Preparation of C60‐functionalized magnetic silica microspheres for the enrichment of low‐concentration peptides and proteins for MALDI‐TOF MS analysis

In this work, for the first time, a novel C60‐functionalized magnetic silica microsphere (designated C60‐f‐MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60‐f‐MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low‐concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60‐f‐MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60‐f‐MS microspheres were successfully applied to the enrichment of low‐concentration peptides in tryptic protein digest and human urine via a MALDI‐TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low‐concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5 min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene‐derivatized magnetic silica materials are superior to those already available in the market. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel C60‐f‐MS microspheres makes it a promising candidate for isolation of low‐concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate.     

Preparation of magnetic core-mesoporous shell microspheres with C8-modified interior pore-walls and their application in selective enrichment and analysis of mouse brain peptidome

In this paper, magnetic mesoporous silica microspheres with C8-modified interior pore-walls were prepared through a facile one-pot sol-gel coating strategy, and were successfully applied for selective enrichment of endogenous peptides in mouse brain for peptidome analysis. Through the one-pot sol-gel approach with surfactant (CTAB) as a template, tetraethyl orthosilicate (TEOS) and n-ctyltriethoxysilane (C8TEOS) as the precursors, C8-modified magnetic mesoporous microspheres (C8-Fe(3)O(4)@mSiO(2)) consisting magnetic core and mesoporous silica shell with C8-groups exposed in the mesopore channels were synthesized. The obtained microspheres possess highly open mesopores of 3.4 nm, high surface area (162.5 m(2)/g), large pore volume (0.17 cm(3)/g), excellent magnetic responsivity (56.3 emu/g) and good dispersibility in aqueous solution. Based on the abundant surface silanol groups, functional C8 groups and the strong magnetic responsivity of the core-shell C8-Fe(3) O(4) @mSiO(2) microspheres, efficient and fast enrichment of peptides was achieved. Additionally, the C8-Fe(3)O(4)@mSiO(2) microspheres exhibit excellent performance in selective enrichment of endogenous peptides from complex samples that are consist of peptides, large proteins and other compounds, including human serum and mouse brain followed by automated nano-LC-ESI-MS/MS analysis. These results indicate C8-Fe(3)O(4)@mSiO(2) microspheres would be a potential candidate for endogenous peptides enrichment and biomarkers discovery in peptidome analysis.     

Facile synthesis of magnetic metal organic frameworks for the enrichment of low‐abundance peptides for MALDI‐TOF MS analysis

In this work, core‐shell magnetic metal organic framework (MOF) microspheres were successfully synthesized by coating magnetite particles with mercaptoacetic acid and subsequent reactions with ethanol solutions of Cu(OAc)₂ and benzene‐1,3,5‐tricarboxylic acid (designated as H₃btc) alternately. The resulting Fe₃O₄@[Cu₃(btc)₂] possess strong magnetic responsiveness. We applied the novel nanocomposites in the enrichment of low‐concentration standard peptides, peptides in MYO and BSA tryptic digests and in human urine in combination with MALDI‐TOF MS analysis for the first time. In addition, the Cu₃(btc)₂ MOF shells exhibit strong affinity to peptides, thus providing a rapid and convenient approach to the concentration of low‐abundance peptides. Notably, peptides at an extremely low concentration of 10 pM could be detected by MALDI‐TOF MS after enrichment with the magnetic MOF composites. In brief, the facile synthesis and efficient enrichment process of the Fe₃O₄@[Cu₃(btc)₂] microspheres make them promising candidates for the isolation of peptides in even complex biological environments.     

Development of oleic acid-functionalized magnetite nanoparticles as hydrophobic probes for concentrating peptides with MALDI-TOF-MS analysis

The oleic acid‐functionalized magnetite nanoparticles (OA‐Fe₃O₄) with mean diameter of about 15 nm were synthesized through a low‐cost, one‐pot method and were designed as hydrophobic probes to realize the convenient, efficient and fast concentration of low‐concentration peptides followed by MALDI‐TOF‐MS analysis. The capability of OA‐Fe₃O₄ nanoparticles in concentration of low‐abundance peptides from simple and complex solutions were evaluated by comparing them with a sort of C8‐modified magnetic microspheres. Samples of standard peptide solution, protein digest solution and human serum were introduced in the evaluating process, and the OA‐Fe₃O₄ nanoparticles exhibited good surface affinity toward low‐concentration peptides     

Designed synthesis of fluorous‐functionalized magnetic mesoporous microspheres for specific enrichment of phosphopeptides with fluorous derivatization

In this work, for the first time, perfluorinated magnetic mesoporous microspheres were designed and synthesized for the highly specific enrichment of fluorous‐derivatized phosphopeptides through the unique fluorine–fluorine interactions. The perfluorinated magnetic mesoporous microspheres were prepared through a surfactant‐mediated one‐pot approach and successfully applied to the selective extraction of fluorous‐derivatized phosphopeptides from β‐casein tryptic digest, protein mixtures, and human serum. Thanks to the hydrophilic silanol groups exposed on the surface, perfluorinated groups modified in the pore channels and the magnetic cores, the flourous‐functionalized magnetic microspheres exhibited excellent dispersibility, specificity toward fluorous‐derivatized phosphopeptides while facilitated separation procedures. The novel composites achieved a high selectivity of 1:1000 toward nonphosphorylated peptides and proved to be practicable in the enrichment of endogenous phosphopeptides in the human serum sample.     

Highly selective and rapid enrichment of phosphorylated peptides using gallium oxide-coated magnetic microspheres for MALDI-TOF-MS and nano-LC-ESI-MS/MS/MS analysis

In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga(2)O(3)-coated Fe(3)O(4) microspheres. The performance of the Ga(2)O(3)-coated Fe(3)O(4) microspheres were further compared with Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga(2)O(3)-coated Fe(3)O(4) microspheres over the other materials. Finally, the Ga(2)O(3)-coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga(2)O(3)-coated Fe(3)O(4) microsphere is an effective material for selective isolation and concentration of phosphopeptides.     

Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shell

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe₃O₄@nSiO₂ Core and perpendicularly aligned mesoporous SiO₂ shell (designated Fe₃O₄@nSiO₂@mSiO₂). The Fe₃O₄@nSiO₂@mSiO₂ microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high‐surface areas of the microspheres were demonstrated to have good size‐exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re‐used Fe₃O₄@nSiO₂@mSiO₂ microspheres was also studied. Large‐scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano‐LC‐ESI‐MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel Fe₃O₄@nSiO₂@mSiO₂ microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.     

Facile preparation of magnetic graphene double‐sided mesoporous composites for the selective enrichment and analysis of endogenous peptides

In this work, magnetic graphene double‐sided mesoporous nanocomposites (mag‐graphene@mSiO₂) were synthesized by coating a layer of mesoporous silica materials on each side of magnetic grapheme. The surfactant (CTAB) mediated sol‐gel coating was performed using tetraethyl orthosilicate as the silica source. The as‐made magnetic graphene double‐sided mesoporous silica composites were treated with high‐temperature calcination to remove the hydroxyl on the surface. The novel double‐sided materials possess high surface area (167.8 cm²/g) and large pore volume (0.2 cm³/g). The highly open pore structure presents uniform pore size (3.2 nm) and structural stability. The hydrophobic interior pore walls could ensure an efficient adsorption of target molecules through hydrophobic–hydrophobic interaction. At the same time, the magnetic Fe₃O₄ particles on both sides of the materials could simplify the process of enrichment, which plays an important role in the treatment of complex biological samples. The magnetic graphene double‐sided nanocomposites were successfully applied to size‐selective and specific enrichment of peptides in standard peptide mixtures, protein digest solutions, and human urine samples. Finally, the novel material was applied to selective enrichment of endogenous peptides in mouse brain tissue. The enriched endogenous peptides were then analyzed by LC‐MS/MS, and 409 endogenous peptides were detected and identified. The results demonstrate that the as‐made mag‐graphene@mSiO₂ have powerful potential for peptidome research.     

Cerium ion-chelated magnetic silica microspheres for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry

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 beta-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 beta-casein in the sample solution, the trace phosphopeptides derived from beta-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.     

Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA

An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.     

An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein

To further improve the selectivity and throughput of phosphopeptide analysis for the samples from real-time cell lysates, here we demonstrate a highly efficient method for phosphopeptide enrichment via newly synthesized magnetite microparticles and the concurrent mass spectrometric analysis. The magnetite microparticles show excellent magnetic responsivity and redispersibility for a quick enrichment of those phosphopeptides in solution. The selectivity and sensitivity of magnetite microparticles in phosphopeptide enrichment are first evaluated by a known mixture containing both phosphorylated and nonphosphorylated proteins. Compared with the titanium dioxide-coated magnetic beads commercially available, our magnetite microparticles show a better specificity toward phosphopeptides. The selectively-enriched phosphopeptides from tryptic digests of β-casein can be detected down to 0.4 fmol μl⁻¹, whereas the recovery efficiency is approximately 90% for monophosphopeptides. This magnetite microparticle-based affinity technology with optimized enrichment conditions is then immediately applied to identify all possible phosphorylation sites on a signal protein isolated in real time from a stress-stimulated mammalian cell culture. A large fraction of peptides eluted from the magnetic particle enrichment step were identified and characterized as either single- or multiphosphorylated species by tandem mass spectrometry. With their high efficiency and utility for phosphopeptide enrichment, the magnetite microparticles hold great potential in the phosphoproteomic studies on real-time samples from cell lysates.     

Novel Fe3O4@TiO2 core-shell microspheres for selective enrichment of phosphopeptides in phosphoproteome analysis

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, conventional IMAC lacks enough specificity for efficient phosphoproteome analysis. In this study, novel Fe 3O 4@TiO 2 microspheres with well-defined core-shell structure were prepared and developed for highly specific purification of phosphopeptides from complex peptide mixtures. The enrichment conditions were optimized using tryptic digests of beta-casein, and the high specificity of the Fe 3O 4@TiO 2 core-shell microspheres was demonstrated by effectively enriching phosphopeptides from the digest mixture of alpha-casein and beta-casein, as well as a five-protein mixture containing nonphosphoproteins (bovine serum albumin (BSA), myoglobin, cytochrome c) and phosphoproteins (ovalbumin and beta-casein). The Fe 3O 4@TiO 2 core-shell microspheres were further successfully applied for the nano-LC-MS/MS analysis of rat liver phosphoproteome, which resulted in identification of 56 phosphopeptides (65 phosphorylation sites) in mouse liver lysate in a single run, indicating the excellent performance of the Fe 3O 4@TiO 2 core-shell microspheres.     

Preparation of Fe3O4@ZrO2 core-shell microspheres as affinity probes for selective enrichment and direct determination of phosphopeptides using matrix-assisted laser desorption ionization mass spectrometry

Fe3O4@ZrO2 microspheres with well-defined core-shell structure were prepared and applied for the highly selective enrichment of phosphopeptides from tryptic digest product of proteins. To successfully coat iron oxide microspheres with uniform zirconia shell, magnetic Fe3O4 microspheres were first synthesized via a solvothermal reaction, followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction. Finally, with the use of the Fe3O4@C microspheres as templates, zirconium isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into zirconia by calcinations. The as-prepared Fe3O4@ZrO2 core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of beta-casein, casein, and five protein mixtures to exemplify their selective enrichment ability of phosphopeptides from complex protein samples. In only 0.5 min, phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe3O4@ZrO2 microspheres. The results demonstrate that Fe3O4@ZrO2 microspheres have the excellent selective enrichment capacity for phosphopeptides from complex samples. The performance of the Fe3O4@ZrO2 microspheres was further compared with commercial IMAC beads for the enrichment of peptides originating from tryptic digestion of beta-casein and bovine serum albumin (BSA) with a molar ratio of 1:50, and the results proved a stronger selective ability of Fe3O4@ZrO2 microspheres over IMAC beads. Finally, the Fe3O4@ZrO2 microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum without any other purification procedures.     

Preparation of sandwich-structured graphene/mesoporous silica composites with C8-modified pore wall for highly efficient selective enrichment of endogenous peptides for mass spectrometry analysis

In this study, sandwich-structured graphene/mesoporous silica composites (C8-modified graphene@mSiO(2) ) were synthesized by coating mesoporous silica onto hydrophilic graphene nanosheets through a surfactant-mediated cocondensation sol-gel process. The newly prepared C8-modified graphene@mSiO(2) nanocomposites possess unique properties of extended plate-like morphology, good water dispersibility, highly open pore structure, uniform pore size (2.8 nm), high surface area (632 m(2) /g), and C8-modified-interior pore walls. The unique structure of the C8-modified graphene@mSiO(2) composite nanosheets not only provide extended planes with hydrophilic surface that prevents aggregation in solution, but also offer a huge number of C8-modified mesopores with high surface area that can ensure an efficient adsorption of peptides through hydrophobic-hydrophobic interaction between C8-moified pore walls and target molecules. The obtained C8-modified graphene@mSiO(2) materials were utilized for size selectively and specifically enriching peptides in standard peptide mixtures and endogenous peptides in real biological samples (mouse brain tissue).     

Facile synthesis of magnetic poly(styrene‐co‐4‐vinylbenzene‐boronic acid) microspheres for selective enrichment of glycopeptides

In this work, the composites of magnetic Fe₃O₄@SiO₂@poly (styrene‐co‐4‐vinylbenzene‐boronic acid) microspheres with well‐defined core–shell–shell structure were facilely synthesized and applied to selectively enrich glycopeptides. Due to the relatively large amount of vinyl groups introduced by 3‐methacryloxy‐propyl‐trimethoxysilane on the core‐shell surface, the poly(styrene‐co‐4‐vinylbenzeneboronic acid) (PSV) was coated with high efficiency, resulting in a large amount of boronic acid on the outermost polymer shell of the Fe₃O₄@SiO₂@PSV microspheres, which is of great importance to improve the enrichment efficiency for glycopeptides. The obtained Fe₃O₄@SiO₂@PSV microspheres were successfully applied to the enrichment of glycopeptides with strong specificity and high selectivity, evaluated by capturing glycopeptides from tryptic digestion of model glycoprotein HRP diluted to 0.05 ng/μL (1.25 × 10^?13 mol, 100 μL), tryptic digest of HRP and nonglycosylated BSA up to the ratio of 1:120 w/w and the real complex sample human serum with 103 unique N‐glycosylation peptides of 46 different glycoproteins enriched.     

Magnetic hydrophilic methacrylate-based polymer microspheres designed for polymerase chain reactions applications

Magnetic hydrophilic non-porous P(HEMA-co-EDMA), P(HEMA-co-GMA) and PGMA microspheres were prepared by dispersion (co)polymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of several kinds of magnetite. It was found that some components used in the preparation of magnetic carriers interfered with polymerase chain reaction (PCR). Influence of non-magnetic and magnetic microspheres, including magnetite nanoparticles and various components used in their synthesis, on the PCR course was thus investigated. DNA isolated from bacterial cells of Bifidobacterium longum was used in PCR evaluation of non-interfering magnetic microspheres. The method enabled verification of the incorporation of magnetite nanoparticles in the particular methacrylate-based polymer microspheres and evaluation of suitability of their application in PCR. Preferably, electrostatically stabilized colloidal magnetite (ferrofluid) should be used in the design of new magnetic methacrylate-based microspheres by dispersion polymerization.     

Concerted experimental approach for sequential mapping of peptides and phosphopeptides using C18-functionalized magnetic nanoparticles

An integrated analytical approach for the enrichment, detection, and sequencing of phosphopeptides using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS) was developed. On the basis of C18-functionalized Fe3O4 nanoparticles, the enrichment method was designed not only to specifically trap phosphopeptides, but also nonphosphorylated peptides, both of which can be subsequently desorbed selectively and directly for MALDI-MS analysis without an elution step. Peptide binding is afforded by the C18-derivatization, whereas the highly selective capture of phosphopeptides is based on higher binding affinity afforded by additional metal chelating interaction between the Fe3O4 nanoparticles and the phosphate groups. Upon binding, the initial aqueous wash allows desalting, while a second and a third wash with high acetonitrile content coupled with diluted sulfuric acid and ammonia removes most of the bound nonphosphorylated peptides. Selective or sequential mapping of the peptides and phosphopeptides can, thus, be effected by spotting the washed nanoparticles onto the MALDI target plate along with judicious choice of matrices. The inclusion of phosphoric acid in a 2,5-dihydroxybenzoic acid matrix allows the desorption and detection of phosphopeptides, whereas an alpha-cyano-4-hydroxy-cinnamic acid matrix with formic acid allows only the desorption of nonphosphorylated peptides. The method used to enrich phosphopeptides prior to MS applications is more sensitive and tolerable to sodium dodecyl sulfate than IMAC. We have demonstrated the applicability of C18-functionalized Fe3O4 nanoparticles in the detection of in vitro phosphorylation sites on the myelin basic protein, and at least 17 phosphopeptides were identified, including one previously uncharacterized site.     

Process and formulation variables in the preparation of injectable and biodegradable magnetic microspheres

The aim of this study was to prepare biodegradable sustained release magnetite microspheres sized between 1 to 2 μm. The microspheres with or without magnetic materials were prepared by a W/O/W double emulsion solvent evaporation technique using poly(lactide-co-glycolide) (PLGA) as the biodegradable matrix forming polymer. Effects of manufacturing and formulation variables on particle size were investigated with non-magnetic microspheres. Microsphere size could be controlled by modification of homogenization speed, PLGA concentration in the oil phase, oil phase volume, solvent composition, and polyvinyl alcohol (PVA) concentration in the outer water phase. Most influential were the agitation velocity and all parameters that influence the kinematic viscosity of oil and outer water phase, specifically the type and concentration of the oil phase. The magnetic component yielding homogeneous magnetic microspheres consisted of magnetite nanoparticles of 8 nm diameter stabilized with a polyethylene glycole/polyacrylic acid (PEG/PAA) coating and a saturation magnetization of 47.8 emu/g. Non-magnetic and magnetic microspheres had very similar size, morphology, and size distribution, as shown by scanning electron microscopy. The optimized conditions yielded microspheres with 13.7 weight% of magnetite and an average diameter of 1.37 μm. Such biodegradable magnetic microspheres seem appropriate for vascular administration followed by magnetic drug targeting.     

Highly efficient and selective enrichment of glycopeptides using easily synthesized magG/PDA/Au/l‐Cys composites

Highly selective and efficient enrichment of glycopeptides from complex biological samples is necessary. In this study, novel zwitterionic hydrophilic polydopamine‐coated magnetic graphene composites (magG/PDA/Au/l ‐Cys) were synthesized and applied to the enrichment of glycopeptides. The size, morphology, and composition of magG/PDA/Au/l ‐Cys composites were investigated by transmission electron microscopy, scanning electron microscopy, FT‐infrared spectroscopy, and X‐ray diffraction. The composites possessed a number of desirable characteristics, including good biocompatibility easy separation property and excellent hydrophilicity. By virtue of the features contributed by different ingredients, the prepared composites demonstrated superior performance for glycopeptide enrichment with high sensitivity (0.1 fmol), efficiency, selectivity (1:100), and repeatability (at least ten times). In addition, the composites were successfully applied to the enrichment of glycopeptides from human serum and 40 unique N‐glycosylation peptides from 31 different N‐linked glycoproteins were identified. The superior hydrophilic material is of great potential for the analysis of glycoproteins.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.