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.     

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.     

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.     

Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes

Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of alpha- and beta-caseins, milk, and egg white to exemplify the enrichment of phosphopeptides from complex samples. Phosphopeptides, in quantities sufficient for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), were enriched by the affinity probes within only 30 s. The affinity probe-target species conjugates were separated from the sample solution simply by applying an external magnetic field. The detection limit for tryptic digest of beta-casein using this approach is approximately 45 fmol. Furthermore, we combined this enrichment method with a rapid enzymatic digestion method, that is, microwave-assisted enzymatic digestion using magnetic particles as the microwave absorbers, to speed up the tryptic digest reactions. Thus, we alternatively enriched phosphoproteins on the zirconia-coated particles followed by mixing with trypsin and heated the mixture in a microwave oven for 1 min. The particles remaining in the mixture were used as affinity probes to selectively enrich phosphopeptides from the tryptic digestion product by pipetting, followed by characterization using MALDI MS. Using the bifunctional zirconia-coated magnetic particles as both the affinity probes and the microwave absorbers could greatly reduce the time for the purification and characterization of phosphopeptides from complex samples.     

Enrichment of phosphopeptides by Fe3+-immobilized mesoporous nanoparticles of MCM-41 for MALDI and nano-LC-MS/MS analysis

Fe3+-immobilized mesoporous molecular sieves MCM-41 with particle size of ca. 600 nm and pore size of ca. 3 nm is synthesized and applied to selectively trap and separate phosphopeptides from tryptic digest of proteins. For the capture of phosphopeptides, typically 10 microL of tryptic digest solution was first diluted to 1 mL by solution of ACN/0.1% TFA (50:50, v/v) and incubated with 10 microL of 0.1% acetic acid dispersed Fe3+-immobilized MCM-41 for 1 h under vibration. Fe3+-immobilized MCM-41 with trapped phosphopeptides was separated by centrifugation. The deposition was first washed with a volume of 300 microL of solution containing 100 mM NaCl in ACN/0.1% TFA (50:50, v/v) and followed by a volume of 300 microL of solution of 0.1% acetic acid to remove nonspecifically bound peptides. The nanoparticles with trapped phosphopeptides are mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and deposited onto the target for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that phosphopeptides from tryptic digest of alpha-casein and beta-casein are effectively and specifically trapped on Fe3+-immobilized MCM-41 with few peptides nonspecifically adsorbed. After the extraction by Fe3+-immobilized MCM-41, the suppression to the detection of phosphopeptides caused by abundant nonphosphopeptides from tryptic digest is effectively eliminated, and the detection of phosphopeptides by MALDI is greatly enhanced with the value of signal-to-noise (S/N) increased by more than an order of magnitude. It is demonstrated that the mechanism of the adsorption of phosphopeptides on Fe3+-immobilized MCM-41 is based on the interaction between the Fe3+ and the phosphate group. Finally, Fe3+-immobilized MCM-41 is applied to extract phosphopeptides from tryptic digest of the lysate of mouse liver for phosphoproteome analysis by nano-LC-MS/MS.     

Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.     

Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.     

Enrichment of phosphopeptides using biphasic immobilized metal affinity-reversed phase microcolumns

Immobilized metal affinity chromatography (IMAC) based on Fe (3+) or Ga (3+) chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of beta-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of beta-casein monophosphorylated peptide in 20 muL of a solution of human serum protein digest (from 200 mug total serum protein after digestion and desalting).     

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.     

Efficient enrichment of phosphopeptides by magnetic TiO₂-coated carbon-encapsulated iron nanoparticles

Titanium dioxide (TiO₂) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO₂ affinity probes. In this report, we present a facile approach to immobilize TiO₂ onto poly(acrylic acid)‐functionalized magnetic carbon‐encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO₂ affinity probes, denoted as TiO₂‐coated Fe@CNPs, rapid and effective MALDI‐TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of β‐casein, and complex β‐casein/BSA mixture. The TiO₂‐coated Fe@CNPs out‐performed the commercial TiO₂‐coated magnetic beads for detection of phosphopeptides from tryptic digests of β‐casein/BSA mixture with a molar ratio of 1:100. The new TiO₂‐coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO₂‐coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out‐performed the commercial magnetic TiO₂ beads in the number of identified phosphopeptides and phosphorylation sites. In a 200‐μg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.     

Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach.     

Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano-powders

The potential of an organic monolith with incorporated titanium dioxide (TiO(2)) and zirconium dioxide (ZrO(2)) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.     

Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography

The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis

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.     

Highly selective enrichment of phosphopeptides by on‐chip indium oxide functionalized magnetic nanoparticles coupled with MALDI‐TOF MS

Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein‐related samples prior to MS‐based analysis. Herein, an on‐chip system coupled magnetic SPE with MALDI‐TOF MS was designed. A metal oxide affinity chromatography material, indium oxide, was coated on the surface of Fe₃O₄ magnetic nanoparticles to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β‐casein and BSA (1:1000) and the mixture of β‐casein, BSA, and ovalbumin (1:100:100). Thanking to the enrichment ability and specificity for phosphopeptides with the adsorbent, the on‐chip magnetic SPE‐MALDI‐TOF MS approach showed high sensitivity with a low detection limit of 4 fmol. In addition, the developed approach was used to analyze phosphopetides in non‐fat milk digests and human serum successfully.     

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.     

Enrichment of phosphopeptides by Fe3+-immobilized magnetic nanoparticles for phosphoproteome analysis of the plasma membrane of mouse liver

Immobilized metal ion affinity chromatography (IMAC) is a commonly used technique for phosphoprotein analysis due to its specific affinity for phosphopeptides. In this study, Fe3+-immobilized magnetic nanoparticles (Fe3+-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe3+-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein alpha-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following Fe3+-IMAN enrichment greatly increased relative to results obtained with direct MALDI-TOFMS analysis. The lowest detectable concentration is 5 x 10(-11) M for 100 microL of pure standard phosphopeptide (FLTEpYVATR) following Fe3+-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe3+-IMAN and also a combined approach of strong cation exchange chromatography and Fe3+-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis.     

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.     

Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature.

Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side-reaction products were observed; however, care was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip.