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High-throughput chemical proteomics workflow for profiling protein citrullination dynamics

ABSTRACT: Citrullination is a post-translational modification implicated in autoimmune and inflammatory diseases but remains challenging to analyze biochemically. Its low stoichiometry and the lack of enrichment tools have hindered its global and site-specific profiling. Here, we developed a high-throughput chemical proteomics workflow for citrullination profiling with enhanced sensitivity and specificity. This workflow employs glyoxal-based derivatization of citrullinated peptides, followed by a click reaction with a biotin conjugate, featuring a cleavable linker that enhances peptide recovery and following identification. We examined the derivatization efficiency using synthetic citrullinated peptides and demonstrated its sensitivity and reproducibility in complex biological samples. Mouse brain digest samples were then used for direct comparison to existent enrichment tools. Dose-dependent ionomycin treatments of isolated neutrophils from fresh blood allowed us to profile citrullination during neutrophil extracellular trap (NET) formation. Known and novel sites on different histone proteins, as well as structural proteins mediating nuclear and cytoskeletal reorganization, were found to be regulated. These findings illuminate citrullination dynamics in NET formation and establish a platform for exploring its roles in diverse systems.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Astral

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Dr. Chien-Yun Lee

PROVIDER: MSV000097617 | MassIVE | Mon Apr 14 05:15:00 BST 2025

SECONDARY ACCESSION(S): PXD062929

REPOSITORIES: MassIVE

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Project description:we aimed to provide an objective roadmap for histone sample preparation wherein the most notable findings and most important metrics were highlighted. This roadmap included a Histone Coverage Tool that allows researchers to make a snapshot of public or in-house data in search of a protocol that suits their specific needs. However, despite the fact that the coverage provided by a sample preparation workflow is pivotal to answer specific research questions, it should be noted that other metrics such as enzyme specificity and workflow variability can be equally important. Moreover, when it comes to histone sample preparation there are multiple additional challenges and pitfalls compared to regular bottom-up proteomics workflows. First there is the abundance and unequal distribution of arginine and lysine cleavage sites, which makes it virtually impossible to cover the entire histone code with a single bottom-up workflow. The most widely spread workaround for the abundance of cleavage sites is derivatization of lysine residues used to block tryptic digest and create longer peptides. However, this also introduces chemical noise, which obscures biological PTMs, while lowering sensitivity due to diluted signal. The most obvious alternative is an arginine specific digest. However, on the one hand, derivatization of lysine residues not only increases peptide length but also increases retention on the LC-system, while decreasing charge state distribution of the precursor ions. The latter presumably reduces instrumental variation of the workflow, which is remarkable since this source of variation is generally assumed to be constant in the field of LC-MS/MS based proteomics. On the other hand, lysine derivatization also increases the singly charged precursor fraction, which is usually not fragmented in regular data dependent acquisitions, thus increasing the risk of losing annotations. It is clear that not one histone sample preparation workflow will fit all research questions. However, we hope that this manuscript will guide researchers in making informed decisions about the many pitfalls and trade-offs involved in optimizing or assessing histone sample preparation workflows.

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High-throughput chemical proteomics workflow for profiling protein citrullination dynamics

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