title: Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry.
abstract: Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.
authors.affiliation: Faculty of Medicine, Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16-18, 04107 Leipzig, Germany.
authors.forename: Ariane
authors.initials: A
authors.lastname: Nimptsch
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schibur
authors.forename: Christian
authors.initials: C
authors.lastname: Ihling
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.forename: Thomas
authors.initials: T
authors.lastname: Riemer
authors.forename: Daniel
authors.initials: D
authors.lastname: Huster
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schiller
mesh_terms: D000818:Animals; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010455:Peptides; D003094:Collagen; D017364:Collagenases