溶菌酶基质辅助激光解吸电离-串联飞行时间质谱分析中的不常见修饰及脱水反应

Uncommon Modifications and Dehydration Reaction in the Analysis of Lysozyme by Matrix-assisted Laser Desorption Ionization Tandem Time of Flight-Mass Spectrometry

利用基质辅助激光解吸电离-串联飞行时间质谱(MALDI-TOF/TOF)分析了溶菌酶标准蛋白,在常规搜库条件下鉴定到6个独立肽段,Mascot得分420,鉴定覆盖率为54%。此外,经人工解析发现,肽段IVSDG-DGMNAWVAWR(98→112)在样品处理过程中发生了天冬酰胺脱氨化、天冬酰胺脱氨化+甲硫氨酸氧化、天冬酰胺脱氨化+甲硫氨酸氧化+色氨酸氧化等修饰,在激光解吸电离过程中发生脱水反应,脱水位点是脱氨后形成的第103位天冬氨酸。此外,还发现了部分肽段的丙酰胺化修饰。本研究表明,选择一级质谱中的极低丰度离子进行串联质谱分析和利用人工解析方法分析数据库未匹配数据,有可能发现一些特殊的蛋白质修饰,可增加数据利用度和分析结果的确定性。

Tryptic digest of lysozyme was analyzed by MALDI-TOF/TOF mass spectrometry.Six unique peptides were identified with high confidence by database searching and the Mascot score was 420 with the protein coverage of 54%.Furthermore,several uncommon modifications including deamidation of asparagines（Asp） residue,deamidation of Asp plus oxidation of methionine（Met）,deamidation of Asp plus oxidation of Met plus oxidation of Trp were observed for peptide 98IVSDGDGMNAWVAWR112.Furthermore,a dehydration reaction was observed in the process of MALDI for this peptide.All above results showed that the Asparagine at 103rd position had a highly chemical reactivity.In addition,the modification of propionamide（Cystine） was observed for another two peptides.This study indicated that,for those of unmatched data by database searching,the data utilization and the certainty of analysis result would be increased by using manual explantation and selecting very low abundant ions in primary spectrum and this method was also helpful for finding potential post-translational modifications.