Liangliang Sun

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 Device for Online Sample Buffer Exchange, Protein Enrichment, and Digestion

An integrated sample treatment device, composed of a membrane interface and a monolithic hybrid silica based immobilized enzymatic reactor (IMER), was developed for the simultaneous sample buffer exchange, protein enrichment, and online digestion, by which for the sample buffer, the acetonitrile content was reduced to approximately 1/10 of the initial one, and the pH value was adjusted from approximately 3.0 to approximately 8.0, compatible for online trypsin digestion. Furthermore, the signal intensity of myoglobin digests was improved by over 10 times. Such an integrated device was successfully applied to the online treatment of three protein eluates obtained by reverse-phase liquid chromatography (RPLC) separation, followed by further protein digest analysis with microreverse-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (microRPLC-ESI-MS/MS). The experimental results showed that the performance of such an integrated sample treatment device was comparable to that of the traditional offline sample treatment method, including lyophilization and in-solution digestion. However, the consumed time was reduced to 1/192. All these results demonstrate that such an integrated sample treatment device could be further online coupled with protein separation, peptide separation, and identification, to achieve high-throughput proteome analysis.

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.

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K=2.65×10(-13) m(2)). With BSA as the model protein, efficient digestion was completed within 20s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.

Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling

A facile integrated platform for proteome profiling was established, in which native proteins were online denatured and reduced within a heater, digested with an immobilized trypsin microreactor, and analyzed by microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (microRPLC-ESI-MS/MS). In comparison to the traditional off-line urea denaturation protocol, even more unique peptides were obtained by online heating in triplicate (14 +/- 2 vs 11 +/- 2 for myoglobin and 16 vs 12 +/- 1 for BSA) within a significantly shortened pretreatment time of approximately 3.5 min (including 1 min of thermal denaturation and reduction and approximately 2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (approximately 6 fmol) to 1 mg/mL (approximately 120 pmol) were positively identified by the online system. Such a platform was further successfully applied for analyzing the soluble fraction of mouse liver extract. Of all the 367 proteins identified from samples pretreated by the urea protocol and online heating, approximately 40% were overlapped, showing the partial complementation of both approaches. All these results demonstrate that the online integrated platform is of great promise for high-throughput proteome profiling and improved identification capacity for low-abundance proteins with a minute sample amount.

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.

Octyl-functionalized hybrid magnetic mesoporous microspheres for enrichment of low-concentration peptides prior to direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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.

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.

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ∼200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.

A facile microdialysis interface for on-line desalting and identification of proteins by nano-electrospray ionization mass spectrometry.

The adverse effect of salts, especially inorganic salts, on electrospray ionization mass spectrometry (ESI-MS) is one of the most serious obstacles that might limit its application. Among the numerous desalting approaches, the microdialysis technique is favorable for large molecules, such as proteins. In this work, employing a hollow fiber membrane of cellulose acetate (MWCO 3000 Da), a simple, facile and efficient microdialysis interface with the dead volume of less than 1 microL was constructed for the on-line desalting and identification of proteins dissolved in high salt concentration buffer by nano-ESI-MS. Furthermore, with counterflow added, the desalting procedure was accelerated, and could be finished within 1 min. This system was successfully applied to the analysis of myoglobin dissolved in either high concentration ammonium acetate or sodium chloride buffer. The experimental results showed that, by using such a microdialysis interface, the salt concentration, even as high as 1 M, could be decreased by at least 2 orders of magnitude, while sample loss was less than 10%, demonstrating the potential of such an interface in broadening the application of nano-ESI-MS in the analysis of large molecules.