Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment*

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive “glycosylations” were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

[1]  M. Dunn.  PROTEOMICS: Ten years in the field , 2010, Proteomics.

[2]  Melanie Y. White,et al.  Modifications of myosin-regulatory light chain correlate with function of stunned myocardium. , 2003, Journal of molecular and cellular cardiology.

[3]  Xinning Jiang,et al.  Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry. , 2009, Journal of proteome research.

[4]  P. Højrup,et al.  Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics. , 2010, Analytical chemistry.

[5]  Steven P Jones,et al.  Cardioprotection by N-Acetylglucosamine Linkage to Cellular Proteins , 2008, Circulation.

[6]  Nichollas E. Scott,et al.  Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni* , 2010, Molecular & Cellular Proteomics.

[7]  H. Wright.  Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins. , 1991, Protein engineering.

[8]  M. Fukayama,et al.  Periostin is essential for cardiac healing after acute myocardial infarction , 2008 .

[9]  H. Chapman,et al.  Mast Cell Cathepsins C and S Control Levels of Carboxypeptidase A and the Chymase, Mouse Mast Cell Protease 5 , 2003, Biological chemistry.

[10]  Paul A Haynes,et al.  Liver Membrane Proteome Glycosylation Changes in Mice Bearing an Extra-hepatic Tumor , 2010, Molecular & Cellular Proteomics.

[11]  M. Lindsey,et al.  Extracellular matrix turnover and signaling during cardiac remodeling following MI: causes and consequences. , 2010, Journal of molecular and cellular cardiology.

[12]  Jakob Bunkenborg,et al.  A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. , 2004, Journal of proteome research.

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

[14]  S. Mohammed,et al.  Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy. , 2007, Journal of proteome research.

[15]  M. Zile,et al.  Myocardial Infarct Expansion and Matrix Metalloproteinase Inhibition , 2003, Circulation.

[16]  P. Carmeliet,et al.  Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction , 2009, The Journal of experimental medicine.

[17]  S. Heymans,et al.  Cardiac extracellular matrix remodeling: fibrillar collagens and Secreted Protein Acidic and Rich in Cysteine (SPARC). , 2010, Journal of molecular and cellular cardiology.

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

[19]  J. Stasch,et al.  Increased levels of laminin and collagen type VI may reflect early remodelling in patients with acute myocardial infarction , 2009, Acta cardiologica.

[20]  N. Packer,et al.  Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture. , 2009, Journal of proteome research.

[21]  J. Marth,et al.  Dietary and Genetic Control of Glucose Transporter 2 Glycosylation Promotes Insulin Secretion in Suppressing Diabetes , 2005, Cell.

[22]  Rebekah L. Gundry,et al.  The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome , 2009, Molecular & Cellular Proteomics.

[23]  A. Helenius,et al.  Intracellular functions of N-linked glycans. , 2001, Science.

[24]  S. Shaw.  Molecular imaging in cardiovascular disease: targets and opportunities , 2009, Nature Reviews Cardiology.

[25]  M. Larsen,et al.  Exploring the Sialiome Using Titanium Dioxide Chromatography and Mass Spectrometry *S , 2007, Molecular & Cellular Proteomics.

[26]  C. Bollensdorff,et al.  Expression pattern of neuronal and skeletal muscle voltage‐gated Na+ channels in the developing mouse heart , 2005, Journal of Physiology.

[27]  R. Beavis,et al.  The identification and characterization of membranome components. , 2008, Journal of proteome research.

[28]  D. Cheresh,et al.  Requirement of vascular integrin alpha v beta 3 for angiogenesis. , 1994, Science.

[29]  J. Post.  Removal of sarcolemmal sialic acid residues results in a loss of sarcolemmal functioning and integrity. , 1992, The American journal of physiology.

[30]  H. Nothaft,et al.  Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry* , 2009, Molecular & Cellular Proteomics.

[31]  G. Langer,et al.  Sialic acid: effect of removal on calcium exchangeability of cultured heart cells. , 1976, Science.

[32]  J. Lowe.  Glycosylation, Immunity, and Autoimmunity , 2001, Cell.

[33]  F. Cappuccio,et al.  Variant of SCN5A Sodium Channel Implicated in Risk of Cardiac Arrhythmia , 2002, Science.

[34]  R. Markwald,et al.  Recruitment of New Cells into the Postnatal Heart , 2006, Annals of the New York Academy of Sciences.

[35]  E. Marbán,et al.  Molecular and cellular mechanisms of myocardial stunning. , 1999, Physiological reviews.

[36]  Yuhong Wang,et al.  Current understanding of klotho , 2009, Ageing Research Reviews.

[37]  R. Barchi,et al.  The voltage-sensitive sodium channel from rabbit skeletal muscle. Chemical characterization of subunits. , 1987, The Journal of biological chemistry.

[38]  Ruedi Aebersold,et al.  Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins , 2009, Nature Biotechnology.

[39]  W. Lederer,et al.  Role of Sodium Channel Deglycosylation in the Genesis of Cardiac Arrhythmias in Heart Failure* , 2001, The Journal of Biological Chemistry.

[40]  S. Cherry,et al.  Cardiac Myocyte-Specific Excision of the &bgr;1 Integrin Gene Results in Myocardial Fibrosis and Cardiac Failure , 2002, Circulation research.

[41]  N. Robinson,et al.  Protein deamidation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Reinout Raijmakers,et al.  Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics , 2009, Nature Protocols.

[43]  O. Ritter,et al.  Fibroblast migration after myocardial infarction is regulated by transient SPARC expression , 2006, Journal of Molecular Medicine.

[44]  J. Marth,et al.  Glycosylation in Cellular Mechanisms of Health and Disease , 2006, Cell.

[45]  J. Judd,et al.  Prolyl hydroxylase and collagen metabolism after experimental mycardial infarction. , 1975, The American journal of physiology.

[46]  M. Mann,et al.  MSQuant, an open source platform for mass spectrometry-based quantitative proteomics. , 2010, Journal of proteome research.

[47]  Jian Yu,et al.  Fibulin-5 suppresses lung cancer invasion by inhibiting matrix metalloproteinase-7 expression. , 2009, Cancer research.

[48]  Florian Gnad,et al.  Precision Mapping of an In Vivo N-Glycoproteome Reveals Rigid Topological and Sequence Constraints , 2010, Cell.

[49]  G. Langer,et al.  Effects of neuraminidase on cellular calcium and contraction in cultured cardiac myocytes. , 1991, Journal of molecular and cellular cardiology.

[50]  J. Cohn,et al.  Cardiac remodeling--concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. Behalf of an International Forum on Cardiac Remodeling. , 2000, Journal of the American College of Cardiology.

[51]  J. Sanderson,et al.  Left and right ventricular collagen type I/III ratios and remodeling post-myocardial infarction. , 1999, Journal of cardiac failure.

[52]  Francis G Spinale,et al.  Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. , 2007, Physiological reviews.

[53]  Martin R Larsen,et al.  Selective enrichment of sialic acid–containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry , 2010, Nature Protocols.

[54]  Madalina M. Drugan,et al.  StatQuant: a post-quantification analysis toolbox for improving quantitative mass spectrometry , 2009, Bioinform..

[55]  J. S. Janicki,et al.  Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function. , 2002, American journal of physiology. Heart and circulatory physiology.

[56]  Y. Yamauchi,et al.  Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site–specific stable isotope tagging , 2006, Nature Protocols.

[57]  M. Entman,et al.  Of mice and dogs: species-specific differences in the inflammatory response following myocardial infarction. , 2004, The American journal of pathology.

[58]  G. Langer,et al.  The Myocardial Cell Surface, Its Histochemistry, and the Effect of Sialic Acid and Calcium Removal on Its Structure and Cellular Ionic Exchange , 1977, Circulation research.

[59]  N. Frangogiannis,et al.  The extracellular matrix as a modulator of the inflammatory and reparative response following myocardial infarction. , 2010, Journal of molecular and cellular cardiology.

[60]  R. Dwek,et al.  Glycosylation and the immune system. , 2001, Science.

[61]  J. Omens,et al.  Early degradation and serum appearance of type I collagen fragments after myocardial infarction. , 2004, Journal of molecular and cellular cardiology.

[62]  Andrew D McCulloch,et al.  Glycated collagen cross-linking alters cardiac mechanics in volume-overload hypertrophy. , 2003, American journal of physiology. Heart and circulatory physiology.

[63]  J. Gebler,et al.  Orthogonality of separation in two-dimensional liquid chromatography. , 2005, Analytical chemistry.

[64]  Thomas K Borg,et al.  Dynamic Interactions between Myocytes, Fibroblasts, and Extracellular Matrix , 2006, Annals of the New York Academy of Sciences.

[65]  M. Entman,et al.  Extracellular matrix remodeling in canine and mouse myocardial infarcts , 2006, Cell and Tissue Research.

[66]  Ten-Yang Yen,et al.  Combining Results from Lectin Affinity Chromatography and Glycocapture Approaches Substantially Improves the Coverage of the Glycoproteome*S , 2009, Molecular & Cellular Proteomics.

[67]  Simon J North,et al.  Regulated and aberrant glycosylation modulate cardiac electrical signaling , 2009, Proceedings of the National Academy of Sciences.

[68]  Stuart J Cordwell,et al.  Technologies for plasma membrane proteomics , 2010, Proteomics.

[69]  T. Ley,et al.  Dipeptidyl Peptidase I Is Essential for Activation of Mast Cell Chymases, but Not Tryptases, in Mice* , 2001, The Journal of Biological Chemistry.

[70]  G. Dorn,et al.  Genetic Manipulation of Periostin Expression Reveals a Role in Cardiac Hypertrophy and Ventricular Remodeling , 2007, Circulation research.