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A workflow towards the reproducible identification and quantitation of protein carbonylation sites in human plasma

ABSTRACT: Protein carbonylation has been consistently used as a marker of excessive oxidative stress and studied in the context of multiple human oxidative stress related diseases. The variety of carbonyl post-translational modifications (PTMs) and their low abundance on easily accessible diagnostic tissues (e.g., plasma) challenges their reproducible identification and quantitation. However, the use of carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allows targeting carbonyl PTMs and enriching proteins and/or peptides carrying these modifications that facilitate their characterization. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma proteins from a healthy individual were derivatized with ARP, digested with trypsin, and ARP-derivatized peptides (ARP-peptides) were enriched using biotin-avidin affinity chromatography prior to RPC-TWIMS-MS/MS analysis. The present workflow highlights previously overlooked analytical challenges, shows the use of ARP specific fragment ions to improve identification reliability of ARP-peptide identifications, and displays an extensive validation of the reproducible recovery and relative quantitation of ARP-peptides. HSA was identified as the most heavily modified protein by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS). Most RCS modification sites were identified in six HSA modification hotspots (Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525).

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Juan Camilo Rojas Echeverri

LAB HEAD: Ralf Hoffmann

PROVIDER: PXD023738 | Pride | 2021-03-02

REPOSITORIES: Pride

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A workflow towards the reproducible identification and quantitation of protein carbonylation sites in human plasma

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