Guijie Zhu

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.

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.

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M-IPG), on-line digestion by trypsin-based immobilized enzyme microreactor (trypsin-IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M-IPG CIEF column and trypsin-IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20  mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M-IPG CIEF, followed by on-line digestion by trypsin-IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE-based on-line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.

Preparation and application of monolithic columns with narrow immobilized pH gradients.

Monolith with immobilized pH gradient (M-IPG) is a novel separation matrix for amphoteric substances, such as peptides and proteins. To improve the properties of the newly designed column, efforts were made to optimize the preparation procedure, including the concentrations of the monomers, glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), as well as the kinds of porogens, which led to a monolith with improved permeability, uniformity and continuity. In addition, different diamines, including ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane and 1,5-diaminopentane, were subjected to aminate the polymer, and the last two exhibited excellent reactivity with epoxy on the polymer surface, which could obviously make the immobilization of pH gradient facilitated. Under the optimal conditions, a simple method to prepare M-IPG column with narrow pH gradient was developed with commercial carrier ampholytes (CAs) solution. Such columns were applied into the analysis of proteins and peptides, and showed the improved resolution compared to the traditional one with a wide pH distribution.

Microchip free flow isoelectric focusing for protein prefractionation using monolith with immobilized pH gradient

Microchip free flow IEF (muFFIEF) with monolithic IPG was proposed for protein prefractionation. The monolithic materials were first prepared by UV irradiation in a microchamber of 43 mm length, 23 mm width, and 38 mum depth. Carrier ampholytes (CAs) were further immobilized on the monolith by chemical bonding, to form a stable pH gradient. By such a technique, the continuous introduction of CAs in traditional muFFIEF could be avoided, not only to decrease the operation cost, but also to avoid the interference of CAs for the further protein identification by MS/MS. With a fluorescence microscope as the detector, under the optimal conditions, the separation of FITC labeled beta-lactoglobulin and carbonic anhydrase, with 0.9 unit difference on pI, was achieved with good reproducibility. The experimental results demonstrated that under the experimental conditions we applied, the separation mechanism of muFFIEF with M-IPG materials might be the cooperative effects of IEF and CZE, and the former one plays a predominant role.

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.

Study of multiple binding constants of dexamethasone with human serum albumin by capillary electrophoresis–frontal analysis and multivariate regression

AbstractStudies into the interactions between drugs and human serum albumin (HSA) are extremely important for drug discovery, since HSA behaves as a carrier for external drugs and internal biological molecules. In this paper, to evaluate the pharmacokinetic and pharmacodynamic properties of dexamethasone (DXM), the interaction between DXM and HSA was studied by capillary electrophoresis–frontal analysis (CE-FA). According to the Klotz equation, four binding sites between DXM and HSA were obtained, and the average binding constant was 1.05 × 103 M−1. Furthermore, according to multiple equilibrium theory, based on the assumption that there are two types of binding site, the binding constant at one site was calculated to be 3.539 × 103 M−1, and the average of the other three was 1.234 × 103 M−1. In addition, to obtain the detailed binding information at each binding site, new equations were deduced by multivariate regression. The four binding constants of DXM and HSA were calculated to be 5.558 × 101 M−1, 2.158 × 104 M−1, 7.312 × 103 M−1 and 2.043 × 103 M−1, respectively, which is helpful for detailed studies into the interactions between drugs and proteins with multiple binding sites. FigureElectropherograms of DXM sodium phosphate and HAS mixtures for different protein to drug concentration ratios, obtained by CE-FA

Single Chain Variable Fragment Displaying M13 Phage Library Functionalized Magnetic Microsphere-Based Protein Equalizer for Human Serum Protein Analysis

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

Fast preparation of monolithic immobilized pH gradient column by photopolymerization and photografting techniques for isoelectric focusing separation of proteins

A new method was developed to prepare monolithic immobilized pH gradient (M‐IPG) columns in UV‐transparent fused‐silica capillaries by the 5‐min photopolymerization of acrylamide and N,N′‐methylenebisacrylamide, followed by the 20‐min photografting of the focused ampholine‐derived glycidylmethacrylate monomer on the monolithic matrix, by which the preparation time was reduced, and the stability of the formed pH gradient was improved, compared with our previous methods. Using the prepared M‐IPG column, the baseline separation of proteins was achieved according to their pIs. Without carrier ampholytes added in the running buffer, the separated components could be detected with high sensitivity by UV at low wavelength.