•The nanocomposite was synthesized by a simple two-step strategy.•It is the first time that molybdenum has ever applied in proteomics.•The as-prepared nanocomposite was successfully applied by metal oxide affinity chromatography.•Enriching phosphopeptides with excellent specificity, high detection sensitivity (1 fmol/mL) and well recovery (91.13%).To promote the development of phosphoproteome analysis in highly selective efficient tracing phosphorylated proteins or peptides, views of researches should not confined with intrinsic materials and their modification. New materials are supposed to be explored for phosphoproteome analysis. In this work, we first introduced Molybdenum (VI) oxide (MoO3) into phosphoproteome, loading on the graphene oxide (GO) nanosheets forming MoO3/GO nanocomposites by a simple two-step strategy. The GO nanosheets offered MoO3 a perfect stable platform for separation and concentration and MoO3 exhibited wonderful property in enriching phosphopeptides with highly selectivity and sensitivity on GO nanosheets. Specifically, the as-synthesized MoO3/GO nanocomposites exhibited excellent specificity (β-casein: BSA = 1:1000), high detection sensitivity (1 fmol/mL) and well recovery (91.13%) in enriching phosphopeptides by metal oxide affinity chromatography (MOAC). Moreover, the as-synthesized MoO3/GO nanocomposites provided effective enrichment of phosphopeptides from nonfat milk (a total of twelve phosphopeptides signals) and human serum (a total of four endogenous phosphopeptides signals), displaying great biological compatibility, which demonstrated that the MoO3/GO nanocomposites is a promising candidate in selectively identifying and determining low-abundance phosphorylated peptides in biological sample.

Magnetic mesoporous carbon composites incorporating hydrophilic metallic nanoparticles were synthesized from resol, ZrO(NO3)2, ferric acetylacetonate, and triblock copolymer F127. The method involves a multi-component co-assembly strategy associated with direct carbonization. The resulting carbon material is shown to be useful as a metal oxide affinity chromatography (MOAC) material for enrichment of phosphopeptides owing to its large mesoporous (4.8 nm) surface area (442 m2 g−1), large pore volume (0.37 cm3 g−1) and excellent hydrophilicity. The metallic iron and ferric oxide particles modified on the mesoporous carbon exert a magnetic force and, in combination with the metallic zirconia, is a viable MOAC material for enrichment of low-abundance phosphopeptides. Because of metal chelation between metallic nanoparticles and the phosphate groups of phosphopeptides, the zirconia/magnetic mesoporous carbon displays high selectivity even at a phosphopeptide/nonphosphopeptide molar ratio of 1:500. As little as 1.5 fmol of phosphopeptides become detectable. The MOAC was successfully applied to the identification by MALDI-TOF MS of phosphopeptides in human serum and nonfat milk.

For in-depth analysis of phosphorylated proteomics, the highly sensitive and selective capture of phosphopeptides from intricate biological samples is extremely significant. In this work, a late-model immobilized metal ion affinity chromatography (IMAC) material was fabricated and applied to enriching phosphopeptides from biological samples by abundant Ti4+ ions, which were chelated to crosslinked chitosan (CS) loaded on the surface of magnetic graphene oxide (GO) (denoted as mag GO–CS–Ti4+). The high amount of crosslinked CS endowed the IMAC material with highly hydrophilic properties and abundant active sites for Ti4+ ions on the surface. These characteristics ensure the great performance of mag GO–CS–Ti4+ in the selective enrichment of phosphopeptides from the tryptic digest of β-casein with high selectivity (phosphopeptides to non-phosphopeptides at a molar ratio of 1 : 400), high sensitivity (0.5 fmol), large enrichment capacity (66.6 mg g−1), prominent phosphopeptide recovery (93.11%), and excellent reusability. What's more, mag GO–CS–Ti4+ was applied to capturing phosphopeptides from real biological samples, human serum from both healthy people and patients and nonfat milk. The results successfully manifested that the mag GO–CS–Ti4+ could be a great affinity material for the enrichment of low-abundant phosphopeptides from biological samples.

•The polymer of ZIC-HILIC nanoparticles were synthesized by one-step reflux-precipitation polymerization (RPP).•Au nanoparticles were decorated on the nanoparticles by in situ reduction, bonding larger amount of zwitterionic groups.•Prepared ZIC-HILIC material performed well in enriching low-abundant glycopeptides from complex biological samples.In consideration of the close connection between glycopeptides and human diseases, the efficient method to separate and enrich glycopeptides from complex biological samples is urgently required. In the work, we developed a magnetic zwitterionic-hydrophilic material for highly effective separation and analysis of glycopeptides from complex samples. The Fe3O4 particles were covered with a thick layer of polymer by one-step reflux-precipitation polymerization (RPP), subsequently decorated by Au nanoparticles (Au NPs) through in situ reduction and finally modified with zwitterionic groups. The abundant zwitterionic sites facilitate the selective enrichment of glycopeptides. Besides, the prepared Fe3O4@PGMA@Au-l-cys showed high detection sensitivity (5 fmol IgG digest), approving enrichment capacity (75 mg g−1), satisfactory enrichment recovery (89.8%), and great performance in the analysis and profiling of low-abundance N-linked glycopeptides. Furthermore, the prepared material was employed in the enrichment of glycopeptides in intricate biological samples, and 774 unique N-glycosylation sites from 411 N-glycosylated proteins were reliably identified in three replicate analyses of a 75 μg protein sample extracted from mouse liver, suggesting wide application prospect in glycoproteomics.

•The mesoporous carbon using CTAB as both sacrificed template and carbon source was prepared.•The magnetic mesoporous nanoparticles own unique pore size, high specific surface area.•A carbon material enrichment method for analyzing peptides from complex samples was developed.Highly sensitive and selective enrichment of endogenous peptides or proteins from complex bio-system takes a significant important place to the proteomic. In this work, a unique Fe3O4@2SiO2@mSiO2-C nanomaterial was synthesized, contributing to the separation and enrichment of low concentration peptides from complex mixture. The highly ordered mesoporous carbon structure render the nanospheres with unique properties of strongly connected pore channels, strong hydrophobic properties, high specific surface area (254.90 m2/g), uniform pore size (3.61 nm). Which made it a promising candidates for the efficient enrichment of peptides through hydrophobic-hydrophobic interaction with low detection limit (0.2 fmol), superb size-exclusion of high molecular weight proteins, highly selectivity for BSA digest (molar ratio of BSA tryptic digests/BSA, 1:400), ideal peptides recovery (about 87.5%), wonderful repeatability (RSD less than 25%). Moreover, the as-prepared Fe3O4@2SiO2@mSiO2-C nanoparticles were successfully enriched 2198 endogenous peptides from human serum, which fully indicated that the mesoporous carbon nanoparticles was a promising candidate for isolating proteins or peptides from complex biologicals.

•A novel bi-enzymatic reactor was prepared and evaluated.•Typsin and chymotrypsin were immobilized onto the mixed support, respectively.•The digestion time was decreased to 50 s.•The bi-enzymatic reactor showed great universality for different proteins.The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24 h to 50 s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight.

A novel adsorbent with high adsorption efficiency and adsorption capacity was synthesized and applied to capture heavy metal ions by coupling with ICP-OES analysis. The adsorbent consists of a magnetite (Fe3O4) core, a shell composed of polymerized 3-(trimethoxysilyl)propyl methacrylate, and another poly(allyl chloride) shell composed of plenty of repetitive units, on which the complexing agent of dithizone was grafted by reaction with side chains of the polymer. Compared with traditional adsorbents, the core–shell–shell magnetic composite microspheres have advantages of quick magnetic separation, excellent mechanical strength and high adsorption capacity. The adsorbent was characterized by scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, vibrating sample magnetometer, X-ray photoelectron spectrum, nitrogen adsorption surface area (BET), and FT-IR. Effects of enrichment conditions including pH, amount of adsorbent and elution conditions, interference of other coexistent ions, and the kinetics characterization and adsorption capacity of the adsorbent were studied. Under the optimized conditions, the limits of detection of Pb(II), Cu(II) and Cr(III) ions were 0.87, 0.079 and 0.029 μg L−1, respectively. The RSDs (c = 10 μg L−1, n = 7) were 2.33%, 5.20% and 1.30%, respectively. This adsorbent was successfully applied to the analysis of certified reference materials, viz. a multi-element stock standard solution [(GBW(E)082083] and tea-leaf [GBW(E)080001], and environmental and biological samples.

•We prepared a novel enzymatic reactor by incorporating SBA-15 nanoparticles into hybrid organic–inorganic monolith.•The enzymatic reactor achieved the sequence coverage of 50%, 93% and 71% for BSA, myoglobin and Cyt-C within about 19 s, respectively.•The enzymatic reactor was applied to digest one fraction of rat liver extract successfully.A novel enzymatic reactor was prepared by incorporating SBA-15 nanoparticles into hybrid organic–inorganic monolith and immobilizing trypsin with glutaraldehyde as bridging reagent. Preparation and operation conditions including nanoparticles percentage and residence time were optimized to improve the digestion efficiency. The digestion products were characterized by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) with sequence coverage of 50%, 93% and 71% for bovine serum albumin, myoglobin and cytochrome C, while consuming only about 19 s in dynamic mode. Compared with enzymatic reactor without nanoparticles incorporated, the enzymatic reactor with SBA-15 nanoparticles embedded achieved higher digestion efficiency by introducing more trypsin, which was originated from combination of SBA-15 nanoparticles and hybrid organic–inorganic monolith.This graph shows the preparation of hybrid organic–inorganic monolith and preparation of enzymatic reactor with trypsin immobilized.

Novel open-tubular capillary electrochromatography (OT-CEC) systems with core/shell magnetic nanoparticles modified by amino or C18 groups as stationary phase were constructed by immobilizing nanoparticles in the capillary with permanent magnets. Influence of preparation method of OT-CEC column with series stationary phases (continuous two-dimension) on column performance and effect of dispersant on capability of OT-CEC column prepared by stationary phases with mixed functionalities (mixed stationary phases) were investigated in details to achieve stable preparation. Organic acids were used to evaluate the OT-CEC systems, and the relative column efficiency of salicylic acid was 420,000 plates/m for series stationary phases, while that of benzoic acid reached 480,000 plates/m for mixed stationary phases. The excellent within-column and between-column repeatability (n=5) testified with the RSDs of retention time were less than 0.44% and 10.20% for series stationary phases and 1.65% and 4.29% for mixed stationary phases. The two OT-CEC systems were further applied to separation of the aqueous extract of Rhizoma gastrodiae. Comparing with normal OT capillary column, the new systems show extra high column efficiency due to large surface areas of nanoparticles and multiple separation mechanisms, and they have great potential in the method development for the analysis of complicated samples.Highlights► We report OT-CEC columns with series/mixed stationary phases constructed by magnetic field. ► The best column efficiencies of both series/mixed OT-CEC columns exceed 400,000 plates/m. ► The stationary phase can be easily regenerated by removing and re-applying the magnets.