Enabling Global Analysis of Protein Citrullination via Biotin Thiol Tag-Assisted Mass Spectrometry

Abstract

Citrullination is a key post-translational modification (PTM) that affects protein structures and functions. Although it has been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize this PTM. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of citrullination via mass spectrometry. We perform global mapping of the citrullination proteome of mouse tissues. In total, we identify 691 citrullination sites from 432 proteins which represents the largest data set to date. We discover novel distribution and functions of this PTM. This study depicts a landscape of protein citrullination and lays the foundation for further deciphering their physiological and pathological roles.

Neutrophil attachment via Mac-1 ( α M β 2 ; CD11b/CD18; CR3) integrins induces PAD4 deimination of profilin and histone H3

“…It is intriguing to observe that PAD4 also plays an important role in the modification of cytoskeletal proteins, as is consistent with previous reports of deiminated β-actin [45] and vimentin [46]. Comprehensive efforts to identify citrullinated proteins, the 'citrullinome', have provided evidence that deimination occurs in neutrophils [15], extends across multiple tissues, and is particularly abundant in the central nervous system [17]. Remarkably, synovial fluids from arthritic joints are a rich biological source of inflammatory cytokines, chemokines [32] and deiminated NET components [18].…”

Recent advances in characterization of citrullination and its implication in human disease research: From method development to network integration

“…This loss was reportedly observed not only in multiple charge states of precursor ions, but also in b‐ and y‐fragmented ions, providing a diagnostic marker for citrullination identification [71]. This observation became the foundation for confident citrullination discovery and profiling, and has been successfully adopted by numerous citrullination identification workflows and applications [51, 52, 72, 73]. For example, Lee and colleagues made tremendous headway by mining the human tissue proteome for protein citrullination evidence from 30 human tissues by MS, using this foundational knowledge as a starting point.…”

“…It should be emphasized that the use of an acidic reaction environment, specifically necessary for citrulline‐containing residues, should be strictly controlled, as carrying out the reaction under neutral or basic conditions can cause arginine to adversely react. This was the first study to introduce a mass probe for easier detection of citrullinated residues and was also a milestone study, as it inspired further citrullination derivatization design [51, 52, 72, 73].…”

“…This was the first study to introduce a mass probe for easier detection of citrullinated residues and was also a milestone study, as it inspired further citrullination derivatization design [51,52,72,73].…”

“…More than 50 citrullinated proteins were identified and significantly enriched by at least 2-fold in a PAD2overexpressing cell line compared to the controls using this probe and platform technology. More recently, Shi et al reported the design and development of a biotin-thiol tag that enabled derivatization, enrichment, and confident identification of citrullination via MS with high specificity and efficiency [73]. They performed proof of principle experiments using recombinant human histone H3 protein with or without in vitro PAD.…”