A method of isolating the collagen (I) alpha2 chain carboxytelopeptide for species identification in bone fragments.
We present a novel method for the isolation and analysis of the bone collagen (I) alpha2 chain carboxytelopeptide as a species biomarker. Conventional methods for the analysis and sequencing of mixtures of proteins and peptides commonly involve using the protease trypsin to cleave proteins present in the sample. However, in the study of collagen, these methods result in very complex mixtures of peptides that are difficult to analyze and the acquired results are not reproducible. Here we use bacterial collagenase (from Clostridium histolyticum) for its ability to cleave the highly unusual Gly-Xaa-Yaa repeating sequence of collagen, where Xaa usually is Pro and Yaa often is Hyp. Followed by a simple isolation step using a reverse phase solid phase extraction cartridge, the alpha2 (I) chain carboxytelopeptide can be readily analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and the results can be used to distinguish between different species of origin.
Buckley
Michael
M
Department of Biology, University of York, York YO10 5YW, UK. mbuckley82@gmail.com
Collins
Matthew
M
Thomas-Oates
Jane
J
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D001842:Bone and Bones / Q000737:chemistry*; D003094:Collagen / Q000737:chemistry* / Q000302:isolation & purification; D024042:Collagen Type I; D016678:Genome; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D010802:Phylogeny; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D024042:Collagen Type I; D010446:Peptide Fragments; D010455:Peptides; D003094:Collagen