Search

Glycopeptidome represents reliable predictors of physiological and pathological status. Obstructions mainly including low abundance of endogenous glycopeptides and varied interference necessitate glycopeptide enrichment prior to MS analysis. Inspired by the prevalence of hydrophilic interaction chromatography for glycopeptide enrichment, a novel magnetic mesoporous silica nanomaterial (Fe 3 O 4 @mSiO 2 -TSG) with strongly hydrophilic property was developed through a one-pot method. In this work, the gluconamide-containing organosilane is innovatively proposed to directly serve as the strongly hydrophilic silica source for fabrication of hydrophilic mesoporous silica nanomaterial for glycopeptidomics research. Apart from excellent hydrophilicity, Fe 3 O 4 @mSiO 2 -TSG also was equipped with large specific surface area, ordered mesopore channels and great magnetic responsiveness. With all the advantages, Fe 3 O 4 @mSiO 2 -TSG displayed remarkable size-exclusion effect and considerable reusability. Moreover, combined with nano-LC-MS/MS, the glycopeptidome of serum from breast cancer patients was analyzed comprehensively, which showed noteworthy difference from healthy serum through gene ontology analysis, indicating great potential of the approach for glycopeptidomics research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Although antibody has played a great role in highly specific recognition of protein biomarkers, it faces poor stability, reproducibility, high-cost and time-consuming preparation, etc. Here, aptamer and molecularly imprinted polymers (MIPs), both as promising substitutes of antibody, were integrated onto magnetic nanoparticles by Au-S bonds and SiO 2 as imprinted layer for preparing a new nanoprobe. Highly specific and sensitive recognition of different protein biomarkers, such as insulin for diabetes and alpha-fetoprotein (AFP) for hepatic carcinoma, were achieved respectively by the system of combining hybrid aptamer-molecularly imprinted magnetic nanoprobe and mass spectrometry. With the double affinities offered by aptamer-MIPs, insulin can be detected at 0.5 ng mL -1 in human serum dilution, the equlibrium dissociation constant between nanoprobe and insulin is measured as 23.61 ± 2.27 μM. Likewise, AFP can be sufficiently detected in human saliva dilution from 1000 ng mL -1 to 20 ng mL -1 , and two patients with hepatic carcinoma are discriminated from healthy person due to the abnormally high expression of AFP in serum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

For the first time, dual metal ions (Ti 4+ -Zr 4+ ) were successfully modified into the channel of magnetic mesoporous silica to obtain an affinity probe for highly selective capture of endogenous phosphopeptides in biological samples. The newly prepared Fe 3 O 4 @mSiO 2 @Ti 4+ -Zr 4+ composites possessed the advantages of ordered mesoporous channels, superparamagnetism, and enhanced affinity properties of dual metal ions of Ti 4+ and Zr 4+ . The phosphopeptide enrichment efficiency of the Fe 3 O 4 @mSiO 2 @Ti 4+ -Zr 4+ composite was investigated, and the result indicated an ultrahigh size exclusive ability (weight ratio of β-casein tryptic digests, BSA, and α-casein protein reached up to 1:1000:1000). Compared to magnetic affinity probes with single metal ions (Fe 3 O 4 @mSiO 2 @Ti 4+ , Fe 3 O 4 @mSiO 2 @Zr 4+ ), the composite possessed stronger specificity, higher sensitivity, and better efficiency; and more importantly, it showed much enhanced enrichment ability towards both mono- and multi-phosphorylated peptides. Additionally, by utilizing the Fe 3 O 4 @mSiO 2 @Ti 4+ -Zr 4+ affinity probe, a total number of 104 endogenous phosphopeptides including 95 mono-phosphopeptides and 9 multi-phosphopeptides were captured and identified from human saliva, indicating the great potential for the application of the novel probe for the peptidome analysis in the future. Graphic abstract.

In this work, for the first time, L -cysteine ( L -Cys), a zwitterionic hydrophilic compound containing abundant amino and carboxyl was modified on silica microspheres for the fabrication of SiO 2 @ L -Cys capillary packed column. Since the SiO 2 @ L -Cys capillary packed column possessed great hydrophilicity brought by L -cysteine, they could be utilized to enrich glycopeptides with a low detection limit of 5 fmol μL -1 HRP digests as well as a good selectivity of the mixture of HRP and BSA digests up to a molar ratio of 1:50. The capillary packed column were further applied into the glycosylation analysis of human saliva and 69 glycopeptides were identified from 2 μL human saliva digests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Peptidomics research is of great significance for discovering potential biomarkers and monitoring human diseases. As a kind of common clinical biofluid, saliva known for its noninvasive collection and easy accessibility has been widely used in peptidomics research. In this article, we combined immobilized metal ions affinity chromotography (IMAC) with mesoporous material and proposed the copper ion doped magnetic mesoporous silica material (denoted as Fe 3 O 4 @mSiO 2 -Cu 2+ ) which had a large surface area of 221 m 2  g -1 and pore volume of 0.20 cm 3  g -1 . By immobilizing copper ions onto the mesopore walls, the standard peptide Angiotensin II could be identified in an extremely low concentration of 0.1 fmol μl -1 and in a mass ratio of 1:500 (Angiotensin II:BSA, m/m), which indicated significant sensitivity and a great size-exclusive ability. In addition, the introduction of polydopamine (PDA) made Fe 3 O 4 @mSiO 2 -Cu 2+ more hydrophilic and biocompatible which could improve the profiling of endogenous peptides in bio-sample. Finally, 131 endogenous peptides were identified in human saliva after enrichment with Fe 3 O 4 @mSiO 2 -Cu 2+ . Therefore, Fe 3 O 4 @mSiO 2 -Cu 2+ nanoparticles provided a promising candidate protocol for biomarker discovery., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Developing an effective strategy for endogenous phosphopeptides enrichment and separation is necessary in phosphopeptidomics analysis due to the serious inference caused by chaotic biological environment. In this work, a size-exclusive magnetic binary metal mesoporous nanocomposites were synthesized to capture phosphorylated peptides for mass spectrometry analysis. The novel Fe 3 O 4 @TiO 2 -ZrO 2 @mSiO 2 nanocomposites possessed the merits of ordered mesoporous channels, superparamagnetism and the integration of dual affinity of Zr-O and Ti-O. Compared with single-metal centered nanocomposites (Fe 3 O 4 @TiO 2 @mSiO 2 and Fe 3 O 4 @ZrO 2 @mSiO 2 ), Fe 3 O 4 @TiO 2 -ZrO 2 @mSiO 2 showing much enhanced enrichment performance towards mono- and multi-phospho-peptides with better sensitivity. With all the advances, the Fe 3 O 4 @TiO 2 -ZrO 2 @mSiO 2 was also successfully applied to capture endogenous phosphorylated peptides from human saliva. And consequently, a total of 30 phosphopeptides containing 2 multi- and 28 mono-phosphopeptides were identified efficiently. These results show that the novel materials have the great potential in peptidome analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel zwitterionic hydrophilic magnetic mesoporous silica was prepared for endogenous glycopeptide enrichment prior to MS analysis. For the first time, the material was successfully applied in capturing endogenous glycopeptides from human saliva, indicating great potential of this strategy for glycopeptidome analysis.

Hydrophilic interaction liquid chromatography (HILIC) is a significant enrichment strategy in glycoproteomics profiling. In this report, hydrophilic magnetic mesoporous silica materials (denoted as Fe 3 O 4 @mSiO 2 -IDA) were designed and synthesized as an outstanding enrichment platform for glycopeptide analysis. By taking advantage of their merits, such as large surface area, excellent hydrophilicity and unbiased affinity toward all types of glycopeptides, the Fe 3 O 4 @mSiO 2 -IDA nanomaterials were successfully applied to capture glycopeptides from complex samples. A total of 25 glycopeptides from horseradish peroxidase (HRP) digests and 33 glycopeptides from IgG were identified, respectively. Especially, as a result, 424 glycopeptides assigned to 140 glycoproteins were identified from only 2 μL human serum.

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 SiO 2 -NH 2 @TiO 2 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 SiO 2 -NH 2 @TiO 2 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). Graphical Abstract Workflow of the simultaneous enrichment strategy for phosphopeptides and glycopeptides by SiO 2 -NH 2 @TiO 2 hollow microspheres.

Efficient and specific enrichment of low-abundant glycopeptides from complex biological samples is essential to glycoproteomics analysis. Herein, novel magnetic zwitterionic hydrophilic nanoparticles based on thiol-ene click chemistry were synthesized. The functionalized magnetic nanoparticles exhibited superior performance in glycopeptide enrichment of HRP tryptic digest, demonstrating low detection limit (0.04ng/μL), high selectivity (a mixture of HRP and BSA at the mass ratio of 1:50) and reproducibility (5 repeating cycles). In addition, the material was successfully applied to glycopeptide enrichment from human serum. The outstanding results indicate the potential of the method in the development of glycoproteomics analysis in real biological samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Taking advantage of fluorine-fluorine interactions, fluorous solid-phase extraction (FSPE) is emerging as a novel approach in proteomics research. Notably, silica gel bound with perfluoroalkyl groups was applied to the FSPE of N-linked glycans. Based on previous studies, mesoporous silica coated magnetic nanoparticles bound with perfluoroalkyl groups were synthesized for the specific enrichment of N-linked glycans in this study. The magnetic nanoparticles-incorporated FSPE strategy successfully identified 22 N-linked glycans from the OVA digest with a concentration of 0.5μg/μL, and achieved a detection limit of 5ng/μL (with 16 N-linked glycans identified). It also showed good day-to-day reproducibility. Its selectivity towards BSA protein is 1:200 (molar ratio), showing excellent size-exclusion effect. In addition, the present method proved to be effective for the analysis of the human serum digest, opening up new prospect for the identification of glycans and proteins with other post-translational modifications in biological environment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets were designed and synthesized for the sequential selective enrichment of phosphopeptides and glycopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the nanosheets were successfully applied to the enrichment of phosphopeptides and glycopeptides sequentially, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine β-casein diluted to 0.02 ng/μL (8 × 10(-16) mol/μL) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 0.1 ng/μL (2.5 × 10(-15) mol/μL). The enrichment selectivity of the bifunctional nanosheets was evaluated by capturing phosphopeptides from a peptide mixture of β-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (8.3 × 10(-12) mol of β-casein and 8.3 × 10(-9) mol of BSA in 100 μL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:50 (5.0 × 10(-11) mol of HRP and 2.5 × 10(-9) mol of BSA in 100 μL). Graphical Abstract A workflow of the sequential enrichment strategy for phosphopeptides and glycopeptides by the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets.

Ti(4+) 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).

In this work, a novel size-exclusive metal oxide affinity chromatography (SE-MOAC) platform was built for phosphoproteome research. The operation for preparing graphene @titania @mesoporous silica nanohybrids (denoted as G@TiO2@mSiO2) was facile and easy to conduct by grafting titania nanoparticles on polydopamine (PD)-covered graphene, following a layer of mesoporous silica was coated on the outermost layer. The G@TiO2@mSiO2 nanohybrids exhibited high sensitivity with a low detection limit of 5 amol/μL (a total amount of 1 fmol) and high selectivity for phosphopeptides at a mass ratio of phosphopeptides to non-phosphopeptides (1:1000). The size-exclusive capability of the nanohybrids were also demonstrated by enriching the phosphopeptides from the mixture of Bovine Serum Albumin (BSA), α-casein, and β-casein digests with a high mass ratio (β-casein digests: α-casein: BSA, 1:500:500), which was attributed to the large surface area and ordered mesoporous channels. In addition, the G@TiO2@mSiO2 nanohybrids were employed to capture the endogenous phosphopeptides from human serum successfully., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glycosylation, which has been confirmed to be associated with many diseases, is an important protein post-translation modification. Taking into account the low abundant of glycan, the purification of complex biological samples is considered to be very significant before mass spectrometry detection. In this work, carbon-functionalized magnetic graphene /mesoporous silica materials (C-Mag G@mSiO2 materials) with high content of carbon were designed and synthesized by using polydopamine as carbon precursor. Taking advantage of the special interaction between carbon and glycan, C-Mag G@mSiO2 materials were successfully applied to enrich N-linked glycans in different complex samples, such as standard glycoprotein digestion, the mixture of standard glycoprotein digestion, glycoprotein and non-glycoprotein, and human serum., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To the best of our knowledge, this study is the first to successfully apply a novel, highly selective enrichment technique based on boronic acid-functionalized core-shell magnetic microspheres (BA-Fe3O4@SiO2-Au@mSiO2) with a large surface area and uniform pore size, to determine the baicalin concentration in rat plasma by HPLC. By taking advantage of the special interaction between boronic acid and baicalin under alkaline conditions, as well as the microspheres' size exclusion ability, baicalin was selectively extracted from protein-rich biosamples, such as plasma, without any other pretreatment procedure except for a 10-min vortexing step. BA-Fe3O4@SiO2-Au@mSiO2 microsphere-adsorbed baicalin was straightforwardly and rapidly isolated from the matrix using a magnet. Baicalin was subsequently eluted from the microspheres under acidic conditions for 2min for further HPLC analysis. The extraction conditions, such as the amount of microspheres added, adsorption time, adsorption pH, and elution time and pH, were also determined. Furthermore, method validation, including the linear range, detection limit, precision, accuracy, and recovery, were determined. This newly developed method based on BA-Fe3O4@SiO2-Au@mSiO2 microspheres is a simple, accurate, selective, and green analytical preparatory technique for analyzing baicalin in rat plasma. This study will be further novel research on the analysis of complex plasma samples and the pharmacokinetics of drugs similar to baicalin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We designed and synthesized aptamer-immobilized magnetic mesoporous silica/Au nanocomposites (MMANs) for highly selective detection of unlabeled insulin in complex biological media using MALDI-TOF MS. The aptamer was easily anchored onto the gold nanoparticles in the mesochannels of MMANs with high capacity for highly efficient and specific enrichment of insulin. With the benefit from the size-exclusion effect of the mesoporous silica shell with a narrow pore size distribution (∼2.9 nm), insulin could be selectively detected despite interference from seven untargeted proteins with different size dimensions. This method exhibited an excellent response for insulin in the range 2-1000 ng mL(-1). Moreover, good recoveries in the detection of insulin in 20-fold diluted human serum were achieved. We anticipate that this novel method could be extended to other biomarkers of interest and potentially applied in disease diagnostics.

Developing an effective separation method is necessary for identifying low-abundant endogenous phosphorylated peptides with the removal of proteins. In this work, we prepared size-exclusive magnetic graphene/mesoporous silica composites with titanium(IV)-immobilized pore walls (denoted as Ti(4+)-MGMSs) for capturing endogenous phosphorylated peptides for mass spectrometry analysis. The introduction of hydrophilic polydopamine simplified the synthetic process of Ti(4+)-MGMSs, and the ordered mesoporous channels are beneficial to the trapping of endogenous phosphopeptides while large-size proteins are excluded. Furthermore, the magnetic performance greatly simplifies the entire process of enrichment. With all of the advances, the novel Ti(4+)-MGMSs present high enrichment efficiency either from the low concentration of β-casein tryptic digest (0.5 fmol/μL) or the mixture of β-casein tryptic digest and α-casein (or plus bovine serum albumin, with a mass ratio up to 1:500). Besides, Ti(4+)-MGMSs have also been successfully applied to enrich endogenous phosphorylated peptides from human serum and human saliva.

Metal oxide affinity chromatography (MOAC) is a powerful technique in phosphoproteome research. However, the achievement of highly specific enrichment and sensitive detection of phosphopeptide by MOAC remains a big challenge since the lack of high specificity and large binding capacity of conventional MOAC materials. In this work, a new MOAC material, TiO2-coated hierarchically ordered macro/mesoporous silica (denoted as HOMMS@TiO2) composites, was prepared via a facile process. The HOMMS@TiO2 composites were demonstrated to have low limit of detection (8 fmol) and great specificity with a very rapid enrichment speed (within 1 min). These experimental results have demonstrated that the HOMMS@TiO2 exhibit great potential in phosphoproteome research.

Abnormal protein glycosylation has been demonstrated to be associated with many diseases; therefore, it is very important to conduct a comprehensive structure analysis of glycan for prognosis and diagnosis of diseases, such as cancer. In this work, for the first time, carbon-functionalized ordered graphene/mesoporous silica composites (denoted as C-graphene@mSiO2) with large surface area and uniform pore size were designed and synthesized. By taking advantage of the special interaction between the carbon and glycans as well as size-exclusion ability, 25 N-linked glycans released from ovalbumin were observed clearly with strong MS signals and increased signal-to-noise (S/N) ratio. In addition, after enrichment with the C-graphene@mSiO2 composites, 48 N-linked glycans (S/N > 10) with sufficient peak intensities were obtained from only 400 nL of healthy pristine human serum. The facile and low-cost synthesis method as well as high selective enrichment ability of the novel C-graphene@mSiO2 composite makes it a promising tool for glycosylation research.

In this study, novel raisin-bread sandwich-structured magnetic graphene/mesoporous silica composites with C18-modified interior pore-walls (mag-graphene@mSiO2-C18) were synthesized by coating mesoporous silica layers onto each side of magnetic graphene through a surfactant-mediated co-condensation sol-gel process. The prepared functionalized nanocomposites possessed marvelous properties of extended plate-like morphology, fine water dispersibility, high magnetic response, large surface area (315.4cm(2)g(-1)), uniform pore size (3.3nm) and C18-modified interior pore-walls. Several kinds of phthalates were selected as model analytes to systematically evaluate the performance of adsorbents in extracting hydrophobic molecules followed by gas chromatography-mass spectrometry analyses. Various extraction parameters, including pH value of sample solution, amounts of adsorbents, adsorption time, species and volume of eluting solvent, and desorption time were optimized. The anti-interference ability to macromolecular proteins was also investigated. Method validations such as linearity, recovery, reproducibility, and limit of detection were also studied. Finally, mag-graphene@mSiO2-C18 composites were successfully applied to analyzing phthalates in environmental water samples. The results indicated that this novel approach offered an attractive alternative for rapid, convenient, efficient and selective magnetic solid-phase extraction for targeted hydrophobic compounds., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Acetaldehyde is regarded as a toxic mainstream cigarette smoke constituent, and measurement of acetaldehyde in complex real samples is difficult owing to its high volatility and reactivity. In this work, phenyl group-functionalized magnetic mesoporous microspheres were developed as the solid-phase extraction sorbents for enrichment and analysis of acetaldehyde in mainstream cigarette smoke. The functional magnetic microspheres were first synthesized through a facile one-pot co-condensation approach. The prepared nanomaterials possessed abundant silanol groups in the exterior surface and numerous phenyl groups in the interior pore-walls, as well as a large surface area (273.5m(2)/g), strong superparamagnetism and uniform mesopores (3.3 nm). Acetaldehyde in mainstream cigarette smoke was collected in water and derivatizated with O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine. The formed acetaldehyde oximes were extracted and enriched by the prepared adsorbents via π-π interactions and subsequently analyzed using GC-MS. Extraction conditions such as amounts of sorbents, eluting solvent, adsorption and desorption time were investigated and optimized to achieve the best efficiency. Method validations including linearity, recovery, repeatability, and limit of detection were also studied. It was found that the suggested methodology provided low detection limit of 0.04 mg/mL, good recovery of 88-92%, intra-day and inter-day RSD values of 4.5% and 10.1%, and linear range of 0.25-4 mg/mL (R(2)=0.999). The results indicated that the proposed method based on phenyl-functionalized magnetic mesoporous microspheres was rapid, efficient and convenient for the enrichment and analysis of acetaldehyde in tobacco., (Copyright © 2013 Elsevier B.V. All rights reserved.)

For the first time, dual-platform graphene@SiO(2)@poly(methyl methacrylate) (PMMA) material was synthesized, and successfully applied to efficiently enrich low-abundance 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)., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, we present a facile approach for the synthesis of zirconium phosphate (ZrP)-functionalized magnetic silica mesoporous microspheres for the selective enrichment of phosphopeptides. At first, magnetic mesoporous silica microspheres were prepared by directly coating mesoporous silica onto Fe3O4 magnetic microspheres, and then addition of phosphate onto the magnetic mesoporous silica microspheres was performed using 3-(trihydroxysilyl)propyl methylphosphate. The obtained phosphate-modified magnetic mesoporous microspheres were monodispersed with a mean diameter of 350 nm, and had an obvious mesoporous silica shell (∼65 nm). Finally, the resultant phosphate-functionalized magnetic mesoporous microspheres were incubated in ZrOCl2 solution with gentle stirring overnight for the loading of Zr4+ cations. The obtained Zr4+-functionalized materials were applied to the selective enrichment of phosphopeptides from both standard protein digestion and real samples. The enriched peptides were analyzed by MALDI-TOF MS and LC-ESI MS. Experimental results demonstrated that zirconium phosphonate-modified magnetic mesoporous silica microspheres show excellent potential for the selective enrichment of phosphopeptides.

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe(3)O(4)@nSiO(2) Core and perpendicularly aligned mesoporous SiO(2) shell (designated Fe(3)O(4)@nSiO(2)@mSiO(2)). The Fe(3)O(4)@nSiO(2)@mSiO(2) 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 Fe(3)O(4)@nSiO(2)@mSiO(2) 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 Fe(3)O(4)@nSiO(2)@mSiO(2) microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.

In this study, novel C8-functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8-functionalized magnetic silica (C8-f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8-f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8-f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.

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.

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.

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.

An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (approximately 300 nm in diameter) and high magnetic responsivity to magnetic field (68.2 emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis.

Peptide mapping analysis, utilizing an easily replaceable and regenerable on-column enzymatic microreactor with metal-ion-chelated adsorption of enzyme on magnetic silica microspheres, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. Firstly, magnetic microspheres of small size and strong magnetism were prepared through solvothermal reaction. Thereafter, by introducing tetraethyl orthosilicate (TEOS), magnetic silica (MS) microspheres were formed. Trypsin could then be immobilized onto the MS microspheres based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which was chemically bound to the microspheres through the introduction of glycidoxypropyltrimethoxysilane (GLYMO). The trypsin-immobilized MS microspheres were then locally packed into the capillary by the application of a strong magnetic field using a magnet. The performance of the method was exemplified with digestion of bovine serum albumin for 5 min at 50 degrees C and the result was comparable to the 12 h in-solution digestion. The ability of regeneration of the prepared on-column microreactor and good reproducibility of microreactor before and after regeneration were also demonstrated., (Copyright 2007 John Wiley & Sons, Ltd.)