title: Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry.
abstract: Collagen IV is the main structural protein that provides a scaffold for assembly of basement membrane proteins. Posttranslational modifications such as hydroxylation of proline and lysine and glycosylation of lysine are essential for the functioning of collagen IV triple-helical molecules. These modifications are highly abundant posing a difficult challenge for in-depth characterization of collagen IV using conventional proteomics approaches. Herein, we implemented an integrated pipeline combining high-resolution mass spectrometry with different fragmentation techniques and an optimized bioinformatics workflow to study posttranslational modifications in mouse collagen IV. We achieved 82% sequence coverage for the α1 chain, mapping 39 glycosylated hydroxylysine, 148 4-hydroxyproline, and seven 3-hydroxyproline residues. Further, we employed our pipeline to map the modifications on human collagen IV and achieved 85% sequence coverage for the α1 chain, mapping 35 glycosylated hydroxylysine, 163 4-hydroxyproline, and 14 3-hydroxyproline residues. Although lysine glycosylation heterogeneity was observed in both mouse and human, 21 conserved sites were identified. Likewise, five 3-hydroxyproline residues were conserved between mouse and human, suggesting that these modification sites are important for collagen IV function. Collectively, these are the first comprehensive maps of hydroxylation and glycosylation sites in collagen IV, which lay the foundation for dissecting the key role of these modifications in health and disease.
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, ‡Center for Matrix Biology, §Department of Biochemistry, and ⊥Department of Biomedical Informatics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: Trayambak
authors.initials: T
authors.lastname: Basak
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, ‡Center for Matrix Biology, §Department of Biochemistry, and ⊥Department of Biomedical Informatics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Vega-Montoto
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, ‡Center for Matrix Biology, §Department of Biochemistry, and ⊥Department of Biomedical Informatics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Zimmerman
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, ‡Center for Matrix Biology, §Department of Biochemistry, and ⊥Department of Biomedical Informatics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: David L
authors.initials: DL
authors.lastname: Tabb
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, ‡Center for Matrix Biology, §Department of Biochemistry, and ⊥Department of Biomedical Informatics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: Billy G
authors.initials: BG
authors.lastname: Hudson
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, ‡Center for Matrix Biology, §Department of Biochemistry, and ⊥Department of Biomedical Informatics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: Roberto M
authors.initials: RM
authors.lastname: Vanacore
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D002460:Cell Line; D056148:Chromatography, Reverse-Phase; D024141:Collagen Type IV / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006900:Hydroxylation; D007903:Lens Capsule, Crystalline / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry
chemical_list: C547475:COL4A1 protein, human; D024141:Collagen Type IV
keywords: MyriMatch; basement membrane; collagen IV; glycosylation; hydroxylysine; hydroxyproline; mass spectrometry; posttranslational modifications