Identification of Species Origin of Collagen Based on Liquid Chromatography-Mass Spectrometry with Peptide Markers

KeyWord:peptide marker  collagen  liquid chromatography-mass spectrometry  species origin

Abstract:

      A liquid chromatography-mass spectrometric method was established for the identification of the species origin of collagen based on peptide markers.The sample was determined by nano electrospray ionization quadrupole time of flight mass spectrometry in positive mode to obtain peptide mass spectrometry information after protein extraction£¬reduction£¬alkylation£¬tryptic digestion and followed by the separation on an Eksigent C18 column£¨75 ¦Ìm¡Á150 mm£¬3 ¦Ìm£© by gradient elution with water-acetonitrile£¨98¡Ã2£© containing 0ª±1%formic acid and acetonitrile-water£¨98¡Ã2£© containing 0ª±1%formic acid as mobile phases.Potential peptide markers were screened by data analysis with ProteinPilotTM and blast.Then the digested sample was separated on an Eclipse Plus C18 column£¨2ª±1 mm¡Á100 mm£¬1ª±8 ¦Ìm£© by gradient elution with acetonitrile-water containing 0ª±1%formic acid as mobile phase.The specificity of peptide markers was further confirmed by electrospray ionization quadrupole/linear ion trap tandem mass spectrometry in positive mode under multiple reaction monitoring information dependent acquisition enhanced product ion£¨MRM-IDA-EPI£© scanning mode.Finally£¬three pig collagen peptide markers£¬four bovine collagen peptide markers and one sheep collagen peptide marker were screened and identified.These peptide markers have a good heat resistance£¬and could provide a specific£¬accurate and reliable method for the identification on species origin of collagen.