Measurement of Homocitrulline, A Carbamylation‐derived Product, in Serum and Tissues by LC‐MS/MS

Abstract

AbstractCarbamylation corresponds to the nonenzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest in evaluating the carbamylation rate of blood or tissue proteins, since carbamylation‐derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaching to the ε‐NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry–based methods. This article describes a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the quantification of homocitrulline, with special emphasis on preanalytical steps that allow quantification of total or protein‐bound homocitrulline in serum or tissue samples. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sample pretreatment for the quantification of homocitrulline by LC‐MS/MSAlternate Protocol: Preanalytical steps for the quantification of homocitrulline in tissue samplesBasic Protocol 2: LC‐MS/MS quantification of homocitrullineBasic Protocol 3: LC‐MS/MS quantification of lysine in hydrolysates