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.

[1]  André M Deelder,et al.  Glycoproteomics based on tandem mass spectrometry of glycopeptides. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[2]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[3]  J. Fujimoto,et al.  Expression of hepatocyte growth factor and its receptor c‐met proto‐oncogene in hepatocellular carcinoma , 1997, Hepatology.

[4]  Yuan Tian,et al.  Solid-phase extraction of N-linked glycopeptides , 2007, Nature Protocols.

[5]  M. Mann,et al.  Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues*S , 2004, Molecular & Cellular Proteomics.

[6]  P. Højrup,et al.  Characterization of Gel-separated Glycoproteins Using Two-step Proteolytic Digestion Combined with Sequential Microcolumns and Mass Spectrometry* , 2005, Molecular & Cellular Proteomics.

[7]  Ruedi Aebersold,et al.  High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry *S , 2005, Molecular & Cellular Proteomics.

[8]  R Apweiler,et al.  On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. , 1999, Biochimica et biophysica acta.

[9]  R. Montesano,et al.  Molecular epidemiology of human liver cancer: insights into etiology, pathogenesis and prevention from The Gambia, West Africa. , 2006, Carcinogenesis.

[10]  G. Glinsky.  Antigen presentation, aberrant glycosylation and tumor progression. , 1994, Critical reviews in oncology/hematology.

[11]  V. Havlíček,et al.  Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin‐resistant boar seminal plasma O‐glycoprotein related to pB1 protein , 1999, Protein science : a publication of the Protein Society.

[12]  John I. Clark,et al.  Shotgun identification of protein modifications from protein complexes and lens tissue , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  L. Deterding,et al.  Characterization of glycopeptides from HIV-ISF2 gp120 by liquid chromatography mass spectrometry , 2004, Journal of the American Society for Mass Spectrometry.

[14]  Fuchu He,et al.  Human Liver Proteome Project , 2005, Molecular & Cellular Proteomics.

[15]  D. Rudnick,et al.  CONCISE REVIEW IN MECHANISMS OF DISEASE Alpha-1-Antitrypsin Deficiency: A New Paradigm for Hepatocellular Carcinoma in Genetic Liver Disease , 2005 .

[16]  Alexey I Nesvizhskii,et al.  Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas , 2006, Genome Biology.

[17]  R. Aebersold,et al.  Mass Spectrometric Detection of Tissue Proteins in Plasma*S , 2007, Molecular & Cellular Proteomics.

[18]  William S Hancock,et al.  Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR). , 2005, Journal of proteome research.

[19]  Anne M. Wang,et al.  Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy. , 1990, The Journal of clinical investigation.

[20]  Eric D. Dodds,et al.  Site determination of protein glycosylation based on digestion with immobilized nonspecific proteases and Fourier transform ion cyclotron resonance mass spectrometry. , 2007, Journal of proteome research.

[21]  M. Tropak,et al.  Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase. , 1989, European journal of biochemistry.

[22]  Joseph A Loo,et al.  Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry. , 2006, Journal of proteome research.

[23]  J. Yates,et al.  Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry. , 2000, Analytical chemistry.

[24]  A. J. Parodi,et al.  Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. , 2000, The Biochemical journal.

[25]  A. Varki,et al.  Biological roles of oligosaccharides: all of the theories are correct , 1993, Glycobiology.

[26]  R. Aebersold,et al.  Mass spectrometry-based proteomics , 2003, Nature.

[27]  Ruedi Aebersold,et al.  Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry , 2003, Nature Biotechnology.

[28]  Ronald J Moore,et al.  Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. , 2005, Journal of proteome research.

[29]  M. Bronner‐Fraser,et al.  N-Cadherin, a cell adhesion molecule involved in establishment of embryonic left-right asymmetry. , 2000, Science.

[30]  T. Endo,et al.  Mutations of the POMT1 gene found in patients with Walker-Warburg syndrome lead to a defect of protein O-mannosylation. , 2004, Biochemical and biophysical research communications.

[31]  R. Cortese,et al.  The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus , 2002, The EMBO journal.

[32]  R. Aebersold,et al.  Mass spectrometry in proteomics. , 2001, Chemical reviews.

[33]  Xinning Jiang,et al.  Large‐scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography , 2008, Proteomics.

[34]  C. Duve,et al.  Digestive activity of lysosomes. II. The digestion of macromolecular carbohydrates by extracts of rat liver lysosomes. , 1968, The Journal of biological chemistry.

[35]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[36]  Jaroslav Koca,et al.  Structures and mechanisms of glycosyltransferases. , 2006, Glycobiology.

[37]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[38]  Xiaogang Jiang,et al.  Capillary trap column with strong cation-exchange monolith for automated shotgun proteome analysis. , 2007, Analytical chemistry.

[39]  R. Siciliano,et al.  Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry. , 2005, Journal of mass spectrometry : JMS.

[40]  G. Lauc,et al.  Glycoproteomics: protein modifications for versatile functions. Meeting on glycoproteomics. , 2005, EMBO reports.

[41]  Y. Ikeda,et al.  Implication of N-acetylglucosaminyltransferases III and V in cancer: gene regulation and signaling mechanism. , 1999, Biochimica et biophysica acta.

[42]  Guanghui Han,et al.  Optimization of filtering criterion for SEQUEST database searching to improve proteome coverage in shotgun proteomics , 2007, BMC Bioinformatics.

[43]  Gordan Lauc,et al.  Glycoproteomics: protein modifications for versatile functions , 2005 .