Mingqi Liu

pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3

Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification; 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides.

First proteomic exploration of protein-encoding genes on chromosome 1 in human liver, stomach, and colon.

The launch of the Chromosome-Centric Human Proteome Project provides an opportunity to gain insight into the human proteome. The Chinese Human Chromosome Proteome Consortium has initiated proteomic exploration of protein-encoding genes on human chromosomes 1, 8, and 20. Collaboration within the consortium has generated a comprehensive proteome data set using normal and carcinomatous tissues from human liver, stomach, and colon and 13 cell lines originating in these organs. We identified 12,101 proteins (59.8% coverage against Swiss-Prot human entries) with a protein false discovery rate of less than 1%. On chromosome 1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot entries, were identified; however, 805 proteins remain unidentified, suggesting that analysis of more diverse samples using more advanced proteomic technologies is required. Genes encoding the unidentified proteins were concentrated in seven blocks, located at p36, q12-21, and q42-44, partly consistent with correlation of these blocks with cancers of the liver, stomach, and colon. Combined transcriptome, proteome, and cofunctionality analyses confirmed 23 coexpression clusters containing 165 genes. Biological information, including chromosome structure, GC content, and protein coexpression pattern was analyzed using multilayered, circular visualization and tabular visualization. Details of data analysis and updates are available in the Chinese Chromosome-Centric Human Proteome Database ( http://proteomeview.hupo.org.cn/chromosome/ ).

Efficient and accurate glycopeptide identification pipeline for high-throughput site-specific N-glycosylation analysis.

Study of site-specific N-glycosylation in complex sample remains a huge analytical challenge because protein glycosylation is structurally diverse in post-translational modifications, resulting in an intricacy of N-glycopeptides. Here we have developed a novel approach for high-throughput N-glycopeptide profiling based on a network-centric algorithm for deciphering glycan fragmentation in mass spectrometry. We performed an extensive validation and a high-throughput N-glycosylation study on serum and identified thousands of N-glycopeptide spectra with high confidence. The results revealed a similar level of glycan microheterogeneity to that of conventional glycomics approach on individual proteins and provided the unique in-depth site-specific information that could only be studied through glycopeptide profiling.

Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease–substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high‐throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio‐informatic tools, several bio‐functions such as tumor cell migration and anti‐apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis.

N-glycosylation pattern of recombinant human CD82 (KAI1), a tumor-associated membrane protein

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn(157) for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.

Proteome atlas of human chromosome 8 and its multiple 8p deficiencies in tumorigenesis of the stomach, colon, and liver

Chromosome 8, a medium-length euchromatic unit in humans that has an extraordinarily high mutation rate, can be detected not only in evolution but also in multiple mutant diseases, such as tumorigenesis, and further invasion/metastasis. The Chromosome-Centric Human Proteome Project of China systematically profiles the proteomes of three digestive organs (i.e., stomach, colon, and liver) and their corresponding carcinoma tissues/cell lines according to a chromosome organizational roadmap. By rigorous standards, we have identified 271 (38.7%), 330 (47.1%), and 325 (46.4%) of 701 chromosome 8-coded proteins from stomach, colon, and liver samples, respectively, in Swiss-Prot and observed a total coverage rate of up to 58.9% by 413 identified proteins. Using large-scale label-free proteome quantitation, we also found some 8p deficiencies, such as the presence of 8p21-p23 in tumorigenesis of the above-described digestive organs, which is in good agreement with previous reports. To our best knowledge, this is the first study to have verified these 8p deficiencies at the proteome level, complementing genome and transcriptome data.

pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification

The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15N/13C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

Hydrazide-functionalized magnetic microspheres for the selective enrichment of digested tryptophan-containing peptides in serum

The extreme complexity of protein samples is becoming a great challenge for proteomic analysis, especially for those having large dynamic range of protein abundance. To solve this problem, and to overcome the limitation of the current proteomic technologies, a new method using hydrazide-functionalized magnetic microspheres was established in this study. With this method, tryptophan (Trp)-containing peptides can be selectively and sensitively enriched from complex and low-volume samples. Furthermore, combined with 1D-LC-MS/MS analysis, the strategy was successfully applied to the proteomic study of mouse serum. The proportion of Trp-containing peptides was increased from 19.4% to 80.2% through enrichment, and the complexity of the sample was reduced more than two times. An additional 113 Trp-containing peptides and 48 novel proteins were detected compared to the conventional method. This enrichment method provides a means for identifying more proteins as potential biomarkers in serum and other complex samples.

Site-specific structural characterization of O-glycosylation and identification of phosphorylation sites of recombinant osteopontin.

Osteopontin (OPN) plays a key role in multiple physiological and pathological processes such as cytokine production, mineralization, inflammation, immune responses, and tumorigenesis. Post-translational modifications (PTMs) of OPN significantly affect its structure and biological properties; however, site-specific characterization of O-glycosylation in human OPN has not been reported. In this work, we profiled the overall glycan pattern of human recombinant OPN using a lectin array and completed detailed structural analysis of O-glycopeptides by mass spectrometry (MS). We detected 28 O-glycopeptides from 7 O-glycosylation regions of human OPN, occupied by highly heterogeneous O-glycans. These O-glycans carried, in part, functionally relevant epitopes such as T antigens (Galβ1-3GalNAcα1-), sialyl-Tn antigens, sialyl-T antigens, and sialyl-Le(x/a) antigens [Neuα2-3Galβ1-4(Fucα1-3)GlcNAc/Neuα2-3Galβ1-3(Fucα1-4)GlcNAc]. MS(3) spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. Furthermore, 26 phosphorylation sites were identified by reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), including a novel one (Y209). We provide a detailed, site-specific structural characterization of O-glycosylation and identify the phosphorylation sites of OPN. These data lay the foundation for further research into the role of oligosaccharides and phosphorylation of recombinant human OPN. This article is part of a Special Issue entitled: Medical Proteomics.

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.