MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide.
Caseinomacropeptide (CMP) is a 64 amino acid polypeptide corresponding to kappa-casein 106-169. CMP naturally exists in several forms due to extensive posttranslational modifications including glycosylation and phosphorylation. The aglycosylated, phosphorylated form of CMP has been shown to exhibit antibacterial activity. The aim of this study was to use matrix assisted laser desorption/ionization post source decay mass spectrometry (MALDI-PSD-MS) to identify the phosphorylation sites in the CMP sequence. CMP was isolated from a chymosin digest of casein by HPLC and then digested with endoproteinase Glu-C to generate peptides suitable for MALDI-PSD-MS analysis. This analysis showed that CMP is fully phosphorylated at Ser(149) and only partially phosphorylated at Ser(127.) Dehydroalanyl residues corresponding to the phosphoserines of CMP were detected upon MALDI-PSD-MS analysis suggesting that the phosphoryl bond in phosphoserine is very labile during PSD analysis such that the phosphoryl group may be lost before backbone fragmentation.
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School of Dental Science, The University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
Suckau
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Malkoski
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Reynolds
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D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D012085:Chymosin / Q000737:chemistry; D006031:Glycosylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D002364:Caseins; D010446:Peptide Fragments; D010455:Peptides; C065989:caseinomacropeptide; D010768:Phosphoserine; D012694:Serine; C015102:dehydroalanine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D012085:Chymosin; D000409:Alanine