Generation of High-Quality Succinyl Spectral Libraries for Improved Proteome-Wide Succinylome Analysis Using...

Protein posttranslational modifications (PTMs) are crucial and dynamic modulators of protein functions, interactions, and localizations, as well as biological pathways and cellular signaling. Lysine succinylation analysis remains challenging, but sophisticated workflows combining succinylated peptide enrichments and quantitative mass spectrometry approaches have revolutionized proteome-wide succinylome analysis. The implementation of data-independent acquisition (DIA)-mass spectrometry has greatly advanced the detection of low-abundance succinylated peptides, succinylome coverage, identification reproducibility, and quantification accuracy. However, the complexity of DIA data requires dedicated data processing algorithms and tools, which typically rely on spectral libraries. These reference libraries can be generated from experimental data-dependent acquisition (DDA) acquisitions of representative study samples submitted to DDA database search engines for confident succinylated peptide identification and precise PTM site localization. Here, we describe how to build DDA PTM spectral libraries using various software tools, specifically Spectronaut, SpectroMine, and MSFragger. The generated libraries were imported into Skyline for the analysis of previously published DIA succinylome data of Sirtuin-5 knocked-out vs wild-type mouse brains in order to accurately quantify and visualize PTM-containing peptides.