Dataset Information

In-depth Analysis of the Sirtuin 5-regulated Mouse Brain Acylome using Library-free Data-Independent Acquisitions

ABSTRACT: Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through combinatory effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. Due to specific challenges of PTM mass spectrometry-based proteomics encountered during data acquisition and processing, novel and innovative workflows have emerged using data-independent acquisition (DIA) to ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach combining antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). The identical DIA data can be used to generate spectral libraries and to comprehensively identify and quantify PTMs, reducing the amount of enriched sample needed, and acquisition time, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock out mice, and discovered and quantified 466 malonylated and 2,211 succinylated peptides. SIRT5 regulation remodeled the acylomes, by targeting 171 malonylated and 640 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We revealed 46 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Birgit Schilling

PROVIDER: MSV000089606 | MassIVE | Sat Jun 04 20:40:00 BST 2022

SECONDARY ACCESSION(S): PXD034275

REPOSITORIES: MassIVE

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Project description:Background: Lysine succinylation is a newly identified PTM, which exists widely from prokaryotes to eukaryotes and participates in various cellular processes, especially in the metabolic processes. Staphylococcus epidermidis is a commensal bacterium in the skin, which attracts more attention as a pathogen, especially in immunocompromised patients and neonates by attaching to medical devices and forming biofilms. However, the significance of lysine succinylation in proteins of Staphylococcus epidermidis has not been investigated. Materials and methods: Using antibody affinity enrichment followed by LC-MS/MS analysis, we examined the succinylome of Staphylococcus epidermidis (ATCC®12228™). Then, bioinformatics analysis was performed, including Gene Ontology, KEGG enrichment, motif characterization, secondary structure, protein-protein interaction, and BLAST analysis. Results: A total of 1557 succinylated lysine sites in 649 proteins were identified in Staphylococcus epidermidis (ATCC 12228). Among these succinylation proteins, GO annotation showed that proteins related to metabolic processes and binding activity accounted for the most based on the analysis of biological process and molecular function, respectively. KEGG pathway characterization indicated that proteins associated with the glycolysis/ gluconeogenesis, and citrate cycle (TCA cycle) pathway were more likely to be succinylated. Moreover, 13 conserved motifs were identified. The specific motif KsuD was conserved in model prokaryotes and eukaryotes. Succinylated proteins with this motif were highly enriched in the glycolysis/gluconeogenesis pathway. One succinylation site(K144) was identified in S-ribosylhomocysteine lyase, a key enzyme in the quorum sensing system, indicating the regulatory role succinylation may play in bacterial processes. Furthermore, 15 succinyltransferases and 18 desuccinylases(erasers) were predicted in S.epidermidis by BLAST analysis. Conclusions: We performed the first comprehensive profile of succinylation in Staphylococcus epidermidis and illustrated the significant role succinylation may play in energy metabolism, QS system, and other bacterial behaviors. This study may be a fundamental basis to investigate the underlying mechanisms of colonization, virulence, and infection of S. epidermidis, as well as provide a new insight into regulatory effects succinylation may lay on metabolic processes.

Dataset Information

In-depth Analysis of the Sirtuin 5-regulated Mouse Brain Acylome using Library-free Data-Independent Acquisitions

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