In‐depth analysis of the Sirtuin 5‐regulated mouse brain malonylome and succinylome using library‐free data‐independent acquisitions

Abstract

Post‐translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry‐based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data‐independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label‐free quantification. In this study, we present a powerful approach that combines antibody‐based enrichment with comprehensive DIA acquisitions and spectral library‐free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, 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 2211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 164 malonylated and 578 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 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.

Protocol for mass spectrometric profiling of lysine malonylation by lysine acetyltransferase in CRISPRi K562 cell lines

“…A detailed step-by-step procedure for building the DIA method is also described in the Supplementary Appendix of Bons et al . 2 Inject samples onto a Dionex UltiMate 3000 system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (both from Thermo Fisher Scientific). Inject 200 ng of peptides for the protein-level analysis ( Figure 2 A).…”

“…A detailed step-by-step procedure for processing the collected DIA data is also described in the Supplementary Appendix of Bons et al . 2 For more details, please refer to the user manual of Spectronaut (latest version: version 18, https://biognosys.com/content/uploads/2023/06/Spectronaut18_UserManual.pdf ).…”

“…The depth of the malonylome coverage yielded by this protocol depends on several factors, including the amount of starting protein material used for the affinity-based PTM enrichment, the amount of peptide material loaded onto the column, and the LC-MS system and settings. It is worth noting that this procedure can be applied to other sample types (e.g., other cell types, tissues 2 , 13 ) and other organisms with some adjustments, such as the protein database.…”

Dicarboxylic Acid Dietary Supplementation Protects against AKI

“…5 Using reversed-phase high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry, succinyl-enriched samples were analyzed by data-independent acquisition (DIA) on an Orbitrap Eclipse mass spectrometer [22][23][24] and site-specific changes in succinylation were quantified using library-free directDIA in Spectronaut (Biognosys, Supplemental Tables 1-4). 22,25 Meanwhile, matching whole lysate liquid chromatography with tandem mass spectrometry analysis conducted on an Orbitrap Exploris 480 mass spectrometer operated by DIA-MS was used to determine total protein abundance changes and to normalize PTM site changes adjusting for the protein-level changes. Further experimental and statistical details are provided in Supplemental Appendix 1.…”

Post‐translational modification and phenotype

“…Among all PTMs, lysine acylation is involved in many diseases such as cancers and diabetes but understudied in the brain. In the second article , Bons et al described an in‐depth analysis of the Sirtuin 5‐regulated mouse brain malonylome and succinylome [11]. They utilized a library‐free, direct data‐independent acquisition mass spectrometry (DIA‐MS) analysis that integrates PTM identification, quantification, and site localization, which was able to detect 466 malonylated and 2211 succinylated sites.…”