[Show abstract][Hide abstract] ABSTRACT: Polymer self-assembly was developed as an epitope imprinting strategy involving facile processes and high recognition site density. As a model, transferrin epitope imprinted polyethersulfone (PES) beads were successfully fabricated using this technique. The imprinted beads demonstrated excellent selectivity toward the transferrin epitope and transferrin even in the real sample.
Chemical Communications 07/2014; 50(67). DOI:10.1039/c4cc03428g · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As low abundance is the great obstacle for glycoprotein analysis, the development of materials with high efficiency and selectivity for glycoprotein enrichment is a prerequisite in glycoproteome research. Herein, we report a new kind of hydrophilic boronate affinity monolith by attaching 4-mercaptophenylboronic acid (MPBA) with 2-mercaptoethylamine (MPA) on the gold nanoparticle-modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate)) monolith for glycoprotein enrichment. With poly(ethylene glycol) diacrylate as the cross-linker and the further modification of gold nanoparticles, the matrix has advantages of good hydrophilicity and enhanced surface area, which are beneficial to improve the enrichment selectivity and efficiency for glycoproteins. The attachment of MPBA and MPA provide intramolecular BN coordination, which could further enhance the specificity of glycoprotein capture. Such a boronate affinity monolith was applied to enrich horseradish peroxidase (HRP) from the mixture of HRP and bovine serum albumin (BSA), and high selectivity was obtained even at a mass ratio of 1:1000. In addition, the binding capacity of ovalbumin on such monolith reached 390 μg g(-1) . Furthermore, the average recovery of HRP on the prepared affinity monoliths was (84.8±1.9) %, obtained in three times enrichment with the same column. Finally, the boronate affinity monolith was successfully applied for the human-plasma glycoproteome analysis. As a result, 160 glycoproteins were credibly identified from 9 μg of human plasma, demonstrating the great potential of such a monolith for large-scale glycoproteome research.
[Show abstract][Hide abstract] ABSTRACT: Due to the extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenges. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference on MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs. Compared with other commonly used additives, such as SDS, Rapigest and methanol, C12Im-Cl showed the best performance. In addition, by a strong cation exchange trap column, it could be easily removed after trypsin digestion, not only beneficial to avoid protein precipitation during digestion, but also had no adverse effect on LC-MS based separation and detection. Such a C12Im-Cl-assisted sample preparation method was further applied to the membrane proteome analysis of rat brain. Compared with SDS-assisted method, 1.4 and 3.5 times improvement on the identified IMP and hydrophobic peptide number was achieved (251 vs 178, and 982 vs 279). All these results demonstrated that C12Im-Cl-assisted sample preparation method is of great promise to promote the large-scale membrane proteome profiling.
[Show abstract][Hide abstract] ABSTRACT: Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic micro-reactor was established to pretreat membrane proteins at microgram, and even nanogram level. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein pre-concentration, pH adjustment, reduction and alkylation, as well as tryptic digestion were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed micro-reactor, a mixture of bovine serum albumin, myoglobin and cytochrome c was pretreated. Compared with the results obtained by traditional in-solution process, the peptide recovery (93% vs. 83%) and analysis throughput (3.5 h vs. 14 h) were obviously improved. The micro-reactor was further applied for the pretreatment of 14 μg membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nano-liquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by traditional in-solution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic micro-reactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.
[Show abstract][Hide abstract] ABSTRACT: The exceptional growth rate of velvet antler makes it a valuable model for studying the development of tissues, such as blood vessels, cartilage and bone. Meanwhile, investigating the activities of extracted proteins from velvet antlers promisingly leads to the discovery of new active factors which regulate the development of above-mentioned tissue types. In this study, a novel sequential protein extraction method was developed for proteome profiling and bioactivity study of velvet antlers. Herein, four antler growing tips were pooled to create a proportional pooled sample, and three aliquots of which were extracted in parallel using the developed extraction method. For each sample, proteins were extracted sequentially by saline solvent (0.15M sodium chloride, pH 7.0), mild acid buffer (0.15M acetate buffer, pH 4.0) and mild alkaline buffer (0.15M glycine-sodium hydroxide buffer, pH 10.0) with good bio-compatibility to prevent proteins denaturation. Then STD lysis buffer, containing 4% SDS, 0.1M Tris-HCl and 0.1M DTT, was used to extract hydrophobic proteins. The tryptic digest of each fraction was analyzed by nanoRPLC-ESI-MS/MS in triplicates, with false discovery rate for peptide identification adjusted to 1% to create filtered protein group list. In total, 1423 protein groups were identified, which expanded up to 3 times of the previous published dataset. The relative standard deviation of identified peptide and protein group number for all analyses indicated the good reproducibility of the developed sequential protein extraction method. Additionally, proteins extracted by acid buffer and alkaline buffer showed obvious promoting effect on the proliferation of human umbilical vein endothelial cells. All these results demonstrate that the developed sequential extraction method is efficient for the comprehensive proteome analysis and activity investigation of velvet antlers.
[Show abstract][Hide abstract] ABSTRACT: Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS-PAGE free protocol. HLMs were solubilized with 5% (v/v) ionic liquid, 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIM BF(4) ), followed by tryptic digestion, and 2D-SCX-RPLC-ESI-MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99% and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.
[Show abstract][Hide abstract] ABSTRACT: In this paper, magnetic Fe₃O₄ nanoparticles modified graphene oxide nanocomposites (GO-CO-NH-Fe₃O₄) were prepared by covalent bonding, via the reaction between the amino groups of fuctionalized Fe₃O₄ and the carboxylic groups of GO, confirmed by Fourier-transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. With GO-CO-NH-Fe₃O₄ as a novel substrate, trypsin was immobilized via π-π stacking and hydrogen bonding interaction, and the binding capacity of trypsin reached as high as 0.275 mg/mg. Since GO-CO-NH-Fe₃O₄ worked as not only support for enzyme immobilization, but also as an excellent microwave irradiation absorber, the digestion efficiency could be further improved with microwave assistance. By such an immobilized enzymatic reactor (IMER), standard proteins could be efficiently digested within 15 s, with sequence coverages comparable or better than those obtained by conventional in-solution digestion (12 h). Since trypsin was immobilized under mild conditions, the enzymatic activity of IMER preserved at least for a month. In addition, due to the good hydrophilicity of GO, no peptide residue was observed in the sequent digestion of bovine serum albumin and myoglobin. To further confirm the efficiency of such an IMER for proteome analysis, it was applied to digest proteins extracted from rat liver, followed by nanoRPLC-ESI-MS/MS analysis. With only 5 min microwave-assisted digestion, in 3 parallel runs, totally 456 protein groups were identified, comparable to that obtained by 12 h in-solution digestion, indicating the great potential of IMERs with GO-CO-NH-Fe₃O₄ as the support for high throughput proteome study.
Journal of Chromatography A 07/2012; 1254:8-13. DOI:10.1016/j.chroma.2012.07.030 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new type of IMAC material, with ATP as the chelating ligand, was synthesized and applied to capture phosphopeptides. For the first time, the approach for phosphopeptide enrichment could provide selectivity under 5000-fold dilution by nonphosphopeptides, and sensitivity of on-target enrichment at 3 amol.
Chemical Communications 05/2012; 48(50):6274-6. DOI:10.1039/c2cc31641b · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies. Herein, we implemented two formats of MS/MS fragment ion intensity, Spectral Index (SI) and Summed MS/MS TIC (SMT), to alleviate this particular deficiency arising from SC. Both were in forms of replacing SC in the Normalized Spectral Abundance Factor (NSAF) formula, resulting in two algorithms, abbreviated as NSI and NSMT, respectively. The necessity of the normalization process was validated using a publicly available dataset. Furthermore, when applied to another well characterized benchmark dataset, both NSI and NSMT showed improved overall accuracy over NSAF for the relative quantification of proteomes. Hereinto, NSI enabled the sensitive detection of differentially expressed proteins, while NSMT ensured accurate calculation for protein abundance fold change. Therefore, the selective use of both algorithms might facilitate the screening and quantification of potential biomarkers on the proteome scale.
The Analyst 05/2012; 137(13):3146-53. DOI:10.1039/c2an35173k · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix. With NVP modification, the hydrophilicity of IMER was greatly improved, resulting in not only the sequence coverage of myoglobin increased from 63% to 73%, but also no residual peptides from BSA observed in myoglobin digests. Although the sequence coverages of proteins obtained by the IMER were comparable to those obtained by in-solution digestion, the digestion time was shortened from 24h to 1 min. By such an IMER, a protein mixture, containing BSA, myoglobin, and cytochrome c (100, 1 and 0.01 μg/mL, respectively), was digested, and all proteins were unambiguously identified with improved sequence coverages than that achieved by in-solution digestion. Furthermore, the hydrophilic IMER was also off-line coupled to nano-RPLC-ESI-MS/MS for the analysis of proteins extracted from yeast. After 1.5 min digestion, 271 protein groups with at least 2 distinct peptides were identified, much more than those obtained by 24h in-solution digestion (192 protein groups), indicating the great potential of such an IMER for proteome analysis.
Journal of Chromatography A 03/2012; 1246:111-6. DOI:10.1016/j.chroma.2012.03.014 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Analysis of integral membrane proteins (IMPs) presents great challenges due to their hydrophobic nature. Recently, much attention has been paid to improve the solubilization of IMPs. However, besides that, the separation of hydrophobic peptides with high recovery is also a dominating factor, but with rare report. Here, the prefractionation of the digests by reverse phase trap column during desalting was presented to efficiently decrease the complexity of samples, with the identified hydrophobic peptides and IMPs increased by more than 43%. Furthermore, the effect of C18 and C8 stationary phases on the separation of membrane protein digests was studied. A total of 301 proteins (536 peptides) with C18 stationary phase and 398 proteins (703 peptides) with C8 stationary phase were identified by μRPLC-ESI-MS/MS using an LCQ instrument in duplicate runs, with false discovery rate (FDR) less than 5% at peptide level. In addition, with C8 stationary phase, the number of identified hydrophobic peptides and IMPs was obviously improved by 29% and 20%, respectively, compared with that identified by C18 stationary phase, indicating that the polarity of stationary phase has evident effect on the analysis of membrane protein digests. All these results show that the prefractionation by reverse phase trap column during desalting and the separation by C8 stationary phases could facilitate the efficient identification of IMPs.
[Show abstract][Hide abstract] ABSTRACT: Octyl-functionalized hybrid magnetic mesoporous (Fe(3)O(4)·nSiO(2)·meso-hybrid-C8) microspheres were synthesized and applied in the isolation and pre-concentration of low-concentration peptides prior to direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Such microspheres possess high surface area (324 m(2)/g), hydrophobic group (C8), relatively large pore volume (0.304 cm(3)/g), uniform pore diameter (~3.7 nm), and magnetic responsivity, which make them a simple and efficient kind of adsorbent for the enrichment of low-concentration peptides. For bovine serum albumin (BSA, 15 fmol μL(-1)) digest, after concentration by Fe(3)O(4)·nSiO(2)·meso-hybrid-C8 microspheres, the enrichment performance was evidently better than those obtained by solvent evaporation and C8-functionalized magnetic particles, and comparable to those obtained by commercial Anchor chip target and ZipTipC18 pipette tip. Such microspheres were further applied in the enrichment of the tryptic digests of rat cerebellum proteins and endogenous peptides of crude human serum, and more peaks with higher signal-to-noise (S/N) ratio were obtained than before pre-concentration. Furthermore, the pre-concentration reproducibility of magnetic microspheres for biological samples was good, and the limit of detection (LOD) for BSA digests by MALDI-TOF MS was decreased by at least one order of magnitude compared with that obtained without pre-concentration. All the above-mentioned results indicate that the synthesized Fe(3)O(4)·nSiO(2)·meso-hybrid-C8 microspheres are promising for the enrichment of low-concentration peptides from complex biosamples.
Rapid Communications in Mass Spectrometry 05/2011; 25(9):1257-65. DOI:10.1002/rcm.4986 · 2.64 Impact Factor