRPLC-ESI-MS/MS). In comparison to the traditional off-line urea denaturation protocol, even more unique peptides were obtained by online heating in triplicate (14 ± 2 vs 11 ± 2 for myoglobin and 16 vs 12 ± 1 for BSA) within a significantly shortened pretreatment time of ∼3.5 min (including 1 min of thermal denaturation and reduction and ∼2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (∼6 fmol) to 1 mg/mL (∼120 pmol) were positively identified by the online system. Such a platform was further successfully applied for analyzing the soluble fraction of mouse liver extract. Of all the 367 proteins identified from samples pretreated by the urea protocol and online heating, ∼40% were overlapped, showing the partial complementation of both approaches. All these results demonstrate that the online integrated platform is of great promise for high-throughput proteome profiling and improved identification capacity for low-abundance proteins with a minute sample amount.
Co-reporter:Huiming Yuan, Lihua Zhang, Chunyan Hou, Guijie Zhu, Dingyin Tao, Zhen Liang and Yukui Zhang
Analytical Chemistry 2009 Volume 81(Issue 21) pp:8708
Publication Date(Web):September 29, 2009
DOI:10.1021/ac900310y
An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by a microcolumn packed with mixed weak anion and weak cation exchange (WAX/WCX) particles under a series of salt steps, online digested by a trypsin immobilized microenzymatic reactor (IMER), trapped and desalted by two parallel C8 precolumns, separated by microreversed-phase liquid chromatography (μRPLC) under a linear gradient of organic modifier concentration, and finally identified by electrospray ionization-MS/MS (ESI-MS/MS). To evaluate the performance of such a platform, a mixture of myoglobin, cytochrome c, bovine serum albumin (BSA), and α-casein, with mass ranging from 25 ng to 2 μg, was analyzed. Compared to the methods by off-line protein fractionation and shotgun based strategy, the analysis time, including sample preparation, digestion, desalting, separation, and detection, was shortened from ca. 30 to 5 h, and cytochrome c with abundance of 25 ng could be identified with improved sequence coverage. Furthermore, such an integrated platform was successfully applied into the analysis of proteins extracted from human lung cancer cells. Compared with the results obtained by the shotgun approach, the identified protein number was increased by 30%. All these results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.
Co-reporter:Junfeng Ma, Lihua Zhang, Zhen Liang, Weibing Zhang, Yukui Zhang
Analytica Chimica Acta 2009 Volume 632(Issue 1) pp:1-8
Publication Date(Web):19 January 2009
DOI:10.1016/j.aca.2007.08.045
Immobilized enzymatic reactors recently have drawn much attention because of the striking advantages, such as high substrate turnover rate and ease in coupling with the separation and detection systems. Carrier materials, which have great effects on the development of the immobilized enzymatic reactors, have always being the focus of study. In this paper, the contributions, mainly in the last 5 years, on the enzymatic reactors and their applications in proteome study are reviewed, with some newly developed inorganic and organic carriers for enzyme immobilization described in details. Moreover, the hyphenation of immobilized enzymatic reactors with the separation and identification systems is also summarized. By reviewing these achievements, it could be seen that enzymatic reactors have very bright future, especially in proteome analysis.
Co-reporter:Huiming Yuan, Yuan Zhou, Lihua Zhang, Zhen Liang, Yukui Zhang
Journal of Chromatography A 2009 Volume 1216(Issue 44) pp:7478-7482
Publication Date(Web):30 October 2009
DOI:10.1016/j.chroma.2009.06.019
An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by μRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.
Co-reporter:Huiming Yuan, Lihua Zhang, Weibing Zhang, Zhen Liang, Yukui Zhang
Journal of Chromatography A 2009 Volume 1216(Issue 42) pp:7024-7032
Publication Date(Web):16 October 2009
DOI:10.1016/j.chroma.2009.08.065
Columns switch recycling size exclusion chromatography (csrSEC) was proposed to achieve high resolution protein separation with good biocompatibility. Proteins were firstly separated by two serially coupled SEC columns, and fractions were in sequence switched back to the first column by two-position valves for further separation in terms of close-loop recycling until satisfactory resolution was achieved. Compared to SEC, the separation window was broadened by increasing column length via cycling without further increase on back pressure. Compared to recycling SEC (rSEC), the overtaking of later eluted components by early eluted ones after several cycles could be avoided for complex sample analysis, by parking fractions in the second SEC column before transferred in turn back to the first one for cycling ordinally. In our experiments, the baseline separation of five proteins with molecular weight ranging from 10 kDa to 80 kDa was achieved by csrSEC. Furthermore, a multidimensional csrSEC–RPLC platform was constructed, and peak capacity up to 3600 was obtained for protein separation. All these results demonstrated that csrSEC is a promising protein separation mode with good biocompatibility, broadened separation window and improved resolution.
Co-reporter:Yongzheng Cong;Yu Liang;Lihua Zhang;Weibing Zhang;Yukui Zhang
Journal of Separation Science 2009 Volume 32( Issue 3) pp:462-465
Publication Date(Web):
DOI:10.1002/jssc.200800514
Abstract
A stepwise gradient of electric field strength was proposed for microchip IEF-based protein separation, by which after focusing at low voltages, IEF was performed by applying higher separation voltages step-by-step. A linear relationship between the focusing time and the inverse of the electric field strength was found. In addition, the conductivity of an established pH gradient showed a negative but nonlinear correlation with the applied voltage. Based on the above-mentioned results, a stepwise gradient of electric field strength, ranging from 160 to 1500 V/cm was applied in the separation of proteins extracted from Escherichia coli in a straight glass microchip channel permanently coated by polyacrylamide. Compared to the conventional separation performed under a constant field strength of 750 V/cm, the increased stepwise gradient of electric field strength resulted in improved resolution and decreased focusing time, while without the negative effects of Joule heat for protein separation. All these results demonstrated that such a method might be of great significance to achieve high resolution and high-throughput analysis of complex protein samples for microchip IEF.
Co-reporter:Bin Han;Pingli Wang;Guijie Zhu;Lihua Zhang;Feng Qu;Yulin Deng;Yukui Zhang
Journal of Separation Science 2009 Volume 32( Issue 8) pp:1211-1215
Publication Date(Web):
DOI:10.1002/jssc.200800572
Abstract
Microchip free flow IEF (μFFIEF) with monolithic IPG was proposed for protein prefractionation. The monolithic materials were first prepared by UV irradiation in a microchamber of 43 mm length, 23 mm width, and 38 μm depth. Carrier ampholytes (CAs) were further immobilized on the monolith by chemical bonding, to form a stable pH gradient. By such a technique, the continuous introduction of CAs in traditional μFFIEF could be avoided, not only to decrease the operation cost, but also to avoid the interference of CAs for the further protein identification by MS/MS. With a fluorescence microscope as the detector, under the optimal conditions, the separation of FITC labeled β-lactoglobulin and carbonic anhydrase, with 0.9 unit difference on pI, was achieved with good reproducibility. The experimental results demonstrated that under the experimental conditions we applied, the separation mechanism of μFFIEF with M-IPG materials might be the cooperative effects of IEF and CZE, and the former one plays a predominant role.
Co-reporter:Pingli Wang;Dingyin Tao;Lihua Zhang;Zhen Liang;Yukui Zhang
Journal of Separation Science 2009 Volume 32( Issue 15-16) pp:2629-2634
Publication Date(Web):
DOI:10.1002/jssc.200900223
Abstract
In this study, microchip free flow planar RP electrochromatography (μFF-PRPEC) was developed by in situ polymerization of monolithic materials in microchamber, and successfully applied for the separation of dyes and proteins. Poly(butyle methyacrylate-co-ethylene dimethacrylate) was prepared by UV-initiated polymerization in a glass microchamber (42 mm long, 23 mm wide, and 28 μm deep). A mixture of 1-propanol, 1,4-butanediol, and water was chosen as porogens, and 1.2% (wt%) 2-acrylamide-2-methyl-propanesulfonic acid (AMPS) was added into the polymerization solution to generate EOF. With 30% v/v ACN-15 mM Tris-HCl as the mobile phase, rhodamine B and methyl green were separated from each other with 400 V transverse voltage applied, and resolution as high as 4.6 was obtained, much higher than that obtained by μFFE under optimal conditions. Furthermore, μFF-PRPEC was also successfully applied into the separation of lysozyme and ribonuclease B, and resolution as high as 9.4 was obtained. All these results demonstrate that μFF-RPPEC might have great potential in the microscale continuous preparation of samples with improved resolution compared to μFFE.
Co-reporter:Xiaoqiang Qiao, Li Wang, Junfeng Ma, Qiliang Deng, Zhen Liang, Lihua Zhang, Xiaojun Peng, Yukui Zhang
Analytica Chimica Acta 2009 640(1–2) pp: 114-120
Publication Date(Web):
DOI:10.1016/j.aca.2009.03.021
Co-reporter:Yongzheng Cong;Lihua Zhang;Dingyin Tao;Yu Liang;Weibing Zhang;Yukui Zhang
Journal of Separation Science 2008 Volume 31( Issue 3) pp:588-594
Publication Date(Web):
DOI:10.1002/jssc.200700444
Abstract
A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 μm and 40 μm, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4×10–9m2·V–1·s–1, about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.
Co-reporter:Hui Wang;Jicheng Duan;Lihua Zhang;Zhen Liang;Weibing Zhang;Yukui Zhang
Journal of Separation Science 2008 Volume 31( Issue 3) pp:480-487
Publication Date(Web):
DOI:10.1002/jssc.200700445
Abstract
The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by μHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of β-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by μHPLC–ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.
Co-reporter:Guijie Zhu;Lihua Zhang;Huiming Yuan;Zhen Liang;Weibing Zhang;Yukui Zhang
Journal of Separation Science 2007 Volume 30(Issue 6) pp:792-803
Publication Date(Web):14 MAR 2007
DOI:10.1002/jssc.200600496
This review summarizes the development of monolithic materials, including both organic and inorganic polymers, according mainly to the papers published in the past two years. Due to their good permeability, fast mass transfer, high stability, and their ease of modification, such materials have been widely used in microcolumn separation systems, not only as stationary phases for CEC and capillary HPLC, but also as substances for sample concentration and enzyme reactor. All the research results demonstrate that monolithic materials in microseparation systems can be expected to play an increasingly important role in the analysis of complex samples.
Co-reporter:Lihua Zhang;Zhen Liang;Junfeng Ma;Weibing Zhang;Yukui Zhang
Journal of Separation Science 2007 Volume 30(Issue 17) pp:3050-3059
Publication Date(Web):21 NOV 2007
DOI:10.1002/jssc.200700362
In the current proteome study, protein digestion is indispensable before proteins could be identified by MS/MS, no matter based on top–down or bottom–up strategies. Compared to the traditional digestion performed in free solution, immobilized enzyme shows great advantages in digestion speed, stability, and longevity, especially with monolithic materials as the supports. Besides the improved digestion capacity, the immobilized enzyme reactors (IMERs) could be further coupled with the separation and detection systems, enabling high-throughput protein identification. In this paper, the latest advances in the monolith-based IMERs and their applications in proteomic study are briefly summarized. By reviewing these achievements, it could be seen that monilith-based IMERs have very bright future in proteome analysis.
Co-reporter:Weibing Zhang;Yushu Huo;Yukui Zhang;Zhen Liang;Jie Zhang;Jicheng Duan;Lihua Zhang
Journal of Separation Science 2006 Volume 29(Issue 16) pp:2514-2522
Publication Date(Web):31 OCT 2006
DOI:10.1002/jssc.200600217
A novel on-line system combining supercritical fluid extraction (SFE) and two-dimensional high performance liquid chromatography (2D-HPLC) was developed. A trap column and two three-port valves were employed to couple SFE and 2D-HPLC system, which was composed of a CN column and a monolithic silica column, connected by a 10-port dual-position valve. The analytes extracted by supercritical CO2 were completely transferred to the 2D-HPLC system. After separation in two orthogonal modes, the eluents were delivered to APCI-tandem-MS for identification of the samples. In this way, sample preparation, separation, detection, and identification were integrated into an on-line system permitting analysis of the fruiting bodies of Ganoderma lucidum, and at least 73 components in the extract were resolved with calculated peak capacity of up to 1643.
Co-reporter:Lukuan Liu, Kaiguang Yang, Lihua Zhang, Yukui Zhang
Science Bulletin (December 2016) Volume 61(Issue 24) pp:1890-1891
Publication Date(Web):1 December 2016
DOI:10.1007/s11434-016-1207-7
Co-reporter:Qi Wu, Huiming Yuan, Lihua Zhang, Yukui Zhang
Analytica Chimica Acta (20 June 2012) Volume 731() pp:1-10
Publication Date(Web):20 June 2012
DOI:10.1016/j.aca.2012.04.010
With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography–mass spectrometry (MDLC–MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including “top-down” and “bottom-up” to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes.Graphical abstractDownload full-size imageHighlights► We discuss progress of MDLC–MS systems in qualitative and quantitative proteomics. ► Both “Top-down” and “bottom-up” strategies are discussed in detail. ► On-line integrations of stable isotope labeling process are highlighted. ► This review gives insights into further directions for higher level integration.
Co-reporter:Shen Zhang, Yichu Shan, Shurong Zhang, Zhigang Sui, Lihua Zhang, Zhen Liang, Yukui Zhang
Journal of Proteomics (10 February 2017) Volume 154() pp:
Publication Date(Web):10 February 2017
DOI:10.1016/j.jprot.2016.12.003
•Non-isobaric peptide termini labeling will help to distinguish both b and y ion series.•The quantitative profiling can be performed simultaneously along with the de novo sequencing.•This strategy can be expanded to peptide mutation analysis with combination of mass-defect based labeling.A simple and effective de novo sequencing strategy assisted by non-isobaric peptide termini labeling, NIPTL-Novo, was established. The y-series ions and b-series ions of peptides can be clearly distinguished according to the different mass tags incorporated in N-terminus and C-terminus. This is helpful for improving the accuracy of peptide sequencing and increasing the sequencing speed. For the spectra commonly identified by both de novo sequencing and database searching software (Mascot or Maxquant), NIPTL-Novo gave identical result to more than 85% of these spectra. Furthermore, the quantitative profiling of the sample can be performed simultaneously along with de novo sequencing. Finally, this strategy can be applied to discover the peptides with potential mutation sites by combining with mass-defect based isotopic labeling.SignificanceThe aim of the research presented in this paper is to establish a simple but effective de novo sequencing strategy based on non-isobaric peptide termini labeling, named NIPTL-Novo. First, different mass tags incorporated in N-terminus and C-terminus generated by non-isobaric peptide termini labeling will help to distinguish both b and y ion series, which significantly simplify the MS/MS spectra and reduce the time consumption for de novo sequencing. Second, the isolation window of this strategy is just 4 Da, much smaller than most existed labeling assisted de novo sequencing methods, which reduces the interferences caused by co-fragmentation ions. Third, the quantitative profiling of the sample can be performed simultaneously along with the de novo sequencing, and the quantitative accuracy is comparable to other chemical labeling methods. Finally, this strategy was expanded to the analysis of peptide mutation with combination of mass-defect based labeling, and two reliable mutated peptides were discovered.
Co-reporter:Senwu Li, Kaiguang Yang, Baofeng Zhao, Xiao Li, Lukuan Liu, Yuanbo Chen, Lihua Zhang and Yukui Zhang
Journal of Materials Chemistry A 2016 - vol. 4(Issue 15) pp:NaN2739-2739
Publication Date(Web):2016/03/30
DOI:10.1039/C6TB90044E
Correction for ‘Epitope imprinting enhanced IMAC (EI-IMAC) for highly selective purification of His-tagged protein’ by Senwu Li et al., J. Mater. Chem. B, 2016, 4, 1960–1967.
Co-reporter:Yanyan Qu, Jianxi Liu, Kaiguang Yang, Qi Wu, Yichu Shan, Lihua Zhang, Zhen Liang and Yukui Zhang
Journal of Materials Chemistry A 2015 - vol. 3(Issue 19) pp:NaN3930-3930
Publication Date(Web):2015/04/15
DOI:10.1039/C5TB00156K
Biocompatible boronate core–shell polymeric particles were grown in a unique polymerization system via a one-pot strategy making full use of the residual soluble boronate oligomer to in situ build the core–shell structure. The obtained submicron particles were shown to exhibit excellent recognition affinity toward glycoproteins with high binding capacity and specificity.
Co-reporter:Senwu Li, Kaiguang Yang, Baofeng Zhao, Xiao Li, Lukuan Liu, Yuanbo Chen, Lihua Zhang and Yukui Zhang
Journal of Materials Chemistry A 2016 - vol. 4(Issue 11) pp:NaN1967-1967
Publication Date(Web):2016/02/17
DOI:10.1039/C5TB02505B
Recombinant protein technology occupies an important position in fields including biopharmaceutics, proteomics, structural and functional biology. However, the purification of His-tagged protein, the majority portion of recombinant protein, is seriously hindered by impurities. These impurities, including host proteins with inherent cysteine and histidine-rich regions or metal centers, are usually beyond the purification ability of commonly used IMAC materials. To remove this barrier, a novel purification material was developed through enhancing the selectivity of IMAC by means of surface epitope imprinting using His-tag, the common terminal of His-tagged protein, as the template. Characterizations including TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy, measurement of DLS size and zeta potential were carried out to prove the fabrication of the imprinted shell. Results exhibited a high imprinting factor of 7.1. Besides, the adsorption kinetics were not affected by the surface imprinted shell and could reach adsorption equilibrium within 15 min. Compared with the substrate IMAC, the novel epitope imprinting enhanced IMAC (EI-IMAC) showed an obvious improvement (5% increase of purity) in the selectivity of His-tagged recombinant protein from crude cell lysis.
Co-reporter:Kaiguang Yang, Jianxi Liu, Senwu Li, Qinran Li, Qi Wu, Yuan Zhou, Qun Zhao, Nan Deng, Zhen Liang, Lihua Zhang and Yukui Zhang
Chemical Communications 2014 - vol. 50(Issue 67) pp:NaN9524-9524
Publication Date(Web):2014/06/30
DOI:10.1039/C4CC03428G
Polymer self-assembly was developed as an epitope imprinting strategy involving facile processes and high recognition site density. As a model, transferrin epitope imprinted polyethersulfone (PES) beads were successfully fabricated using this technique. The imprinted beads demonstrated excellent selectivity toward the transferrin epitope and transferrin even in the real sample.
Co-reporter:Jianxi Liu, Kaiguang Yang, Yanyan Qu, Senwu Li, Qi Wu, Zhen Liang, Lihua Zhang and Yukui Zhang
Chemical Communications 2015 - vol. 51(Issue 18) pp:NaN3898-3898
Publication Date(Web):2015/01/30
DOI:10.1039/C4CC10004B
Stimuli-responsive core–shell nanoparticles, with 3-acrylamidophenyl boronic acid (APBA) as the functional group, were synthesized via combined distillation precipitation polymerization and RAFT media precipitation polymerization. The well-defined boronic acid-polymer branch on the nanoparticle surface displayed high binding capacity and good selectivity towards cis-diol-containing molecules.
Co-reporter:Liyuan Zhang, Qun Zhao, Zhen Liang, Kaiguang Yang, Liangliang Sun, Lihua Zhang and Yukui Zhang
Chemical Communications 2012 - vol. 48(Issue 50) pp:NaN6276-6276
Publication Date(Web):2012/05/01
DOI:10.1039/C2CC31641B
A new type of IMAC material, with ATP as the chelating ligand, was synthesized and applied to capture phosphopeptides. For the first time, the approach for phosphopeptide enrichment could provide selectivity under 5000-fold dilution by nonphosphopeptides, and sensitivity of on-target enrichment at 3 amol.
Co-reporter:Jinxiang Liu, Kaiguang Yang, Qiliang Deng, Qinran Li, Lihua Zhang, Zhen Liang and Yukui Zhang
Chemical Communications 2011 - vol. 47(Issue 13) pp:NaN3971-3971
Publication Date(Web):2011/02/24
DOI:10.1039/C0CC05317A
A new approach, combining metal-coordination with molecular imprinting technology, was developed to prepare protein-affinity materials, which showed higher specific recognition ability towards the target protein than those prepared using either metal-coordination or molecular imprinting technology.
