Rojas Echeverri Juan Camilo, Milkovska-Stamenova Sanja, Hoffmann Ralf
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, 04103 Leipzig, Germany.
Center for Biotechnology and Biomedicine, Universität Leipzig, 04103 Leipzig, Germany.
Antioxidants (Basel). 2021 Mar 1;10(3):369. doi: 10.3390/antiox10030369.
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.
蛋白质羰基化是氧化应激过度的一个标志物，已在与氧化应激相关的多种人类疾病背景下进行了研究。翻译后羰基修饰（羰基PTMs）种类繁多，且它们在血浆中的浓度较低，这对其可重复鉴定和定量提出了挑战。然而，羰基特异性生物素化衍生化标签（如醛反应探针，ARP）可通过富集携带这些修饰的蛋白质和肽来靶向羰基PTMs。在本研究中，用ARP对氧化人血清白蛋白蛋白模型（OxHSA）和健康供体的血浆进行衍生化，用胰蛋白酶消化，并在与行波离子淌度质谱联用的纳米反相色谱-电喷雾电离串联质谱（nRPC-ESI-MS/MS-TWIMS）之前，使用生物素-抗生物素蛋白亲和色谱进行富集。所提出的工作流程通过使用ARP特异性碎片离子可靠地鉴定ARP肽，解决了几个分析难题。此外，还验证了ARP肽的可重复回收率和相对定量。血浆中的人血清白蛋白（HSA）被多种直接氨基酸氧化产物和来自活性羰基物种（RCS）的加合物大量修饰，大多数RCS修饰在成熟HSA的六个热点位置被检测到，即Lys10、Lys190、Lys199、Lys281、Lys432和Lys525。
