Mingliang Ye

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

The study of protein glycosylation has lagged far behind the progress of current proteomics because of the enormous complexity, wide dynamic range distribution and low stoichiometric modification of glycoprotein. Solid phase extraction of tryptic N-glycopeptides by hydrazide chemistry is becoming a popular protocol for the analysis of N-glycoproteome. However, in silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. Herein, we describe a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion. The lysate of human liver tissue was digested with three proteases, that is, trypsin, pepsin and thermolysin, with different specificities, separately. Use of trypsin alone resulted in identification of 622 N-glycosites, while using pepsin and thermolysin resulted in identification of 317 additional N-glycosites. Among the 317 additional N-glycosites, 98 (30.9%) could not be identified by trypsin in theory because the corresponding in silico tryptic peptides are either too small or too big to detect in mass spectrometer. This study clearly demonstrated that the coverage of N-glycosites could be significantly increased due to the adoption of multiple enzyme digestion. A total number of 939 N-glycosites were identified confidently, covering 523 noredundant glycoproteins from human liver tissue, which leads to the establishment of the largest data set of glycoproteome from human liver up to now.

Systematic Analysis of Protein Phosphorylation Networks From Phosphoproteomic Data

In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human liver and experimentally identified 9719 p-sites in 2998 proteins. Using iGPS, we predicted a human liver protein phosphorylation networks containing 12,819 potential site-specific kinase-substrate relations among 350 PKs and 962 substrates for 2633 p-sites. Further statistical analysis and comparison revealed that 127 PKs significantly modify more or fewer p-sites in the liver protein phosphorylation networks against the whole human protein phosphorylation network. The largest data set of the human liver phosphoproteome together with computational analyses can be useful for further experimental consideration. This work contributes to the understanding of phosphorylation mechanisms at the systemic level, and provides a powerful methodology for the general analysis of in vivo post-translational modifications regulating sub-proteomes.

Separation of acidic compounds by strong anion-exchange capillary electrochromatography.

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240000 to 460000/m, and the relative standard deviation for t0 and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated.

A Fully Automated System with Online Sample Loading, Isotope Dimethyl Labeling and Multidimensional Separation for High-Throughput Quantitative Proteome Analysis

Multidimensional separation is often applied for large-scale qualitative and quantitative proteome analysis. A fully automated system with integration of a reversed phase-strong cation exchange (RP-SCX) biphasic trap column into vented sample injection system was developed to realize online sample loading, isotope dimethyl labeling and online multidimensional separation of the proteome samples. Comparing to conventionally manual isotope labeling and off-line fractionation technologies, this system is fully automated and time-saving, which is benefit for improving the quantification reproducibility and accuracy. As phosphate SCX monolith was integrated into the biphasic trap column, high sample injection flow rate and high-resolution stepwise fractionation could be easily achieved. Approximately 1000 proteins could be quantified in approximately 30 h proteome analysis, and the proteome coverage of quantitative analysis can be further greatly improved by prolong the multidimensional separation time. This system was applied to analyze the different protein expression level of HCC and normal human liver tissues. After three times replicated analysis, finally 94 up-regulated and 249 down-regulated (HCC/Normal) proteins were successfully obtained. These significantly regulated proteins are widely validated by both gene and proteins expression studies previously. Such as some enzymes involved in urea cycle, methylation cycle and fatty acids catabolism in liver were all observed down-regulated.

Improvement of the Quantification Accuracy and Throughput for Phosphoproteome Analysis by a Pseudo Triplex Stable Isotope Dimethyl Labeling Approach

Accurately quantifying the changes of phosphorylation level on specific sites is crucial to understand the role of protein phosphorylation in physiological and pathological processes. Here, a pseudo triplex stable isotope dimethyl labeling approach was developed to improve the accuracy and the throughput of comprehensive quantitative phosphoproteome analyses. In this strategy, two identical samples are labeled with light and heavy isotopes, respectively, while another comparative sample is labeled with an intermediate isotope. Two replicated quantification results were achieved in just one experiment, and the relative standard deviation (RSD) criterion was used to control the quantification accuracy. Compared with the conventional duplex labeling approach, the number of quantified phosphopeptides increased nearly 50% and the experimental time was reduced by 50% under the same quantification accuracy. Combined with the automated online reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional separation system, a comparative phosphoproteome analysis of hepatocellular carcinoma (HCC) and normal human liver tissues was performed. Over 1800 phosphopeptides corresponding to ~2000 phosphorylation sites were quantified reliably in a 42 h multidimensional analysis. The pro-directed motifs, which were mainly associated with the extracellular signal-regulated kinases (ERKs), were observed as being overrepresented in the regulated phosphorylation sites, and some quantification results of phosphorylation sites were validated by the other studies. Therefore, this pseudo triplex labeling approach was demonstrated as a promising alternative for the comprehensive quantitative phosphoproteome analysis.

STUDY OF PHYSICALLY ADSORBED STATIONARY PHASES FOR OPEN TUBULAR CAPILLARY ELECTROCHROMATOGRAPHY

A novel method based on the adsorption of positively charged compounds on the wall of a fused‐silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run‐to‐run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590 000/m and the average one above 250 000/m. Stored at 2—8°C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.

Effects of organic modifiers on retention mechanism and selectivity in micellar electrokinetic capillary chromatography studied by linear solvation energy relationships

The effects of six organic modifiers (urea, methanol, dioxane, tetrahydrofuran, acetonitrile and 2-propanol) on the retention mechanism and separation selectivity of the bulk buffer in micellar electrokinetic capillary chromatography (MECC) with sodium dodecyl sulfate (SDS) micelles as pseudo-stationary phase have been investigated through linear solvation energy relationships (LSERs). It is found that the retention value in MECC systems with or without organic modifier is primarily dependent on the solvophobic interaction and the hydrogen bonding interaction with the solute as proton acceptor, while the dipolar interaction and the hydrogen bonding interaction with the solute as proton donor play minor roles. The effects of the organic modifiers on the solvophobic, dipolar and hydrogen bonding interactions are evaluated in terms of the relationship between regression coefficient of the LSER equations and the modifier concentration. The variations of the solvophobic interaction and the dipolar interaction with change of the modifier concentration can be approximately explained using the solubility parameter and the dipolarity/polarizability parameter of the organic modifier, respectively. However, the relationships between the hydrogen bond acidity and basicity of the bulk buffer and the organic modifiers are rather complicated. Those results may be caused from the displacement of organic modifiers to the water adsorbed on the micellar surface as well as changes in the acidity and basicity of the bulk buffer with the addition of organic modifiers. In addition, it is found that the phase ratio is influenced significantly by the use of organic modifier.

Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach.

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.

Capture and Dimethyl Labeling of Glycopeptides on Hydrazide Beads for Quantitative Glycoproteomics Analysis

Incorporation of isotopic tag onto peptides via chemical labeling is a popular approach for quantitative proteomics. Chemical labeling via solution based methods usually lead to a tedious process and sample loss because several sample preparation steps including buffer exchange and desalting are performed. In this study, a solid phase based labeling approach by integration of glycopeptide enrichment and stable isotope labeling on hydrazide beads was developed for relative quantification of protein glycosylation, by which enrichment, washing, labeling, and release of the glycopeptides were all performed on the hydrazide beads sequentially. This approach was proved to be accurate in quantitative glycoproteome analysis and have good linearity range with 2 orders of magnitude for quantification of glycopeptides. Compared with dimethyl labeling conventionally performed in solution, the developed approach has better enrichment recovery (10-330% improvement) and high detection sensitivity in which 42% of annotated glycosites (vs 26%) still can be quantified using only 10 μg of four standard glycoprotein mixtures and 400 μg of bovine serum album interference as starting sample. The applicability of the approach for quantitative glycopeptide profiling was also explored by differential analysis of glycoproteome between human normal serum and liver cancer serum.