MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide

Caseinomacropeptide (CMP) is a 64 amino acid polypeptide corresponding to K -casein 106-169. CMP naturally exists in several forms due to extensive pos ttranslational modifications including glycosylation and phosphorylation. T he aglycosylated, phosphorylated form of CMP has been shown to exhibit anti bacterial 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 f rom a chymosin digest of casein by HPLC and then digested with endoproteina se Glu-C to generate peptides suitable for MALDI-PSD-MS analysis. This anal ysis showed that CMP is fully phosphorylated at Ser(149) and only partially phosphorylated at Ser(127) Dehydroalanyl residues corresponding to the pho sphoserines of CMP were detected upon MALDI-PSD-MS analysis suggesting that the phosphoryl bond in phosphoserine is very labile during PSD analysis su ch that the phosphoryl group may be lost before backbone fragmentation. (C) 2001 Elsevier Science inc. All rights reserved.