Identification of acidic residues in proteins by selective chemical labeling and tandem mass spectrometry

Abstract

**Please note that the full text is embargoed until 08/01/2025** Proteins play important role in carrying out a wide range of cellular activities. They are subjected to various post-translational modifications (PTMs) by the addition of functional groups such as acetyl, phosphoryl, and methyl which leads to changes in their biological functions, localization, activity, and structure. The growth of MS technology has immensely facilitated the identification of PTMs comprehensively. This dissertation focuses on mapping these changes using covalent labeling techniques by tagging carboxylic acid residues, which undergo various kinds of PTMs. The second chapter focuses on the methods designed for Affinity Purification Mass Spectrometry (AP-MS) chemical cross-linking (CXL) of protein complexes. There are numerous methods available for bioconjugation which target cysteine, lysine, and arginine residues. Although carboxyl residues, which are prevalent in proteins, are essential for preserving the protein's functionality, there are currently few accessible selective labeling procedures. We have described a novel reactive probe that allows for the chemoselective modification of acidic residues in peptides and proteins. We evaluated the reactivity of diphenyldiazomethane (DPDAM) in peptides and proteins in the third chapter. The fragmentation patterns of these labeled peptides have been investigated using tandem mass spectrometry.