Dingyin Tao

Organic−Inorganic Hybrid Silica Monolith Based Immobilized Trypsin Reactor with High Enzymatic Activity

A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (microRPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by in-solution digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.

Organic-Inorganic Hybrid Silica Monolith Based Immobilized Titanium Ion Affinity Chromatography Column for Analysis of Mitochondrial Phosphoproteome

A novel kind of immobilized metal affinity chromatography (IMAC) column based on organic-inorganic hybrid silica monolith has been developed. The monolithic support was prepared in a 250 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, amine groups were functionalized by glutaraldehyde, and then activated with (aminomethyl) phosphonic acid, followed by Ti(4+) chelation. By such a hybrid silica monolithic Ti(4+)-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of alpha-casein and BSA with the molar ratio as low as 1:200, illustrating its superior selectivity. With a synthetic phosphorylated peptide, YKVPQLEIVPNSpAEER, as the sample, the loading capacity and recovery of the Ti(4+)-IMAC monolithic column were measured to be 1.4 micromol/mL and 69%, respectively. Such an IMAC monolithic column was further applied to enrich phosphopeptides from rat liver mitochondria. In total, 224 unique phosphopeptides, corresponding to 148 phosphoprotein groups, were identified by duplicate nanoRPLC-LTQ MS/MS/MS runs with a false-positive rate of less than 1% at the peptide level. These results demonstrate that the hybrid silica monolith based Ti(4+)-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.

Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12s to 71 s. With μRPLC-ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12 h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.

Integrated Platform for Proteome Analysis with Combination of Protein and Peptide Separation via Online Digestion

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by a microcolumn packed with mixed weak anion and weak cation exchange (WAX/WCX) particles under a series of salt steps, online digested by a trypsin immobilized microenzymatic reactor (IMER), trapped and desalted by two parallel C8 precolumns, separated by microreversed-phase liquid chromatography (muRPLC) under a linear gradient of organic modifier concentration, and finally identified by electrospray ionization-MS/MS (ESI-MS/MS). To evaluate the performance of such a platform, a mixture of myoglobin, cytochrome c, bovine serum albumin (BSA), and alpha-casein, with mass ranging from 25 ng to 2 microg, was analyzed. Compared to the methods by off-line protein fractionation and shotgun based strategy, the analysis time, including sample preparation, digestion, desalting, separation, and detection, was shortened from ca. 30 to 5 h, and cytochrome c with abundance of 25 ng could be identified with improved sequence coverage. Furthermore, such an integrated platform was successfully applied into the analysis of proteins extracted from human lung cancer cells. Compared with the results obtained by the shotgun approach, the identified protein number was increased by 30%. All these results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.

Coupling Formic Acid Assisted Solubilization and Online Immobilized Pepsin Digestion with Strong Cation Exchange and Microflow Reversed-Phase Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for Integral Membrane Proteome Analysis

In this study, a facile system for membrane proteome profiling was established, in which membrane proteins were solubilized by formic acid, online digested by a pepsin-based immobilized enzyme reactor (pepsin-IMER), and analyzed by strong cation exchange and microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (SCX-μRPLC-ESI-MS/MS). Under optimized conditions, such a system showed excellent compatibility between all crucial steps and was successfully applied for analyzing integral membrane proteins extracted from rat liver microsomes. Out of the 235 unique proteins positively identified, 39% (91/235) were annotated as membrane proteins with one or more transmembrane domains (TMDs). It is anticipated that the efficient sample treatment and the relevant online analytical system might provide a promising tool for automated and comprehensive profiling of membrane proteomes.

Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research

High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS) is regarded as one of the most powerful techniques for separation and identification of proteins. Recently, much effort has been made to improve the separation capacity, detection sensitivity, and analysis throughput of micro- and nano-HPLC, by increasing column length, reducing column internal diameter, and using integrated techniques. Development of HPLC columns has also been rapid, as a result of the use of submicrometer packing materials and monolithic columns. All these innovations result in clearly improved performance of micro- and nano-HPLC for proteome research.

Miniaturized two-dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.

Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum

Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g−1. And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

Large-scale N-glycoproteome map of rat brain tissue: Simultaneous characterization of insoluble and soluble protein fractions

The large‐scale N‐glycosylation analysis is critical for biomedical research, since a variety of diseases are found to be associated with glycoproteins. By a combination of glycoprotein analysis in insoluble protein fraction solubilized with 1% v/v 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMIM BF4) and those in soluble fraction, a total number of 462 non‐redundant N‐glycoprotein groups, including 316 transmembrane glycoproteins, were successfully identified. Correspondingly, 849 unique N‐glycosites were confidently recognized. The data set could provide a support for the further in‐depth research of brain N‐glycosylation, such as for the discovery of candidate drug targets and biomarkers.

Serially coupled microcolumn reversed phase liquid chromatography for shotgun proteomic analysis

Microcolumn RPLC (μRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of μRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by μRPLC‐ESI MS/MS, and similar distributions of Mw and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of μRPLC in shotgun proteomic analysis.