Cyanogen Bromide Peptides of the Fibrillar Collagens I, III, and V and Their Mass Spectrometric Characterization:  Detection of Linear Peptides, Peptide Glycosylation, and Cross-Linking Peptides Involved in Formation of Homo- and Heterotypic Fibrils

Abstract

The network of the fibrillar collagens I, III, and V, extracted from fetal calf skin and cleaved with cyanogen bromide, was studied by means of ultraviolet matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UV-MALDI MS). Nearly all of the expected cyanogen bromide peptides of the different alpha chains were detected. Distinct peptides are identified that can serve as a reference signal for the individual alpha-chains. Homo- and heterotypic cross-linking patterns, some of which have not been described before for bovine collagen, are indicated by comparison of the mass spectrometric data with documented amino acid sequences. Potential cross-linking mechanisms are discussed. For example, the mass spectrometric data suggest that the formation of heterotypic I/III and I/V fibrils is substantially determined by the telo-regions of type I collagen, which are covalently connected to the corresponding helical and nonhelical cross-linking domains of adjacent molecules either by 4D or 0D-stagger bonds. The chemical nature of the cross-links can be concluded. The data also indicate a disturbed formation of heterotypic fibrils. Finally, collagen glycosylation can also be identified.

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

“…The ␣1(V) chain contains extensive post-translational modifications (PTMs) relative to other fibrillar collagen chains consisting of 4-hydroxyproline (4-Hyp) and 3-hydroxyproline (3-Hyp) residues and of hydroxylysine (Hyl) residues, most of which are decorated with di-and monosaccharides (14,15). Functionally, 4-Hyp residues have well characterized effects in stabilizing triple helices (16), whereas Hyl residues are important to the formation of stable covalent cross-links between collagen chains (17), and Hyl glycosylation may be important in assembly and secretion of at least some collagens (18).…”

“…Specifically, human skin ␣1(V) chains (15) were estimated to comprise ϳ39 hydroxylysines, 29 in the form of Glc-Gal-Hyl residues and 5 in the form of Gal-Hyl residues, suggesting Glc-Gal and Gal to be frequently and infrequently occurring PTMs, respectively. Henkel and Dreisewerd (14), employing MALDI MS analysis of fetal calf skin ␣1(V) chains, estimated that most, if not all, Hyl residues are likely glycosylated, with ϳ87% predicted to be GlcGal-Hyl. However, they lacked MS n sequence data to support their assignments of PTMs, making it difficult to confidently determine the nature of saccharide species (e.g.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Other studies include the identification of glycosylated peptides obtained by cyanogen bromide cleavage of collagen from fetal calf skin (Henkel & Dreisewerd, 2007), the identification of cellotriose, produced from rapidly growing plant tissue and characterization of commercial samples, for example .…”

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

“…The degree of glycosylation varies among different collagens (24,25). For type I collagen, the degree of glycosylation also varies between different tissues.…”