Characterization of Whole Fibril-Forming Collagen Proteins of Types I, III, and V from Fetal Calf Skin by Infrared Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Abstract

Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IR-MALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 degrees C for 10 min, resulting in the dissociation of the triple helix, the single alpha-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single alpha-chains were detected with significantly reduced sensitivity.

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.…”

Fragility of Reconstituted Type V Collagen Fibrils with the Chain Composition of α1(V)α2(V)α3(V) Respective of the D-Periodic Banding Pattern

“…The difference between the two subtypes resides on the third chain, since both subtypes have α1(V) and α2(V) chains in their molecules. In type V collagen, lysine residues in the Yaa position are highly glycosylated [44,45]. No direct evidence for the thermal stability is currently available on glycosylated hydroxylysine.…”

Proteomic analysis of cartilage‐ and bone‐associated samples

“…The difficulties in the MS analysis of large PGs and glycoproteins have been discussed [65]. Crosslinkages, such as observed in trimerization of collagen, create another pitfall; improperly cleaved disulphide bridges result in nonlinear telomer peptides complicating the database search [66]. Other similar possible caveats include formation of hydroxylysyl pyridinoline cross-links [67], or formation LysArg cross-links (glucosepane via Maillard reactions) [68].…”