Talbo G H, Suckau D, Malkoski M, Reynolds E C
School of Dental Science, The University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
Peptides. 2001 Jul;22(7):1093-8. doi: 10.1016/s0196-9781(01)00426-0.
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.
酪蛋白巨肽（CMP）是一种由64个氨基酸组成的多肽，对应于κ-酪蛋白的106-169位氨基酸。由于存在广泛的翻译后修饰，包括糖基化和磷酸化，CMP天然存在多种形式。已证明去糖基化、磷酸化形式的CMP具有抗菌活性。本研究的目的是使用基质辅助激光解吸/电离源后衰变质谱（MALDI-PSD-MS）来鉴定CMP序列中的磷酸化位点。通过高效液相色谱（HPLC）从酪蛋白的凝乳蛋白酶消化物中分离出CMP，然后用内肽酶Glu-C消化，以产生适合MALDI-PSD-MS分析的肽段。该分析表明，CMP在Ser(149)处完全磷酸化，而在Ser(127)处仅部分磷酸化。在MALDI-PSD-MS分析中检测到与CMP的磷酸丝氨酸相对应的脱氢丙氨酰残基，这表明在PSD分析过程中，磷酸丝氨酸中的磷酰键非常不稳定，以至于在主链断裂之前磷酰基可能会丢失。
