Zhen Sun

Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO2-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC-MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO2@single-MSA and nonuniform multi-layer PMSA brushes coated SiO2@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO2-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO2-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.

Comprehensive Mapping of Protein N‑Glycosylation in Human Liver by Combining Hydrophilic Interaction Chromatography and Hydrazide Chemistry

Although glycoproteomics is greatly developed in recent years, our knowledge about N-glycoproteome of human tissues is still very limited. In this study, we comprehensively mapped the N-glycosylation sites of human liver by combining click maltose-hydrophilic interaction chromatography (HILIC) and the improved hydrazide chemistry. The specificity could be as high as 90% for hydrazide chemistry and 80% for HILIC. Altogether, we identified 14,480 N-glycopeptides matched with N-!P-[S|T|C] sequence motif from human liver, corresponding to 2210 N-glycoproteins and 4783 N-glycosylation sites. These N-glycoproteins are widely involved into different types of biological processes, such as hepatic stellate cell activation and acute phase response of human liver, which all highly associate with the progression of liver diseases. Moreover, the exact N-glycosylation sites of some key-regulating proteins within different human liver physiological processes were also obtained, such as E-cadherin, transforming growth factor beta receptor and 29 members of G protein coupled receptors family.

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.

Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation

Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS(3) in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.

A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method

Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome.

In-depth research of multidrug resistance related cell surface glycoproteome in gastric cancer

UNLABELLED Human gastric cancer is a big public health problem. Multidrug resistance is a main obstacle to successful chemotherapeutic treatment in gastric cancers and the underlying mechanism is not clear. Glycosylation, one of the most important post translational modifications of proteins, plays a vital role in diverse aspects of tumor progression. In the present study, we applied two multidrug resistance cell lines and their parental drug sensitive gastric cancer cell line to a modified cell surface capturing strategy with triplex labeling to characterize MDR related cell surface glycoproteome. Finally, 56 cell membrane glycoproteins were successfully identified via combination of identification by glycopeptides and quantitation by non-glycopeptides, and 11 of them were found to be differentially expressed with the same trend in both drug resistant cell lines compared with that in sensitive cell line. The further analysis by western blot and in vitro drug sensitivity assay demonstrated that our approach is reliable and accurate and suggested that these glycoproteins may represent as biomarkers for multidrug resistance in gastric cancer. BIOLOGICAL SIGNIFICANCE In this study, we performed a cell surface glycoproteomics research of multidrug resistance in gastric cancer using a modified CSC approach. Totally we identified and quantified 11 membrane N-glycoproteins which were significantly changed in MDR gastric cancer cells. These glycoproteins are quite possible to be biomarkers for predicting MDR or key regulators for targeted therapy, and are also helpful for better interpreting the sophisticated mechanisms of MDR in gastric cancer. In addition to that, this approach used in this study can be well applied to screen aberrantly glycosylated biomarkers associated with other malignant phenotypes of various kinds of cancers.

Integration of Cell Lysis, Protein Extraction, and Digestion into One Step for Ultrafast Sample Preparation for Phosphoproteome Analysis

Conventional sample preparation protocols for phosphoproteome analysis require multiple time-consuming and labor-intensive steps, including cell lysis, protein extraction, protein digestion, and phosphopeptide enrichment. In this study, we found that the presence of a large amount of trypsin in the sample did not interfere with phosphopeptide enrichment and subsequent LC-MS/MS analysis. Taking advantage of fast digestion achieved with high trypsin-to-protein ratio, we developed a novel concurrent lysis-digestion method for phosphoproteome analysis. In this method, the harvested cells were first placed in a lysis buffer containing a huge amount of trypsin. After ultrasonication, the cells were lysed and the proteins were efficiently digested into peptides within one step. Thereafter, tryptic digest was subjected to phosphopeptide enrichment, in which unphosphorylated peptides, trypsin, and other components incompatible with LC-MS/MS analysis were removed. Compared with conventional methods, better phosphoproteome coverage was achieved in this new one-step method. Because protein solubilization and cell lysis were facilitated by fast protein digestion, the complete transformation of cell pellets into the peptide mixture could be finished within 25 min, while it would take at least 16 h for conventional methods. Hence, our method, which integrated cell lysis, protein extraction, and protein digestion into one step, is rapid and convenient. It is expected to have broad applications in phosphoproteomics analysis.

A new method for quantitative analysis of cell surface glycoproteome

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface‐capturing strategy where the glycopeptides were submitted to LC‐MS/MS analysis directly for identification of glycoproteins and the non‐glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.

Trypsin-Catalyzed N-Terminal Labeling of Peptides with Stable Isotope-Coded Affinity Tags for Proteome Analysis

An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography-tandem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis.

Large-Scale Proteome Quantification of Hepatocellular Carcinoma Tissues by a Three-Dimensional Liquid Chromatography Strategy Integrated with Sample Preparation

Hepatocellular carcinoma is one of the most fatal cancers worldwide. In this study, a reversed-phase-strong cation exchange-reversed-phase three-dimensional liquid chromatography strategy was established and coupled with mass spectrometry to investigate the differential proteome expression of HCC and normal liver tissues. In total, 2759 proteins were reliably quantified, of which, 648 proteins were dysregulated more than 3-fold in HCC liver tissues. Some important proteins that relate to HCC pathology were significantly dysregulated, such as NAT2 and AKR1B10. Furthermore, 2307 phosphorylation sites from 1264 phosphoproteins were obtained in our previous phosphoproteome quantification, and the nonphosphorylated counterparts of 445 phosphoproteins with 983 phosphorylation sites were reliably quantified in this work. It was observed that 337 (34%) phosphorylation sites exhibit significantly different expression trends from that of their corresponding nonphosphoproteins. Some novel phosphorylation sites with important biological functions in the progression of HCC were reliably quantified, such as the significant downregulation of pT185 for ERK2 and pY204 for ERK1.