付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22697号 / 医科博第112号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 竹内 理, 教授 髙折 晃史, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA.