Co-reporter:Shuang Lin, Dong Yun, Dawei Qi, Chunhui Deng, Yan Li and Xiangmin Zhang
Journal of Proteome Research March 2008 Volume 7(Issue 3) pp:1297-1307
Publication Date(Web):February 8, 2008
DOI:10.1021/pr700586j
In this study, a novel microwave-assisted protein digestion method was developed using trypsin-immobilized magnetic nanoparticles (TIMNs). The magnetic nanoparticles worked as not only substrate for enzyme immobilization, but also excellent microwave irradiation absorber and, thus, improved the efficiency of microwave-assisted digestion greatly. Three standard proteins, bovine serum albumin (BSA), myoglobin, and cytochrome c, were used to optimize the conditions of this novel digestion method. With the optimized conditions, peptide fragments produced in very short time (only 15 s) could be identified successfully by MALDI-TOF-MS. When it was compared to the conventional in-solution digestion (12 h), equivalent or better digestion efficiency was observed. Even when protein quantity was as low as micrograms, this novel digestion method still could digest proteins successfully, while the same samples by conventional in-solution digestion failed. Moreover, with an external magnetic field, the enzyme could be removed easily and reused. It was verified that, after 4 replicate runs, the TIMNs still kept high activity. To further confirm the efficiency of this rapid digestion method for proteome analysis, it was applied to the protein extract of rat liver. Without any preparation and prefractionation processing, the entire proteome digested by TIMNs in 15 s went through LC-ESI−MS/MS direct analysis. The whole shotgun proteomic experiment was finished in only 1 h with the identification of 313 proteins (p < 0.01). This new application of TIMNs in microwave-assisted protein digestion really opens a route for large-scale proteomic analysis.Keywords: microwave-assisted digestion; proteome analysis; shotgun; Trypsin immobilized magnetic nanoparticles;
Co-reporter:Xi Shao;Min Chen;Da Wu;Baizhan Liu;Xiangmin Zhang;Chaoying Chen
Analytical Methods (2009-Present) 2017 vol. 9(Issue 5) pp:761-767
Publication Date(Web):2017/02/02
DOI:10.1039/C6AY02762H
In this work, we developed a system for the qualitative and quantitative analysis of four tobacco-specific N-nitrosamines (TSNAs). This system was based on strong cation exchange/reversed phase liquid chromatography-tandem mass spectrometry (SCX/RPLC-MS/MS). Benefiting from an efficient two-dimensional separation, the system could well circumvent tremendous matrix effects and provide excellent detection of ultra-low concentrations of TSNAs (ng mL−1 level, after pretreatments) in Chinese Virginia cigarette mainstream smoke samples. Based on the features of TSNAs, one heart-cutting was used to transfer fractions from the first dimension to the second. A side flow was added between the two dimensions to gain retention in RPLC of TSNAs from SCX fractions. The system was evaluated using standard TSNA samples and mainstream cigarette smoke samples. The total separation time was within 20 minutes. The limits of quantitation (LOQs) for N′-nitrosonornicotine (NNN), 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), (R,S)-N-nitrosoanatabine (NAT) and (R,S)-N-nitrosoanabasine (NAB) were 39, 47, 58 and 9 pg mL−1, respectively, which could well meet the requirements of all kinds of commercial cigarette samples. With Kentucky reference cigarettes as a standard sample, the relative deviations (r) of the measured values of TSNAs from the standard values were between −1.6% and 4.6%. And the relative standard deviations (RSD) of intra- and inter-day analysis were below 4.9% and 5.9%, respectively. These results revealed that the SCX/RPLC-MS/MS system we built could efficiently achieve minimal sample matrix effects and make highly sensitive analysis of pg mL−1 level TSNAs in extremely complicated samples easy, fast and accurate. Finally, we successfully applied the system to the evaluation of TSNA yields in Chinese Virginia cigarettes.
Co-reporter:Dongpo Xu;Guoquan Yan;Mingxia Gao;Chunhui Deng
Analytical and Bioanalytical Chemistry 2017 Volume 409( Issue 6) pp:1607-1614
Publication Date(Web):2017 February
DOI:10.1007/s00216-016-0101-0
Glycosylation and phosphorylation as the commonest and most important posttranslational modifications of proteins play critical roles in biological processes. Specific and sensitive strategies have been developed to identify the glycosylation and phosphorylation of proteins by mass spectrometry but most of these methods have mainly focused on enriching glycopeptides or phosphopeptides separately, and few of them can enrich both of them. In this work, highly selective SiO2–NH2@TiO2 hollow microspheres were designed and synthesized for the simultaneous selective enrichment of both phosphopeptides and glycopeptides. Because of the bifunctionalized property of the titanium dioxide and the amino group, the hollow microspheres were successfully applied for the simultaneous enrichment of phosphopeptides and glycopeptides. We evaluated the enrichment selectivity of the SiO2–NH2@TiO2 hollow microspheres by capturing phosphopeptides and glycopeptides from a peptide mixture of bovine β-casein, horseradish peroxidase, and bovine serum albumin in a molar ratio of 1:1:500 (1.7 × 10−12 mol β-casein and horseradish peroxidase, 8.5 × 10−10 mol bovine serum albumin in 100 μL).
Co-reporter:Zhi Huang, Guoquan Yan, Mingxia Gao, and Xiangmin Zhang
Analytical Chemistry 2016 Volume 88(Issue 4) pp:2440
Publication Date(Web):January 19, 2016
DOI:10.1021/acs.analchem.5b04553
In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.
Co-reporter:Dongpo Xu, Mingxia Gao, Chunhui Deng and Xiangmin Zhang
Analyst 2016 vol. 141(Issue 11) pp:3421-3427
Publication Date(Web):18 Apr 2016
DOI:10.1039/C6AN00361C
Ti4+ immobilized SiO2 graphene-like multilayer nanosheets were designed and synthesized for the ultrasensitive enrichment of phosphopeptides with the detection limit of 1.0 pg μL−1 (4 × 10−17 mol μL−1). The enrichment selectivity of the nanosheets was evaluated by capturing phosphopeptides from a peptide mixture of bovine β-casein (β-casein) and bovine serum albumin (BSA) with the molar ratio of 1:1000 (1.7 × 10−12 mol of β-casein and 1.7 × 10−9 mol of BSA in 100 μL).
Co-reporter:Qian Qi;Guoquan Yan;Chunhui Deng
Analytical and Bioanalytical Chemistry 2016 Volume 408( Issue 29) pp:8437-8445
Publication Date(Web):2016 November
DOI:10.1007/s00216-016-9964-3
The process of protein digestion is a critical step for successful protein identification in proteomic analysis. Many efforts have been dedicated to enhancing the digestion efficiency for sufficient digestion. Among these approaches, protein complete denaturation with denaturants is a common process for better digestion. However, the removal of denaturants was tedious or would cause protein loss and other problems. In this work, a feasible digestion approach, immobilized protein digestion (IPD), based on covalent binding has been developed. Proteins can be completely denatured and immobilized on the surface of functional materials by covalent binding to form a monolayer. Subsequently, varieties of denaturants or contaminants would be removed thoroughly by washing. To achieve fast immobilization and high digestion efficiency, different functional materials and denaturants were selected. Compared with traditional in-solution digestion, the method achieved a prominent increase in identified peptides numbers and sequence coverage of proteins. Data analysis also showed that covalent binding could evidently decrease enzymatic missed cleavage for various protein sequences. Furthermore, possible peptide losses due to covalent binding were also investigated. Also, it has been proved to be efficient for complex biological sample digestion.
Co-reporter:Yiying Liu;Guoquan Yan;Mingxia Gao
Analytical and Bioanalytical Chemistry 2016 Volume 408( Issue 13) pp:3495-3502
Publication Date(Web):2016 May
DOI:10.1007/s00216-016-9427-x
An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4 % SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells.
Co-reporter:Huili Yang, Chunhui Deng, Xiangmin Zhang
Talanta 2016 Volume 153() pp:285-294
Publication Date(Web):1 June 2016
DOI:10.1016/j.talanta.2016.03.012
•We have developed Ti4+-immobilized capillary trapping column (250 μm i.d.) for on-line enrichment of phosphopeptides.•It minimizes the sample loss and improves the effectiveness of phosphopeptide detection that the limit of detection is as low as 1 fmol/μL.•It is successfully applied to the detection of phosphopeptides fromskim milk, human serum and mouse brain.In this work, we have developed Ti4+-immobilized capillary trapping column (250 μm i.d.) for highly specific on-line enrichment of phosphopeptides in the bio-samples. It minimizes the sample loss and improves the effectiveness of phosphopeptide detection that the limit of detection is as low as 1 fmol/μL. It is successfully applied to the detection of phosphopeptides from complex biological samples, such as skim milk, human serum and mouse brain. The results indicate that the Ti4+-immobilized capillary trapping column is time-effective and has the great potential of application in low-abundance phosphopeptides on-line analysis. The prepared Ti4+-immobilized capillary trapping column will be further used in LC/MS platform for phosphoproteome analysis.Herein, we present Ti4+-immobilized capillary trapping column (250 μm i.d.) for highly specific on-line enrichment of phosphopeptides in the bio-samples.
Co-reporter:Ling Zhang, Ren Zhang, Mingxia Gao, Xiangmin Zhang
Talanta 2016 Volume 158() pp:315-321
Publication Date(Web):1 September 2016
DOI:10.1016/j.talanta.2016.05.064
•One-step synthetic method of thiol and alkynyl contained bioorthogonal SERS reporter is environmentally benign, high yield and easy in operation.•Au@PBAT@PDA nanoparticles exhibit strong SERS signal intensity, excellent stability in complex biological environment.•Au@PBAT@PDA nanoparticles show good imaging results for live cancer cell SERS imaging in Raman-silent region.•The rich functional groups (i.e., catechol and amine) on PDA surface provide a broad platform for further modification in biomedical application.•A Raman-silent region nanoprobe was synthesized for live cell SERS imaging.A bioorthogonal Raman reporter-embedded Au-core and polydopamine-shell nanoprobe was initially designed and synthesized for live cell surface-enhanced Raman scattering (SERS) imaging in Raman-silent region. The firstly synthetic bioorthogonal Raman reporter (E)-2- ((4-(phenylethynyl) benzylidene)amino)ethanethiol (PBAT) provided intense Raman signal at 2220 cm−1 in Raman-silent region. And its synthetic method was environmentally benign, high yield and easy in operation. In addition, the hydrophobicity of PBAT led to slightly aggregation of gold nanoparticles, which enhance the SERS intensity of the nanoprobe through hot spots. Furthermore, owing to the remarkable biocompatibility of polydopamine (PDA), the SERS nanoprobe can be smoothly internalized into cancer cells to realize SERS imaging in Raman-silent region. Finally, the SERS mapping results confirmed that nanoprobe exhibited strong SERS signal intensity, excellent stability in complex biological environment and low toxicity inside live cancer cells. Therefore, the reporter-embedded core-shell nanoprobe has enormous potential of applications in biomedical diagnostics in the near future.
Co-reporter:Jiaxi Wang, Yanan Wang, Mingxia Gao, Xiangmin Zhang, and Pengyuan Yang
ACS Applied Materials & Interfaces 2015 Volume 7(Issue 29) pp:16011
Publication Date(Web):July 10, 2015
DOI:10.1021/acsami.5b04295
Capturing glycopeptides selectively and efficiently from mixed biological samples has always been critical for comprehensive and in-depth glycoproteomics analysis, but the lack of materials with superior capture capacity and high specificity still makes it a challenge. In this work, we introduce a way first to synthesize a novel boronic-acid-functionalized magnetic graphene@phenolic-formaldehyde resin multilayer composites via a facile process. The as-prepared composites gathered excellent characters of large specific surface area and strong magnetic responsiveness of magnetic graphene, biocompatibility of resin, and enhanced affinity properties of boronic acid. Furthermore, the functional graphene composites were shown to have low detection limit (1 fmol) and good selectivity, even when the background nonglycopeptides has a concentration 100 fold higher. Additionally, enrichment efficiency of the composites was still retained after being used repeatedly (at least three times). Better yet, the practical applicability of this approach was evaluated by the enrichment of human serum with a low sample volume of 1 μL. All the results have illustrated that the magG@PF@APB has a great potential in glycoproteome analysis of complex biological samples.Keywords: boronic-acid-functionalized magnetic graphene; enrichment; glycoproteome; MALDI-TOF mass spectrometry; phenolic-formaldehyde resin;
Co-reporter:Qi Chen, Guoquan Yan, Mingxia Gao, and Xiangmin Zhang
Analytical Chemistry 2015 Volume 87(Issue 13) pp:6674
Publication Date(Web):June 10, 2015
DOI:10.1021/acs.analchem.5b00808
Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.
Co-reporter:Changlong Sun, Ling Zhang, Ren Zhang, Mingxia Gao and Xiangmin Zhang
RSC Advances 2015 vol. 5(Issue 88) pp:72369-72372
Publication Date(Web):25 Aug 2015
DOI:10.1039/C5RA12628B
A novel and straightforward strategy for SERS probe fabrication using polydopamine as both the protection and the conjugation layer was proposed. Synthesized SERS probes showed significantly enhanced stability and acted as efficient probes for multiplex tumor associated cell surface antigen detection using SERS imaging.
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:Lanting Li, Guoquan Yan, Xiangmin Zhang
Talanta 2015 Volume 144() pp:122-128
Publication Date(Web):1 November 2015
DOI:10.1016/j.talanta.2015.05.068
•We developed a protein N-termini enrichment method based on TFM.•TFM was efficient in binding internal peptides.•Both acetylated and free N-termini were isolated from the standard proteins.•122 acetylated N-termini and 107 free N-termini were identified in mouse liver.Analysis of protein N-termini is of great importance in helping to figure out important posttranslational modifications (PTMs) occurred in N-termini. Those PTMs include initial methionine removal, proteolytic cleavage, peptide signal processing, or N-terminal acetylation, which are usually neglected by conventional shotgun proteomics strategies. Herein, we develop a protein N-terminal peptides enrichment method based on commercial tresyl-functionalized microspheres (TFM). TFM could specifically immobilize the non-N-terminal peptides (internal peptides) from the supernatant. We demonstrated the isolation by TFM was more fast and efficient than formyl or epoxy-functionalized microspheres. Furthermore, this method could simultaneously isolate not only naturally free but acetylated blocked N-terminus. That facilitates a more comprehensive acquisition of N-terminus. After being verified by three standard proteins, cytochrome C, ribonuclease B and bovine serum albumin, this method was applied to mouse liver protein sample. We identified 122 naturally acetylated N-terminus and 107 free N-terminus in the sample. With the good performance of TFM, this method is efficient and useful for N-termini recovery.Schematic diagram of tresyl-functionalized microspheres based protein N-termini enrichment method. (a) Overall flow chart. (b) Dimethylation reaction of amino. (c) Internal peptides depletion. The nucleophilic substitution reaction between primary amine and the hydrophilic vinyl polymer. The primary amine comes from peptides.
Co-reporter:Peng Zhang, Ren Zhang, Mingxia Gao, and Xiangmin Zhang
ACS Applied Materials & Interfaces 2014 Volume 6(Issue 1) pp:370
Publication Date(Web):December 9, 2013
DOI:10.1021/am404406c
The capture and detection of circulating tumor cells (CTCs) in the bloodstream of patients with cancer is crucial for the clinical diagnosis and therapy. In the present work, a facile and integrated approach based on novel nitrocellulose membrane substrate and large-scale surface-enhanced Raman scattering (SERS) imaging technology has been developed for CTCs’ sensitive detection and enumeration. The system mainly consists of three aspects: capture of CTCs in bloodstream, SERS probes labeling of the captured CTCs and large-scale SERS imaging readout of CTCs enumeration. The NC membrane was used to prepare the novel CTC-capture substrate through antibody self-assembled. It was low-cost, easily prepared and completely nontoxic. Furthermore, excellent capture efficiency of the substrate was demonstrated using nonsmall-cell lung cancer (NSCLC) cells (NCI-H1650) as target cells. As the most sensitive detection technology, SERS holds huge potential in CTCs analysis. Large-scale SERS imaging was employed in CTCs enumeration for the first time, instead of the conventional fluorescence imaging. Our SERS probes, with a simplified structure, offered highly enough sensitivity to recognize every single cell clearly. In the simulation experiment of spiking 100 cancer cells into 1 mL of human whole blood, 34 cells were captured and counted successfully according to the SERS imaging result. Our experimental results demonstrate the potential feasibility of novel NC membrane substrate coupled with large-scale SERS imaging technology for the accurate enumeration of CTCs in human whole blood.Keywords: circulating tumor cells; large-scale surface-enhanced Raman scattering imaging; nitrocellulose membrane substrate;
Co-reporter:Dan Wan;Qi Chen;MingXia Gao;XiangMin Zhang;PengYuan Yang
Science China Chemistry 2014 Volume 57( Issue 5) pp:703-707
Publication Date(Web):2014 May
DOI:10.1007/s11426-014-5084-0
In our work, a new extraction tip with gold-modified polymer is developed. The simple, self-made and extremely economical tips were successfully applied to capture cysteine-containing peptides. The loading capacity of a tip (column bed: 0.3 mm diameter, 5 mm length) is 2–4 μg peptides. We can make one tip in 30 s and each costs less than 0.1 cent. The use of these tips can achieve a stable analysis with less background interference, even for 10 ng target peptides. Compared with other separation techniques, our method can save much time and energy while providing a means to selectively capture cysteine-containing peptides from complex analyte due to the strong interaction. All results showed that our new extraction tips have minimal cost and perfect selectivity; thus they have great potential in sample pretreatment systems for proteomics.
Co-reporter:Chaofeng Wang;Mingxia Gao;Peng Zhang;Xiangmin Zhang
Chromatographia 2014 Volume 77( Issue 5-6) pp:413-418
Publication Date(Web):2014 March
DOI:10.1007/s10337-013-2622-4
A hydrophilic immobilized enzyme reactor (IMER) containing trypsin was prepared and applied in the proteolysis of glycoproteins. Glycoproteins including horseradish peroxidase, asialofetuin, and fetuin were used to evaluate the performance of the hydrophilic IMER for the glycoprotein digestion. The digested products were detected by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight mass spectrometry and micro-high-performance liquid chromatography. The hydrophilic IMER showed higher enzymatic digestion efficiency compared with conventional in-solution digestion. The digestion time could be reduced from 16 h to several minutes. Furthermore, using microwaves as a heat source, the reproducibility of the hydrophilic IMER was evaluated and this IMER could be recycled for at least ten times without obvious loss of enzyme activity. The hydrophilic IMER provides a promising tool for high-throughput glycoproteome analysis.
Co-reporter:Dan Wan;Mingxia Gao;Yuhua Wang;Peng Zhang ;Xiangmin Zhang
Journal of Separation Science 2013 Volume 36( Issue 3) pp:629-635
Publication Date(Web):
DOI:10.1002/jssc.201200766
In this study, we present a rapid and simple method for the separation and direct detection of glutathione by combining gold nanoparticles and MALDI–TOF-MS with graphene as matrix. Gold nanoparticles enable the selective capture of thiol-containing compounds. Gold nanoparticles bound with analytes can be mixed with graphene matrix for direct analysis by MALDI–TOF-MS, which can avoid sample loss and contamination during transfer process. Compared with a conventional matrix, α-cyano-4-hydroxycinnamic acid, graphene exhibits an excellent desorption/ionization efficiency, thermal and mechanical properties. The use of graphene as matrix avoids the fragmentation of analytes. Stable analysis was achieved with less background interference even at the concentration of 0.625 ng/μL. To further confirm its efficiency, the optimized approach was applied to the separation and detection of glutathione in mouse liver extraction. This result showed the great potential of detection of biologically important thiols in biochemical and biomedical research.
Co-reporter:Jie Lei ;Xiangmin Zhang
Journal of Separation Science 2013 Volume 36( Issue 12) pp:1996-2002
Publication Date(Web):
DOI:10.1002/jssc.201201146
Columns for open tubular capillary electrochromatography, coated with a mixed-mode (RP/ion-exchange) stationary phase, were prepared by using the sol–gel method. The synthetic procedure was optimized by changing the ratios of tetraethoxysilane, octyltriethoxysilane, and 3-aminopropyltriethoxysilane in the initial sol. SEM studies reveal that a coating with about 400 nm thickness can be obtained. The inner surface properties of these capillaries were probed by measuring the EOF as a function of pH. The surface of this stationary phase contains octyl, amine, and residual silanol moieties; the amine and silanol groups determine the net charge on the inner surface of the capillary and can produce a switchable EOF (anodal/cathodal). The performances of the columns were evaluated by open tubular capillary electrochromatography using a wide range of compounds (polycyclic aromatic hydrocarbons, aromatic acids, and aromatic amines).
Co-reporter:Xueyang Zhang;Shaochun Zhu;Ya Xiong;Dr. Chunhui Deng;Dr. Xiangmin Zhang
Angewandte Chemie International Edition 2013 Volume 52( Issue 23) pp:6055-6058
Publication Date(Web):
DOI:10.1002/anie.201300566
Co-reporter:Chaofeng Wang, Mingxia Gao, Zhi Huang, Xiangmin Zhang
Talanta 2013 Volume 117() pp:229-234
Publication Date(Web):15 December 2013
DOI:10.1016/j.talanta.2013.09.005
•The micro-analysis method for trace amounts of saccharide was developed.•High fluorescent labeling reagent was used in one-step derivatization of saccharide.•High sensitive Capillary-HPLC-LIF and MALDI-TOF-MS were applied as the detectors.The new approach to one-step derivatization of saccharide with 5-(((2-(carbohydrazino)methyl)thio)acetyl)-aminofluorescein (C356) was described. In this approach, high fluorescent C356 was applied to label saccharide to enhance the response of derivative saccharide and high sensitive capillary high performance liquid chromatography with laser-induced fluorescence (Capillary-HPLC-LIF) associated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to characterize C356 labeled saccharide. The effect of derivatization conditions was evaluated and discussed. The limit of detection (LOD) of neutral saccharide in our method attained the level of femtomolar. As a result, this method could be successfully applied to determine the structure of N-glycans of glycoprotein.
Co-reporter:Xueyang Zhang, Shaochun Zhu, Chunhui Deng and Xiangmin Zhang
Chemical Communications 2012 vol. 48(Issue 21) pp:2689-2691
Publication Date(Web):20 Jan 2012
DOI:10.1039/C2CC17997K
An aptamer microarray was directly fabricated on a MALDI target plate for high-throughput insulin detection. High sensitivities were observed both in standard solutions (5 ng mL−1, 0.86 nM) and serum sample (20 ng mL−1, 3.4 nM). This method shows great promise in the field of biomarker detection.
Co-reporter:Xueyang Zhang, Shaochun Zhu, Chunhui Deng, Xiangmin Zhang
Talanta 2012 Volume 88() pp:295-302
Publication Date(Web):15 January 2012
DOI:10.1016/j.talanta.2011.10.044
Here, we describe a sensitive and specific method for thrombin detection with aptamer functionalized core–shell Fe3O4@C@Au magnetic microspheres (Au-MMPs). Firstly, Au-MMPs were synthesized through surface adsorption of gold nanoparticles onto PDDA functionalized Fe3O4@C magnetic microspheres. Then, the as-synthesized Au-MMPs were developed as new substrate for immobilization of thrombin binding aptamer (TBA) through easy formation of Au–S bond. After that, the prepared aptamer functionalized Au-MMPs (TBA@Au-MMPs) were used as effective magnetic absorbent to extract trace level of thrombin from dilute solutions. Finally, enriched thrombin was digested by trypsin and analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the efficient affinity extraction ability of our TBA@Au-MMPs and the sensitive mass readout of MALDI-TOF, highly sensitive detection of thrombin was achieved. The limit of detection was as low as 18 fmol, corresponding to 0.36 nM thrombin in 50 μL original solution. Linear relation was observed within a concentration range from 0.5 nM to 10 nM with linear correlation R2 = 0.998. Other proteins including human serum albumin (HSA), Ig G, transferrin, oval albumin (OVA) and fetal calf serum did not interfere with thrombin detection. This simple method holds great potential for analyzing, sensing, purification and preconcentration of proteins in biological fluids.Highlights► We developed a novel method for thrombin detection with sensitivity as low as 0.36 nM. ► Aptamer was immobilized on core–shell gold coated magnetic microspheres. ► MALDI-TOF mass spectrometry was applied for high-throughput and sensitive mass readout. ► Successful recovery from serum was achieved.
Co-reporter:Junyan Liu;Yang Liu;Mingxia Gao
Journal of The American Society for Mass Spectrometry 2012 Volume 23( Issue 8) pp:1424-1427
Publication Date(Web):2012 August
DOI:10.1007/s13361-012-0400-4
In this work, a new pre-analysis method for tetracyclines (TCs) detection from the milk samples was established. As a good accomplishment for the existing accurate quantification strategies for TCs detection, the new pre-analysis method was demonstrated to be simple, sensitive, fast, cost effective, and high throughput, which would do a great favor to the routine quality pre-analysis of TCs from milk samples. Graphene or graphene oxide was utilized, for the first time, as a duel-platform to enrich and detect the TCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All together, four TCs were chosen as models: tetracycline, oxytetracycline, demeclocycline, and chlortetracycline. Due to the excellent electronic, thermal, and mechanical properties, graphene and graphene oxide were successfully applied as matrices for MALDI-TOF MS with free background inference in low mass range. Meanwhile, graphene or graphene oxide has a large surface area and strong interaction force with the analytes. By taking the advantage of these features, TCs were effectively enriched with the limit of detection (LOD) as low as 2 nM.
Co-reporter:Peng Zhang, Mingxia Gao, Shaochun Zhu, Jie Lei, Xiangmin Zhang
Journal of Chromatography A 2011 Volume 1218(Issue 47) pp:8567-8571
Publication Date(Web):25 November 2011
DOI:10.1016/j.chroma.2011.09.084
In this report, laser radiation (808 nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60 s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.Highlights► The near-IR laser radiation can accelerate the proteolysis efficiency through IMER significantly. ► The whole process can be completed in 1 min and room temperature. ► Satisfying analysis of human serum sample using laser-assisted IMER proteolysis.
Co-reporter:Cheok In Ng, Xiangmin Zhang
Talanta 2011 Volume 85(Issue 4) pp:1766-1771
Publication Date(Web):30 September 2011
DOI:10.1016/j.talanta.2011.06.074
In this work, an analytical method for GC using direct solid sample introduction was developed to tackle the problem regarding quick detection of pesticide residue in crops and large-scale screening of samples. 10 mg of the crop solid sample without sample pre-treatment was directly introduced into a modified split/splitless injector for GC analysis. A split/splitless injector was modified to quickly remove oxygen and low boiling-point matrices of the sample. The whole sampling procedure was simple and it required less than 5 min. The experimental parameters including injector-port temperature, removal of oxygen and low boiling point matrices, size and the amount of the solid sample, oven temperature program were studied. Satisfactory recoveries of 6 pesticides (methyl parathion, fenitrothion, aldrin, dieldrin, endosulfan, o,p′-DDT) were obtained in maize and rice sample. Relative standard deviation was less than 15%. Experimental results showed that the proposed method was quick and reliable for pesticide residues analysis in crops.
Co-reporter:Qian Tao, Ming-Xia Gao, Guang-Feng Hong, Qi Chen, Xiang-Min Zhang
Talanta 2011 Volume 84(Issue 2) pp:457-461
Publication Date(Web):15 April 2011
DOI:10.1016/j.talanta.2011.01.046
A new method based on solid-support reaction is described to realize fluorescent derivatization of proteins at concentrations as low as 10−8 M. A simple, low-cost homemade capillary C18 cartridge was fabricated as the solid-support reactor. Using bovine serum albumin (BSA) as a test protein, we demonstrated that the protein can be captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. The analysis by nano-HPLC with laser-induced fluorescence (LIF) detection was described. The effect of reaction conditions on the derivatization has been evaluated and discussed. The use of the solid-support reactor allows easy handling of as little as 8.5 pmol of BSA. A fraction from weak anion-exchange chromatography (WAX) of human liver extract was used as an illustrative example of application to real samples.
Co-reporter:Yang Liu;Yan Li;Junyan Liu;Chunhui Deng
Journal of The American Society for Mass Spectrometry 2011 Volume 22( Issue 12) pp:2188-2198
Publication Date(Web):2011 December
DOI:10.1007/s13361-011-0231-8
In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R2 = 0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R2 = 0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.
Co-reporter:Qian Tao, Qian Wu and Xiangmin Zhang
Analytical Chemistry 2010 Volume 82(Issue 3) pp:842
Publication Date(Web):January 5, 2010
DOI:10.1021/ac901855t
A thermal expansion pump (TEP) based on a principle of liquid thermal expansion for capillary high-performance liquid chromatography has been developed. The novel pump is capable of generating a continuous flow at high pressure for constant and stable delivery of binary solvents from nanoliters to microliters per minute without splitting. Theoretical equations for controlling fluidic output of this pump have been established and validated by a series of experiments. Factors affecting flow rate, such as density discrepancy, liquid compressibility, and mass loss in output, were taken into account. An assembly of the pump system employing two groups of thermal expansion pumps (TEPs) working in turns were fabricated, and a controlling strategy for the pump system to maintain a continuous delivery without pressure fluctuation even at switching points was also developed. Both isocratic and gradients of binary solvent delivery by the TEPs were performed. Reproducibility and standard deviation at different flow rates were determined. A capillary high-performance liquid chromatography (μ-HPLC) system consisting of the TEPs, an injection valve, a homemade packed capillary column (20 cm × 100 μm i.d. with 5 μm C18), and a laser-induced fluorescence detector was set up, and sample separations were carried out. Results of RSD = 4% for flow and RSD = 2% for retention times at 500 nL/min were achieved. Such a pump system has almost no moving parts except for the solvent switches. Its overall costs of manufacture and running are very low. It is proven that the TEPs system has great potential and competitive capabilities in capillary liquid chromatography.
Co-reporter:Xia Guan, Guoquan Yan, Mingxia Gao, Guangfeng Hong, Chunhui Deng, Xiangmin Zhang
Journal of Chromatography A 2010 Volume 1217(Issue 44) pp:6875-6881
Publication Date(Web):29 October 2010
DOI:10.1016/j.chroma.2010.08.051
A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol–gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Monolithic trapping columns were fabricated by entrapping C8 reversed-phase particles into the capillary columns through a sol–gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to 300 bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8 monolithic trapping columns was 99.3% on average. The loading capacity of 5 mm × 320 μm i.d. C8 trapping columns for the protein mixtures was 30 μg. Day-to-day relative standard deviation (RSD) values for recoveries of protein mixtures on the same C8 trapping column ranged from 2.34 to 5.87%, column-to-column RSD values were from 3.01 to 6.81%. The C8 trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.
Co-reporter:Dawei Qi, Yu Mao, Jin Lu, Chunhui Deng, Xiangmin Zhang
Journal of Chromatography A 2010 Volume 1217(Issue 16) pp:2606-2617
Publication Date(Web):16 April 2010
DOI:10.1016/j.chroma.2009.10.084
In this work, we developed phosphate functionalized magnetic Fe3O4@C microspheres to immobilize Zr4+ ions for selective extraction and concentration of phosphopeptides for mass spectrometry analysis. Firstly, we synthesized Fe3O4@C magnetic microspheres as our previous work reported. Then, the microspheres were functionalized with phosphate groups through a simple hydrolysis reaction using 3-(trihydroxysilyl)propyl methylphosphate. And the Zr4+ ions were immobilized on phosphate-functionalized magnetic microspheres by using phosphate chelator. Finally, we successfully employed Zr4+-phosphate functionalized magnetic microspheres to selectively isolate the phosphopeptides from tryptic digests of standard protein and real samples including rat brain. All the experimental results demonstrate the enrichment efficiency and selectivity of the method we reported here.
Co-reporter:Jia Tang, Peng Yin, Xiaohui Lu, Dawei Qi, Yu Mao, Chunhui Deng, Pengyuan Yang, Xiangmin Zhang
Journal of Chromatography A 2010 Volume 1217(Issue 15) pp:2197-2205
Publication Date(Web):9 April 2010
DOI:10.1016/j.chroma.2010.02.008
In this study, mesoporous TiO2 microspheres were synthesized by simple hydrothermal reaction, and successfully developed for phosphopeptides enrichment from both standard protein digestion and real biological sample such as rat brain tissue extract. The mesoporous TiO2 microspheres (the diameter size of about 1.0 μm) obtained by simple hydrothermal method were found to have a specific surface area of 84.98 m2/g, which is much larger than smooth TiO2 microspheres with same size. The surface area of mesoporous TiO2 microspheres is almost two times of commercial TiO2 nanoparticle (a diameter of 90 nm). Using standard proteins digestion and real biological samples, the superior selectivity and capacity of mesoporous TiO2 microspheres for the enrichment of phosphorylated peptides than that of commercial TiO2 nanoparticles and TiO2 microspheres was also observed. It has been demonstrated that mesoporous TiO2 microspheres have powerful potential for selective enrichment of phosphorylated peptides. Moreover, the preparation of the mesoporous TiO2 microspheres obtained by the hydrothermal reaction is easy, simple and low-cost. These mesoporous TiO2 microspheres with the ability of large scale synthesis can widely be applied for phosphorylated proteomic research.
Co-reporter:Dawei Qi, Huaiyuan Zhang, Jia Tang, Chunhui Deng and Xiangmin Zhang
The Journal of Physical Chemistry C 2010 Volume 114(Issue 20) pp:9221-9226
Publication Date(Web):May 3, 2010
DOI:10.1021/jp9114404
In this work, we reported an alternative strategy to the synthesis of core−shell structure Fe3O4@C@Au magnetic microspheres by a self-assembly approach. The as-synthesized Fe3O4@C@Au magnetic microspheres were functionalized with 4-mercaptophenylboronic acid and successfully applied for selective enrichment of glycopeptides and glycoproteins. At first, the Fe3O4@C magnetic microspheres were synthesized by two-step reactions including solvothermal and hydrothermal reactions. Then, Fe3O4@C@Au magnetic microspheres with core−shell structure were obtained by a self-assembly approach. Finally, the Fe3O4@C@Au magnetic microspheres were modified with 4-mercaptophenylboronic acid. The 4-mercaptophenylboronic acid-modified Fe3O4@C@Au magnetic microspheres were successfully applied to selective enrichment of glycoproteins and glycopeptides.
Co-reporter:Mingxia Gao, Peng Zhang, Guangfeng Hong, Xia Guan, Guoquan Yan, Chunhui Deng, Xiangmin Zhang
Journal of Chromatography A 2009 Volume 1216(Issue 44) pp:7472-7477
Publication Date(Web):30 October 2009
DOI:10.1016/j.chroma.2009.05.003
In this work, a novel and facile monolithic enzymatic microreactor was prepared in the fused-silica capillary via a two-step procedure including surface acryloylation and in situ aqueous polymerization/immobilization to encapsulate a single enzyme, and its application to fast protein digestion through a direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis was demonstrated. At first, vinyl groups on the protein surface were generated by a mild acryloylation with N-acryloxysuccinimide in alkali buffer. Then, acryloylated enzyme was encapsulated into polyacrylates by free-radical copolymerization with acrylamide as the monomer, N,N′-methylenebisacrylamide as the cross-linker, and N,N,N′,N′-tetramethylethylenediamine/ammonium persulfate as the initiator. Finally, polymers were immobilized onto the activated inner wall of capillaries via the reaction of vinyl groups. Capability of the enzyme-immobilized monolithic microreactor was demonstrated by myoglobin and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF-MS with sequence coverage of 94% and 29% observed. This microreactor was also applied to the analysis of fractions through two-dimensional separation of weak anion exchange/reversed-phase liquid chromatography of human liver extract. After a database search, 16 unique peptides corresponding to 3 proteins were identified when two RPLC fractions of human liver extract were digested by the microreactor. This opens a route for its future application in top–down proteomic analysis.
Co-reporter:Dawei Qi, Jin Lu, Chunhui Deng, Xiangmin Zhang
Journal of Chromatography A 2009 Volume 1216(Issue 29) pp:5533-5539
Publication Date(Web):17 July 2009
DOI:10.1016/j.chroma.2009.05.049
Protein phosphorylation is one of the most important post-translational modifications. Due to the dynamic nature and low stoichiometry of the protein phosphorylation, enrichment of phosphopeptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Many metal oxides such as titanium dioxide and zirconium dioxide have been successfully applied to isolation and enrichment of phosphopeptides. Recently, niobium pentoxide was proved to have the ability for selective enrichment of phosphopeptides. Considering the proximity of tantalum to niobium, we supposed that Ta2O5 can be used as affinity probes for phosphopeptide enrichment. In the work, we synthesized Fe3O4@Ta2O5 magnetic microspheres with core–shell structure for selective enrichment of phosphopeptides. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe3O4@Ta2O5 magnetic microspheres to isolation and enrichment of the phosphopeptides from tryptic digestion of standard proteins and real samples, and then the enriched peptides were analyzed by matrix-assisted laser desorption mass spectrometry analysis (MALDI-MS) or liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS). Experiment results demonstrate that Ta2O5 coated-magnetic microspheres show the excellent potential for selective enrichment of phosphopeptides.
Co-reporter:Dawei Qi, Jin Lu, Chunhui Deng and Xiangmin Zhang
The Journal of Physical Chemistry C 2009 Volume 113(Issue 36) pp:15854-15861
Publication Date(Web):August 14, 2009
DOI:10.1021/jp902959d
In this work, we synthesized core−shell magnetic tin dioxide (Fe3O4@C@SnO2) microspheres. The Fe3O4@C@SnO2 microspheres were applied to enrich phosphopeptides for mass spectrometry analysis. Different from titanium and zirconium,the regular sol−gel process is not effective in generating tin dioxide on the magnetite particles because the tin oxide tends to crystallize during the reaction. Herein, we reported a simple but effective method combining solvothermal and hydrothermal reactions to synthesize Fe3O4@C@SnO2 microspheres. The obtained Fe3O4@C@SnO2 microspheres have a mean diameter of 340 nm, well-crystallized SnO2 shell (∼30 in thickness), and high magnetization (63.8 emu/g). We have also demonstrated that Fe3O4@C@SnO2 microspheres are the promising materials for convenient, efficient enrichment of phosphopeptides by the analysis of phosphopeptides in standard protein digestion and real samples using MALDI MS and LC-ESI MS.
Co-reporter:Fengli Hu, Chunhui Deng, Yang Liu, Xiangmin Zhang
Talanta 2009 Volume 77(Issue 4) pp:1299-1303
Publication Date(Web):15 February 2009
DOI:10.1016/j.talanta.2008.09.003
The chlorogenic acid (CA) in Honeysuckle is determined and identified by nano-liquid chromatography-electrospray ionization mass spectrometry (nano-LC-ESI/MS) after extraction with microwave-assisted extraction (MAE). As a new sample preparation method for Honeysuckle, the MAE procedure is optimized, validated and compared with conventional methods including reflux extraction (RE) and ultrasonic extraction (USE). It is found that MAE gives the best result due to the highest extraction efficiency within shortest extraction time (only 4 min). Here, CA is determined by nano-LC-ESI/MS based on the calibration curve of its authentic standard. The method linearity, detection limit, precision and recovery are studied. The results show that the combined MAE and nano-LC-ESI/MS method has a linearity (R2 0.991, 0.8–20 ng mL−1), a low limit of detection (0.5 ng mL−1), good precision (R.S.D. = 2.54%) and a recovery (84.8%). The experiment has demonstrated that the nano-LC-ESI/MS following MAE is a fast and reliable method for quantitative analysis of CA in Honeysuckle.
Co-reporter:Yan Li, Jinsong Wu, Dawei Qi, Xiuqing Xu, Chunhui Deng, Pengyuan Yang and Xiangmin Zhang
Chemical Communications 2008 (Issue 5) pp:564-566
Publication Date(Web):20 Nov 2007
DOI:10.1039/B716055K
A novel approach is proposed to synthesize Fe3O4@TiO2 microspheres with a well-defined core–shell structure, and the synthesized Fe3O4@TiO2 core–shell microspheres were successfully applied for the simple and fast enrichment of phosphopeptides via direct MALDI-TOF mass spectrometry analysis.
Co-reporter:Shuang Lin, Guoping Yao, Dawei Qi, Yan Li, Chunhui Deng, Pengyuan Yang and Xiangmin Zhang
Analytical Chemistry 2008 Volume 80(Issue 10) pp:3655
Publication Date(Web):April 11, 2008
DOI:10.1021/ac800023r
A fast and efficient proteolysis approach of microwave-assisted protein digestion was developed by using trypsin-immobilized magnetic silica (MS) microspheres. In the work, immobilization of the enzyme onto MS microspheres was very simple and only through a one-step reaction with 3-glycidoxypropyltrimethoxysilane (GLYMO) which provides the epoxy group as a reactive spacer. Considering that the magnetic particles are excellent microwave absorbers, we developed a novel microwave-assisted digestion method based on the easily prepared trypsin-immobilized MS microspheres. This novel digestion method combined the advantages of immobilized trypsin and the rapid-fashion of microwave-assisted digestion, which resulted in high digestion efficiency. BSA and myoglobin were used as model proteins to optimize the conditions of this method. Peptide fragments produced in 15 s could be confidently identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Equivalent or better digestion efficiency was observed comparing to current in-solution digestion. Besides, because of the unique magnetic responsivity, the immobilized trypsin can be isolated easily with the help of an external magnet and thus used repeatedly. High activity was obtained even after seven runs of the trypsin-immobilized MS microspheres. To further verify its efficiency in proteome analysis, one reversed-phase liquid chromatography (RPLC) fraction of rat liver extract was applied. After 15 s incubation, 16 totally unique peptides corresponding to two proteins were identified. Finally, the rat liver sample was used to evaluate its worth for the application. With analysis by liquid chromatography−electrospray-tandem mass spectrometry (LC−ESI-MS/MS), comparable digestion efficiency was observed with typical in-solution digestion but the incubation time was largely shortened. This new microwave-assisted digestion method will hasten the application of the proteome technique to biomedical and clinical research.
Co-reporter:Yan Li, Dawei Qi, Chunhui Deng, Pengyuan Yang and Xiangmin Zhang
Journal of Proteome Research 2008 Volume 7(Issue 4) pp:1767-1777
Publication Date(Web):2017-2-22
DOI:10.1021/pr070385l
In this study, we employed, for the first time, the Ce4+-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before. After enrichment, the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet. With the optimized enrichment conditions, the performance of the Ce4+-chelated magnetic microspheres was compared with the Fe3+-chelated microspheres using tryptic digested peptides originating from ovalbumin, a five protein mixture containing phosphoproteins and nonphosphoproteins, as well as a mixture of β-casein and BSA with a molar ratio of 1:50. Compared to Fe3+, Ce4+-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures. Even when the amount of the tryptic digest product of BSA is 50 times higher than that of β-casein in the sample solution, the trace phosphopeptides derived from β-casein can still be concentrated effectively by the Ce4+-chelated magnetic microspheres in only 30 s. Furthermore, we initially utilized the Ce4+-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment, which opens up a possibility for their further application in phosphoproteomics.
Co-reporter:Huaqing Lin, Qing Ye, Chunhui Deng, Xiangmin Zhang
Journal of Chromatography A 2008 Volumes 1198–1199() pp:34-37
Publication Date(Web):11 July 2008
DOI:10.1016/j.chroma.2008.05.050
Acetaldehyde is generated in the mainstream tobacco smoke mainly from the pyrolysis (and oxidative pyrolysis) of carbohydrates that are present in tobacco plant, cigarette paper, and also used as additives in tobacco. Acetaldehyde has been classified as an animal carcinogen, and may be cytotoxic or genetoxic. Owing to its high volatility and reactivity, it is difficult to accurately measure it, especially on site. In this work, a novel analytical method based on collection and simultaneous derivatization in water, solid-phase microextraction (SPME), and portable gas chromatography (GC) analysis has been developed for the field and rapid analysis of acetaldehyde in the mainstream tobacco smoke. In the proposed method, acetaldehyde in mainstream tobacco smoke was collected in 1 ml water, and derivatizated with O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine. The formed acetaldehyde oximes were headspace extracted by a divinylbenzene/carboxen/polydimethylsiloxane fiber at 30 °C for 10 min, with a stirring rate of 1100 rpm. The acetaldehyde oximes extracted on the fiber were desorbed and analyzed rapidly by portable GC. The method validations including detection limit, recovery, precision and linearity were studied. It was found that the proposed method required the whole analysis time 27 min, and provided low detection limit of 0.04774 mg/ml, good recovery of 85%, RSD value 6.7%, and linear range 0.0716–0.7160 mg/ml (r2 = 0.997). The obtained results demonstrated that SPME-portable GC is a simple, rapid and solvent-free method for the field analysis of acetaldehyde. Finally, the proposed method was further applied to the quantification of acetaldehyde in mainstream tobacco smoke of five different brands.
Co-reporter:Mingxia Gao, Wenjia Yu, Yang Zhang, Guoquan Yan, Chunhui Deng, Pengyuan Yang and Xiangmin Zhang
Analyst 2008 vol. 133(Issue 9) pp:1261-1267
Publication Date(Web):30 Jul 2008
DOI:10.1039/B803388A
We present a comprehensive method for proteome analysis that integrates both intact protein separation and proteolytic fragment characterization mass spectrometric approaches. Strong cation exchange chromatography (SCX) was used as the first separation dimension and capillary reversed-phase liquid chromatography (cRPLC) was integrated as the second separation dimension. Fractions from SCX were collected offline and loaded onto cRPLC. Effluents from cRPLC were directly deposited onto the MALDI target plates and further digested by using a rapid on-probe tryptic digestion technique. This approach minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and transfer. MALDI-TOF/TOF was used for subsequent analyses. The sensitivity of on-target digestion is showed by analyzing 0.07 ng of myoglobin, 0.07 ng of cytochrome c and 0.7 ng BSA. The high efficiency of the overall system was demonstrated by the analysis of intact proteins extracted from normal human liver tissue. In total, 458 proteins were identified, which proved the system's promising potential for analysis and application in proteomics.
Co-reporter:Shuang Lin, Dong Yun, Dawei Qi, Chunhui Deng, Yan Li and Xiangmin Zhang
Journal of Proteome Research 2008 Volume 7(Issue 3) pp:1297-1307
Publication Date(Web):February 8, 2008
DOI:10.1021/pr700586j
In this study, a novel microwave-assisted protein digestion method was developed using trypsin-immobilized magnetic nanoparticles (TIMNs). The magnetic nanoparticles worked as not only substrate for enzyme immobilization, but also excellent microwave irradiation absorber and, thus, improved the efficiency of microwave-assisted digestion greatly. Three standard proteins, bovine serum albumin (BSA), myoglobin, and cytochrome c, were used to optimize the conditions of this novel digestion method. With the optimized conditions, peptide fragments produced in very short time (only 15 s) could be identified successfully by MALDI-TOF-MS. When it was compared to the conventional in-solution digestion (12 h), equivalent or better digestion efficiency was observed. Even when protein quantity was as low as micrograms, this novel digestion method still could digest proteins successfully, while the same samples by conventional in-solution digestion failed. Moreover, with an external magnetic field, the enzyme could be removed easily and reused. It was verified that, after 4 replicate runs, the TIMNs still kept high activity. To further confirm the efficiency of this rapid digestion method for proteome analysis, it was applied to the protein extract of rat liver. Without any preparation and prefractionation processing, the entire proteome digested by TIMNs in 15 s went through LC-ESI−MS/MS direct analysis. The whole shotgun proteomic experiment was finished in only 1 h with the identification of 313 proteins (p < 0.01). This new application of TIMNs in microwave-assisted protein digestion really opens a route for large-scale proteomic analysis.
Co-reporter:Fengli Hu;Huiying Zhang;Huaqing Lin
Journal of The American Society for Mass Spectrometry 2008 Volume 19( Issue 6) pp:865-873
Publication Date(Web):2008 June
DOI:10.1016/j.jasms.2008.02.016
In this study, a novel technique for screening inhibitors by electrospray mass spectrometry (ESI-MS) with immobilized enzyme on magnetic microspheres has been demonstrated. First, the model enzyme acetylcholinesterase (AChE) is immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic silica microspheres. AChE activity was monitored by biochemical assay that is based on mixing of AChE immobilized microspheres and model substrate acetylcholine, separating and detecting the product through ESI-MS. Stability of the enzyme-immobilized microspheres was investigated. No apparent loss of enzyme activity was observed after fivefold reuse of AChE-immobilized microspheres. The enzyme-immobilized bioassay was used to effectively identify AChE inhibitors among two standard samples, huperzine A and huperzine B, and their source herbal Huperzia serrata, all of which were spiked into the substrate. The inhibition was determined by measuring a decrease of product formation using ESI-MS.
Co-reporter:Chunhui Deng, Ning Liu, Mingxia Gao, Xiangmin Zhang
Journal of Chromatography A 2007 Volume 1153(1–2) pp:90-96
Publication Date(Web):15 June 2007
DOI:10.1016/j.chroma.2007.01.081
Traditional Chinese medicines (TCMs) have a long history dating back thousands of years. Recently, there has been increasing interest worldwide in the use of TCMs for the prevention and treatment of various illnesses. In China, a large number of analytical tools, especially chromatographic techniques have been used to analyze the constituents of TCMs in order to control their quality and discover new bioactive compounds. In this paper, recent developments in sample preparation techniques for the extraction, clean-up, and concentration of analytes from TCMs are compared. These techniques include headspace solid-phase microextraction (HS-SPME), headspace liquid-phase microextraction (HS-LPME), microwave-assisted extraction (MAE), supercritical-fluid extraction (SFE), pressurized-liquid extraction (PLE), and microwave distillation (MD).
Co-reporter:Chunhui Deng, Yu Mao, Fengli Hu, Xiangmin Zhang
Journal of Chromatography A 2007 Volume 1152(1–2) pp:193-198
Publication Date(Web):8 June 2007
DOI:10.1016/j.chroma.2006.08.074
In this work, for the first time, microwave distillation (MD) coupled with simultaneous headspace single-drop microextraction (HS-SDME) was developed for the determination of the volatile components in the Chinese herb, Artemisia capillaris Thunb. The volatile components were rapidly isolated by MD, and simultaneously extracted and concentrated by using a dodecane microdrop. The volatile oil extracted in the microdrop solvent was analyzed by gas chromatography–mass spectrometry (GC–MS). The experimental parameters of solvent selection, microdrop volume, microwave power, irradiation time and sample amount were investigated, and the method precision was also studied. The optimal parameters were extraction solvent of dodecane, solvent volume of 2.0 μL, microwave power of 400 W, irradiation time of 4 min, and sample amount of 2.0 g. Thirty-five volatile compounds present in Artemisia capillaris Thunb. were identified by using the proposed method, which were identical with those obtained by the conventional steam distillation method. The experimental results showed that MD–HS-SDME is a simple, rapid, reliable, and solvent-free technique for the determination of volatile compounds in Chinese herbs.
Co-reporter:Chunli Liu, Xiangmin Zhang
Journal of Chromatography A 2007 Volume 1139(Issue 2) pp:191-198
Publication Date(Web):19 January 2007
DOI:10.1016/j.chroma.2006.11.019
A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150 min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.
Co-reporter:Yan Li, Yingchao Liu, Jia Tang, Huaqing Lin, Ning Yao, Xizhong Shen, Chunhui Deng, Pengyuan Yang, Xiangmin Zhang
Journal of Chromatography A 2007 Volume 1172(Issue 1) pp:57-71
Publication Date(Web):16 November 2007
DOI:10.1016/j.chroma.2007.09.062
Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe3O4@Al2O3 magnetic core–shell microspheres with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe3O4@Al2O3 magnetic core–shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe3O4@Al2O3 magnetic core–shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins β-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe3O4@Al2O3 magnetic core–shell microspheres over Fe3+-immobilized magnetic silica microspheres, commercial Fe3+–IMAC (immobilized metal affinity chromatography) resin, and TiO2 beads. Finally, the Al2O3 coated Fe3O4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe3O4@Al2O3 magnetic core–shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.
Co-reporter:Mingxia Gao;Chunhui Deng;Shuang Lin;Fengli Hu;Jia Tang;Ning Yao;Xiangmin Zhang
Journal of Separation Science 2007 Volume 30(Issue 6) pp:785-791
Publication Date(Web):9 MAR 2007
DOI:10.1002/jssc.200600372
The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists in protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yet the 2-D PAGE-mass spectrometry (MS) approach has its drawbacks with regard to automation, sensitivity, and throughput. Consequently, considerable effort has been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings of 2-D PAGE. In addition, traditional Chinese medicines (TCMs), due to their long period of clinical testing and reliable therapeutic efficacy, are attracting increased global attention. However, hundreds or even thousands of components are usually present in TCMs, which results in great difficulties of separation. As a mainstream separation tool, multidimensional liquid separation systems have shown powerful separation ability, high peak capacity, and excellent detectability in the analysis of complex samples including biological samples and TCMs, etc. Therefore, this review emphasizes the most recent advances in multidimensional liquid chromatography and capillary electrophoresis-based separation techniques, and the corresponding applications in proteomics and TCMs. In view of the significant contributions from Chinese scientists, this review focuses mainly on the work of Chinese scientists in the above fields.
Co-reporter:Jie Ji, Chunhui Deng, Huiqin Zhang, Yunyun Wu, Xiangmin Zhang
Talanta 2007 Volume 71(Issue 3) pp:1068-1074
Publication Date(Web):28 February 2007
DOI:10.1016/j.talanta.2006.05.087
In this work, microwave-assisted steam distillation (MASD) extraction method followed by gas chromatography/electron capture detection (GC/ECD) was developed for the determination of organochlorine pesticides (OCPs) and pyrethroids in the Chinese teas. MASD is a combination of microwave-assisted extraction (MAE) and steam distillation techniques. Water vapor generated by microwave irradiation is used to accelerate desorption of the analytes from the sample, and the nonpolar organic solvent used for trapping the analytes is kept from direct contact with the sample by the water. Therefore, relatively clean extracts were obtained compared to the method directly using organic solvent as extraction solvent, such as ultrasonic extraction (USE). Microwave power of 200 W and irradiation time of 2 min was found to be the optimum conditions for the MASD process, and n-heptane was chosen as the analyte-trapping solvent in the study. Five OCPs (α-HCH, γ-HCH, dicofol, p,p′-DDE, p,p′-DDT) and two pyrethroids (bifenthrin, fenvalate) were determined using this extraction method in the tea samples. The relative standard deviation (R.S.D.) of the analytes varied from 2.2 to 8.4%, and the method detection limits (MDLs) found were lower than 0.23 μg/kg. The recoveries of the seven compounds in the Jasmine tea sample were between 84.04 and 110.1%. Comparative results obtained by MASD and USE were also discussed in the study.
Co-reporter:X. Xu;M. Gao;C. Deng;W. Yu;P. Yang;X. Zhang
Advanced Materials 2006 Volume 18(Issue 24) pp:3289-3293
Publication Date(Web):11 DEC 2006
DOI:10.1002/adma.200601546
Core/shell structured magnetic silica microspheres with immobilized Fe3+ (see image) are prepared with a simple method. Using a magnetic field, the microspheres are quickly, efficiently, and specifically enriched. Phosphopeptide identification can then be achieved through mass spectroscopy. This is a new method for the enrichment of phosphopeptides and opens up the possibility of other new applications using the microspheres.
Co-reporter:Chunhui Deng, Yu Mao, Ning Yao, Xiangmin Zhang
Analytica Chimica Acta 2006 Volume 575(Issue 1) pp:120-125
Publication Date(Web):4 August 2006
DOI:10.1016/j.aca.2006.05.073
In the work, microwave-assisted extraction (MAE) followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC–MS) was developed for quantitative analysis of the bioactive components of camphor and borneol in a traditional Chinese medicines (TCM) of Flos Chrysanthemi Indici. After systematical investigation, the optimal experimental parameters microwave power (400 W), irradiation time (4 min), fiber coating (PDMS/DVB fiber), extraction temperature (40 °C), extraction time (20 min), stirring rate (1100 rpm), and salt effect (no salt added) were investigated. The optimized method provided satisfactory precision (RSD values less than 12%), good recovery (from 86% to 94%), and good linearity (R2 > 0.999). The proposed method was applied to quantitative analysis of camphor and borneol in Flos Chrysanthemi Indici samples from 11 different growing areas. To demonstrate the method feasibility, steam distillation was also used to analyze camphor and borneol in Flos Chrysanthemi Indici samples from these different growing areas. The very close results were obtained by the two methods. It has been shown that the proposed ME–HS-SPME–GC–MS is a simple, rapid, solvent-free and reliable method for quantitative analysis of camphor and borneol in TCM, and a potential tool for quality assessment of Flos Chrysanthemi Indici.
Co-reporter:Jie Zhang, Mingxia Gao, Jia Tang, Pengyuan Yang, Yinkun Liu, Xiangmin Zhang
Analytica Chimica Acta 2006 Volume 566(Issue 2) pp:147-156
Publication Date(Web):4 May 2006
DOI:10.1016/j.aca.2006.03.045
Recently, matrix-assisted laser desorption ionization (MALDI) technique has been shown to be complementary to electrospray ionization (ESI) with respect to the population of peptides and proteins that can be detected. In this study, we tried to hyphenate MALDI–TOF–TOF–MS and ESI–QUADRUPOLE–TOF–MS with a single 2D liquid chromatography for complicated protein sample analysis. The effluents of RPLC were split into two parts for the parallel MS/MS detection. After optimizing the operation conditions in LC separation and MS identification, a total of 1149 proteins were identified from the global lysate of normal human liver (NHL) tissue. Compared to the single MS/MS detection, the combined analysis increased the number of proteins identified (more than 25%) and enhanced the protein identification confidence. Proteins identified were categorized and analyzed based upon their cellular location, biological process and molecular function. The identification results demonstrated the application potential of a parallel MS/MS analysis coupled with multi-dimensional LC separation for complicated protein sample identification, especially for proteome analysis, such as human tissues or cells extracts.
Co-reporter:Chunhui Deng, Jie Ji, Ning Li, Yingjia Yu, Gengli Duan, Xiangmin Zhang
Journal of Chromatography A 2006 Volume 1117(Issue 2) pp:115-120
Publication Date(Web):9 June 2006
DOI:10.1016/j.chroma.2006.03.066
Curcumol, germacrone and curdione are the main active ingredients in a common traditional Chinese medicine (TCM) of Rhizoma Curcuma, and commonly used as the TCM quality control markers. In the present work, microwave-assisted extraction (MAE) followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC–MS) was developed for the quantitative analysis of curcumol, curdione and germacrone in Rhizoma Curcuma. The MAE and HS-SPME parameters were studied, and the method was validated. The optimal MAE conditions obtained were: microwave power of 700 W and irradiation time of 4 min, and HS-SPME optimal conditions were: fiber coating of 100 μm PDMS, extraction temperature of 80 °C, extraction time of 20 min, stirring rate of 1100 rpm, and salt concentration of 30% NaCl. The proposed method provided good precision (RSD less than 12%) and recoveries between 86% and 93%. The proposed method was applied to the determination of the three marker compounds in three species of Curcuma rhizomes (Curcuma wenyujin, Curcuma phaeocaulis, and Curcuma kwangsiensis). To demonstrate the proposed method reliability, a conventional technique of steam distillation was also used for the analysis of curcumol, germacrone and curdione in the TCMs. The results show that MAE–HS-SPME is a simple, rapid, solvent-free and reliable method for the determination of curdione, curcumol and germacrone in TCM, and also a potential and powerful tool for quality assessment of Rhizoma Curcuma.
Co-reporter:Chunhui Deng, Ning Yao, Ben Wang, Xiangmin Zhang
Journal of Chromatography A 2006 Volume 1103(Issue 1) pp:15-21
Publication Date(Web):20 January 2006
DOI:10.1016/j.chroma.2005.11.023
Paeonol is an important active component present in traditional Chinese medicines (TCMs), which was used for the treatment of many diseases such as eczema. In this work, microwave-assisted extraction (MAE) was firstly combined with headspace single-drop microextraction (HS-SDME), and applied to rapid determination of paeonol in two TCMs of Cynanchum paniculatum and Paeonia suffruticosa. In the proposed method, paeonol in TCMs was isolated by using MAE, followed by extraction and concentration by HS-SDME, and detected by gas chromatography–mass spectrometry (GC–MS). The experiment parameters of MAE and HS-SDME were discussed, and the method precision, recovery and detection limit were also studied. To further demonstrate the reliability of the quantification, both the proposed method and a standard method of steam distillation (SD) were simultaneously applied to quantitative analysis of paeonol in TCM samples from different growing areas. The experimental results show that MAE–HS-SDME is a simple and rapid method for the quantitative analysis of paeonol in TCMs, and is also a potential and alternative tool for quality monitoring for the two TCMs of C. paniculatum and P. suffruticosa.
Co-reporter:Mingxia Gao, Na Li, Jie Zhang, Pengyuan Yang, Xiangmin Zhang
Separation and Purification Technology 2006 Volume 52(Issue 1) pp:170-176
Publication Date(Web):November 2006
DOI:10.1016/j.seppur.2006.04.006
Efficient and reproducible sample preparation methods are key to successful separation in proteome. Meanwhile, efficient sample preparation also enriches target sample to some extent and plays a pre-separation role. In this study, for the first time we put forward using acidic and basic extraction buffer as complementary methods of standard ways to extract complex proteins in rat liver tissue. We also compared the effect of three extraction methods on sample pre-separation and enrichment. The acidic or basic condition was obtained by adding 0.1% trifluoroacetic acid or 40 mM Tris, respectively. The rat liver samples were divided into three equal groups and different extraction buffers were used for each group. The concentration of protein from different methods was measured and the corresponding sample was separated using two-dimensional gel electrophoresis, followed by detection of MALDI-TOF-TOF mass spectrometry. Three extraction lysates were also separated by reversed-phase high performance liquid chromatography. The images in gel and mass spectrum indicated that the different methods can pre-separate and enrich some special proteins. Experimental results also showed that the three methods had good reproducibility and could be applied in proteomic analysis.
Co-reporter:Xiuqing Xu;Huali Shen;Xiangmin Zhang;Jie Zhang
Journal of Separation Science 2006 Volume 29(Issue 17) pp:2635-2646
Publication Date(Web):25 OCT 2006
DOI:10.1002/jssc.200600065
The analysis of whole cell or tissue extracts is too complex for current protein identification technology and not suitable for the study of proteins with low copy levels. To concentrate and enrich low abundance proteins, organelle proteomics is a promising strategy. This approach can not only reduce the protein sample complexity but also provide information about protein location in cells, organs, or tissues under analysis. Nano-flow two-dimensional strong-cation exchange chromatography (SCX)–RPLC–ESI-MS/MS is an ideal platform for analyzing organelle extracts because of its advantages of sample non-bias, low amounts of sample required, powerful separation capability, and high detection sensitivity. In this study, we apply nano-scale multidimensional protein identification technology to the analysis of C57 mouse liver nuclear proteins. Organelle isolation has been optimized to obtain highly pure nuclei. Evaluation of nucleus integrity and purity has been performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by five independent nano-flow on-line SCX–RPLC–ESI-MS/MS analyses to improve the proteome coverage. Finally, a total of 462 proteins were identified. Corresponding analyses of protein molecular mass and pI distribution and biological function categorization have been undertaken to further validate our identification strategy.
Co-reporter:Xiaochuan Wang;Xiangmin Zhang;Xiuhan Yang
Journal of Separation Science 2006 Volume 29(Issue 5) pp:677-683
Publication Date(Web):14 MAR 2006
DOI:10.1002/jssc.200500381
Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 μM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2–2 μg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.
Co-reporter:Jie Ji, Chunhui Deng, Wenwen Shen, Xiangmin Zhang
Talanta 2006 Volume 69(Issue 4) pp:894-899
Publication Date(Web):15 June 2006
DOI:10.1016/j.talanta.2005.11.032
In this work, portable gas chromatography–microflame ionization detection (portable GC–μFID) coupled to headspace solid-phase microextraction (HS-SPME) was developed for the field analysis of benzene, toluene, ethylbenzene and xylene (BTEX) in water samples. The HS-SPME parameters such as fiber coating, extraction times, stirring rate, the ratio of headspace volume to sample volume, and sodium chloride concentration were studied. A 65 μm poly(dimethylsiloxane)-divinylbenzene (PDMS-DVB) SPME fiber, 900 rpm, 3.0 ml of headspace (1.0 ml water sample in 4.0 ml vial), and 35% sodium chloride concentration (w/v) were respectively chosen for the best extraction response. An extraction time of 1.0 min was enough to extract BTEX in water samples. The relative standard deviation (R.S.D.) for the procedure varied from 5.4% to 8.3%. The method detection limits (MDLs) found were lower than 1.5 μg/l, which was enough sensitive to detect the BTEX in water samples. The optimized method was applied to the field analysis of BTEX in wastewater samples. These experiment results show that portable GC–μFID combined with HS-SPME is a rapid, simple and effective tool for field analysis of BTEX in water samples.
Co-reporter:Yonghui Deng, Chunhui Deng, Dong Yang, Changchun Wang, Shoukuan Fu and Xiangmin Zhang
Chemical Communications 2005 (Issue 44) pp:5548-5550
Publication Date(Web):06 Oct 2005
DOI:10.1039/B511683J
Magnetic silica nanoparticle functionalized multi-walled carbon nanotubes (MS-MWNTs) were prepared, characterized and used for the convenient, rapid and efficient separation of trace aromatic compounds.
Co-reporter:Chunhui Deng, Ning Yao, Aiqin Wang, Xiangmin Zhang
Analytica Chimica Acta 2005 Volume 536(1–2) pp:237-244
Publication Date(Web):22 April 2005
DOI:10.1016/j.aca.2004.12.044
In this paper, for the first time, two sample techniques of pressurized hot water extraction (PHWE) and liquid-phase microextraction (LPME) were combined and applied to the analysis of essential oil in a traditional Chinese medicine (TCM), Fructus amomi. Essential oil in F. amomi samples (50.0 mg) were extracted by PHWE equipment in dynamic mode, followed by extraction and concentration with headspace (HS)-LPME and detection by gas chromatography–mass spectrometry (GC–MS). The PHWE and HS-LPME parameters were optimized and the method repeatability was studied. The three active compounds of camphor, borneol and borneol acetate in the F. amomi sample from five different growing areas were quantitatively analyzed by internal standard method. Compared to steam distillation (SD), the proposed method required simple sample preparation, little sample mass and whole analysis time less than 40 min. The present method provided good repeatability (R.S.D. <12.0%). It has been demonstrated that PHWE–LPME–GC–MS is a simple, rapid and low-cost method for determination of essential oils in TCMs and is a potential tool for TCM quality assessment.
Co-reporter:Ming-xia Gao, Jin Hong, Peng-yuan Yang, Xing-min Zhang
Analytica Chimica Acta 2005 Volume 553(1–2) pp:83-92
Publication Date(Web):30 November 2005
DOI:10.1016/j.aca.2005.07.060
In this paper, we describe an approach for fractionating complex protein samples from rat liver prior to two-dimensional gel electrophoresis (2-DE) using reversed-phase high-performance liquid chromatography (RP-HPLC). The whole lysate of liver tissue was prefractionated by RP-HPLC with an optimal multi-stage linear gradient elution. Successive fractions were analyzed using 2-DE and selected spots were identified by MALDI-TOF-TOF mass spectrometry. The reproducibility of this prefractionation technology allows pooling of several consecutive runs of the same sample, resulting in a highly enrichment of low abundance proteins. Computer-assisted calculation showed that the total spot number of samples prefractionated by RP-HPLC was nearly five times as many as that of unfractionated sample. The choice of Coomassie Blue staining rather than silver staining indicated that RP-HPLC prefractionation can provide strong enrichment effect which enabled us to visualize additional and less abundance proteins. Chromatographic enrichment was also demonstrated by the peptide mass fingerprint data, which gave mass spectra with increased number of peptide detected and improved signal intensity.
Co-reporter:Xiuhan Yang, Xiaochuan Wang, Xiangmin Zhang
Analytica Chimica Acta 2005 Volume 549(1–2) pp:81-87
Publication Date(Web):6 September 2005
DOI:10.1016/j.aca.2005.06.008
Capillary zone electrophoresis separation with laser-induced fluorescence (LIF) detection for the determination of fluorescein isothiocyanate (FITC) derivatized stimulants (ephedrine, phenylpropanolamine and heptaminol) was demonstrated. A simple electrophoretic buffer system composed of 30 mM triethylamine was selected and high voltage of 16 kV was applied for the capillary electrophoresis separations. Stimulants in human urine were extracted by organic solvents and then derivatized with FITC. By employing a semiconductor laser-induced fluorescence detector, low concentration stimulants of 22 ng/mL can be detected within 10 min. Operation conditions for derivatization and separation were discussed. This simple CZE–LIF method with sensitive detection provided the possibility to develop the on-chip stimulants screening methods for doping control.
Co-reporter:Chunhui Deng, Aiqin Wang, Shun Shen, Daxi Fu, Jiakuan Chen, Xiangmin Zhang
Journal of Pharmaceutical and Biomedical Analysis 2005 Volume 38(Issue 2) pp:326-331
Publication Date(Web):15 June 2005
DOI:10.1016/j.jpba.2004.12.027
In this paper, a simple, rapid, solvent-free and low-cost method of pressurized hot water extraction (PHWE) followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC–MS) was developed for the analysis of essential oil in a traditional Chinese medicine (TCM) of the dried ripe fruit of Fructus Amomi (Sha Ren). The essential oil in the TCM (0.050 g) was extracted by water at 50 bar and 150 °C, followed by extraction and concentration by SPME fibers at 80 °C for15 min and analysis by GC–MS. The PHWE and HS-SPME parameters were optimized. Thirty-five compounds in the TCM were identified by PHWE–HS-SPME. Among them, camphor, an active compound, in the TCM samples was quantitatively analyzed. The proposed method required little time to prepare the sample. Moreover, little sample mass and no organic solvent was needed. The precision of the present method was found to be good (R.S.D. <10.0%). It is shown that PHWE–SPME–GC–MS is an alternative method for the determination of volatile components in TCMs and can be used as a powerful tool for TCM quality assessment.
Co-reporter:Chunhui Deng;Weimin Zhu;Ji Qian;Xiangmin Zhang;Ning Li;Xiaofeng Yang
Journal of Separation Science 2005 Volume 28(Issue 2) pp:172-176
Publication Date(Web):1 FEB 2005
DOI:10.1002/jssc.200401912
A simple, rapid, sensitive, and solvent-free method was developed for determination of plant-signalling compounds, the three C6-aldehydes hexanal, (Z)-3-hexenal, and (E)-2-hexenal, in tomato plant emission by gas chromatography–mass spectrometry (GC-MS) and solid-phase microextraction (SPME) with on-fiber derivatization. In this method, O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine (PFBHA) in aqueous solution was first headspace adsorbed onto a 65 μm poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber at 25°C for 5 min, and then the fiber with adsorbed PFBHA was used for headspace extraction of tomato plant emission at 25°C for 6 min. Finally, the resulting oximes adsorbed on the fiber were desorbed and analyzed by GC-MS. Extraction conditions and method validation were studied. The proposed method had low detection limit values for the three aldehydes from 0.1 to 0.5 ng/L and good precision (RSD less than 10%). In this work, the method was applied to investigation of tomato plant defense response to Helicoverpa armigera.
Co-reporter:Chunhui Deng;Jie Ji;Xiaochuan Wang;Xiangmin Zhang
Journal of Separation Science 2005 Volume 28(Issue 11) pp:1237-1243
Publication Date(Web):28 JUN 2005
DOI:10.1002/jssc.200400108
In this work, a simple, rapid, solvent-free, and low-cost method was developed for the determination of ligustilides in traditional Chinese medicines (TCMs), which was based on pressurized hot water extraction (PHWE) followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The two bioactive compounds Z-ligustilide and E-ligustilide in two common TCMs, viz. Ligusticum chuanxiong and Angelica sinensis, were extracted by water at 150°C and 40 bar, followed by concentration with HS-SPME and detection by GC-MS. PHWE and HS-SPME parameters were investigated and method validation (precision and recovery) was studied. It has been shown that the proposed method provides a powerful approach for quantitative analysis of ligustilides in TCMs. The method was applied to determination of ligustildes in the TCMs from different growing areas. The results indicate that PHWE-HS-SPME-GC-MS is a potential tool for TCM quality assessment.
Co-reporter:Chunhui Deng;Ji Qian;Weimin Zhu;Xiaofeng Yang;Xiangmin Zhang
Journal of Separation Science 2005 Volume 28(Issue 11) pp:1137-1142
Publication Date(Web):14 JUL 2005
DOI:10.1002/jssc.200401891
Many plants infested by herbivores or viruses can rapidly produce and accumulate a plant-signaling compound, methyl salicylate (MeSA), in their leaves to activate disease resistance. In the present work, a simple, rapid, and sensitive method was developed for the determination of MeSA in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS. Results show that the proposed method has a low detection limit (0.08 ng mg–1) and good precision (RSD = 8.9%). The present method was applied to the investigation of tomato plant defense response to tobacco mosaic virus (TMV) by rapid analysis of volatile compounds in plant leaves. It was found that tomato plants can produce large amounts of MeSA as a defense response to TMV. This indicates that MeSA may be a plant-signaling compound in tomato plant defense response to TMV.
Co-reporter:Chunhui Deng;Ning Yao;Ning Li;Xiangmin Zhang
Journal of Separation Science 2005 Volume 28(Issue 17) pp:2301-2305
Publication Date(Web):15 NOV 2005
DOI:10.1002/jssc.200500085
A new technique, headspace single-drop microextraction (HS-SDME) with in-drop derivatization, was developed. Its feasibility was demonstrated by analysis of the model compounds, aldehydes in water. A hanging microliter drop of solvent containing the derivatization agent of O-2,3,4,5,6-(pentaflurobenzyl)hydroxylamine hydrochloride (PFBHA) was shown to be an excellent extraction, concentration, and derivatization medium for headspace analysis of aldehydes by GC-MS. Using the microdrop solvent with PFBHA, acetaldehyde, propanal, butanal, hexanal, and heptanal in water were headspace extracted and simultaneously derivatized. The formed oximes in the microdrop were analyzed by GC-MS. HS-SDME and in-drop derivatization parameters (extraction solvent, extraction temperature, extraction time, stirring rate microdrop volume, and the headspace volume) and the method validations (linearity, precision, detection limit, and recovery) were studied. Compared to liquid–liquid extraction and solid-phase microextraction, HS-SDME with in-drop derivatization is a simple, rapid, convenient, and inexpensive sample technique.
Co-reporter:Shun Shen, Yunfei Sha, Chunhui Deng, Xiangmin Zhang, Daxi Fu, Jiakuan Chen
Journal of Chromatography A 2004 Volume 1047(Issue 2) pp:281-287
Publication Date(Web):27 August 2004
DOI:10.1016/j.chroma.2004.06.129
Flos Chrysanthemi Indici is a common traditional Chinese medicine (TCM). In this paper, headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was developed for quality assessment of Flos Chrysanthemi Indici from different growing areas in China. SPME parameters such as extraction fibers, extraction temperature, extraction time and sample mass were investigated to achieve identical results to those obtained by the steam distillation (SD). The selected SPME conditions were as follows: SPME fiber coated with 65-μm PDMS/DVB, extraction temperature of 60 °C, extraction time of 30 min and sample mass of 1.0 g. Furthermore, four active compounds (eucalyptol, camphor, borneol and bornyl acetate) presented in the TCM were applied to evaluating the quality of Flos Chrysanthemi Indici from 20 various areas. The quality assessment was successfully performed to compare the similarity value (S) between different sample vector of Flos Chrysanthemi Indici and the standard profile vector (SPV). The results showed that the proposed HS-SPME-GC-MS was an alternative technique for quality assessment of Flos Chrysanthemi Indici samples.
Co-reporter:Chunhui Deng, Wei Zhang, Jie Zhang, Xiangmin Zhang
Journal of Chromatography B 2004 Volume 805(Issue 2) pp:235-240
Publication Date(Web):15 June 2004
DOI:10.1016/j.jchromb.2004.03.001
Acetone is an important volatile disease marker. Due to its nature of activity and volatility, it is a difficult task to measure the concentration of acetone in biological samples with accuracy. In this paper, we developed a novel method for determination of trace amount acetone in human plasma by solid-phase microextraction technique with on-fiber derivatization. In this method, the poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber was used and O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was first loaded on the fiber. Acetone in plasma sample was agitated into headspace and extracted by solid-phase microextraction (SPME) fiber and subsequently derivatized with PFBHA on the fiber. Acetone oxime was analyzed by gas chromatography–mass spectrometry (GC–MS). Quantitative analysis of acetone in plasma was carried out by using external standard method. The SPME conditions (extraction temperature and time) and the method validation were studied. The present method was tested by determination of acetone in diabetes plasma and normal plasma. Acetone concentration in diabetes plasma was found to be higher than 1.8 mM, while in normal plasma was lower than 0.017 mM. The results show that the present method is a potential tool for diagnosis of diabetes.
Co-reporter:Xiangmin Zhang;Hua-Ling Hu;Shaoying Xu;Xiuhan Yang;Jie Zhang
Journal of Separation Science 2001 Volume 24(Issue 5) pp:385-391
Publication Date(Web):20 JUN 2001
DOI:10.1002/1615-9314(20010501)24:5<385::AID-JSSC385>3.0.CO;2-U
Comprehensive two-dimensional separation by capillary liquid chromatography coupled with capillary micellar electrokinetic chromatography was investigated. Fast micellar electrokinetic chromatography was achieved by speeding up electroosmotic flow and improving solute mass transfer in micellar pseudo-stationary phase. High efficiency separation was achieved by selecting operating conditions based on theoretical relationships and experimental data. A new gating interface from the first dimension to the second dimension based on effluent stacking-injection was developed. The reproducibility of the interface for sample transfer efficiency, migration time, and peak height was studied. The overall system performance was verified in the separation of complex neutral components. Resolution of hundreds of compounds present in traditional Chinese medicines was demonstrated.
Co-reporter:Xueyang Zhang, Shaochun Zhu, Chunhui Deng and Xiangmin Zhang
Chemical Communications 2012 - vol. 48(Issue 21) pp:NaN2691-2691
Publication Date(Web):2012/01/20
DOI:10.1039/C2CC17997K
An aptamer microarray was directly fabricated on a MALDI target plate for high-throughput insulin detection. High sensitivities were observed both in standard solutions (5 ng mL−1, 0.86 nM) and serum sample (20 ng mL−1, 3.4 nM). This method shows great promise in the field of biomarker detection.
Co-reporter:Yan Li, Jinsong Wu, Dawei Qi, Xiuqing Xu, Chunhui Deng, Pengyuan Yang and Xiangmin Zhang
Chemical Communications 2008(Issue 5) pp:NaN566-566
Publication Date(Web):2007/11/20
DOI:10.1039/B716055K
A novel approach is proposed to synthesize Fe3O4@TiO2 microspheres with a well-defined core–shell structure, and the synthesized Fe3O4@TiO2 core–shell microspheres were successfully applied for the simple and fast enrichment of phosphopeptides via direct MALDI-TOF mass spectrometry analysis.
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![2,6-Epoxy-2H-naphthaceno[1,2-b]oxocin-14-carboxylicacid, 11-[(6-deoxy-3-C-methyl-2,3,4-tri-O-methyl-a-L-mannopyranosyl)oxy]-4-(dimethylamino)-3,4,5,6,9,11,12,13,14,16-decahydro-3,5,8,10,13-pentahydroxy-6,13-dimethyl-9,16-dioxo-,methyl ester, (2R,3S,4R,5R,6R,11S,13S,14R)-](/data/chemimg/470500/1404-15-5.png)
![2,6-Epoxy-2H-naphthaceno[1,2-b]oxocin-14-carboxylicacid, 11-[(6-deoxy-3-C-methyl-2,3,4-tri-O-methyl-a-L-mannopyranosyl)oxy]-4-(dimethylamino)-3,4,5,6,9,11,12,13,14,16-decahydro-3,5,8,10,13-pentahydroxy-6,13-dimethyl-9,16-dioxo-,methyl ester, (2R,3S,4R,5R,6R,11S,13S,14R)-](/data/chemimg/470500/1404-15-5_b.png)
![Acetamide,N-(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-yl)-2-iodo-](/data/chemimg/941800/63368-54-7.png)
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