Novel high-resolution ion mobility mass spectrometry for site-specific quantification of the sirtuin-5 regulated...

Protein post-translational modifications (PTMs) dynamically regulate essential biological and cellular processes. Lysine succinylation changes the amino acid charge, potentially affecting protein structures and functions, and dysregulation of protein succinylation may lead to metabolic disorders. Proteome-wide succinylation quantification using proteomic tools remains challenging, especially due to the low abundance of succinylated peptides and the frequent presence of isomeric PTM forms. Ion mobility spectrometry workflows that can differentiate peptidoforms with different PTM distributions represent a powerful strategy to alleviate these challenges. Recently, a new Parallel Accumulation with Mobility Aligned Fragmentation (PAMAF™) operating mode for high-resolution ion mobility-mass spectrometry (HRIM-MS) analysis based on the structures for lossless ion manipulation (SLIM) technology was introduced. Here, we first assessed the performance of PAMAF mode for protein succinylation analysis using synthetic succinylated peptides, demonstrating residue-level differentiation of co-eluting isomers and isobars and precise PTM site localization. We leveraged this novel approach to investigate succinylome remodeling in kidney tissues from wild-type and Sirtuin-5 (Sirt5) knock-out mice, a NAD⁺-dependent lysine de-succinylase. PAMAF acquisitions yielded ∼1000 confidently identified and accurately quantified succinylated peptides and sites from mouse kidney. Sirt5 regulated succinylation of mitochondrial proteins involved in metabolic processes, including fatty acid oxidation, the tricarboxylic acid cycle, and propionate metabolism. SIGNIFICANCE: Understanding the dynamic remodeling of the protein post-translational modification landscape is critical to gain insights into the underlying molecular mechanisms of biological systems. Lysine succinylation is a recently discovered reversible post-translational modification (PTM), that regulates various biological processes and associates with diverse diseases. However, this PTM is poorly characterized, partly due to analytical barriers. Here, we present a novel mass spectrometry (MS) methodology leveraging high-resolution ion mobility (HRIM) spectrometry and Parallel Accumulation with Mobility Aided Fragmentation (PAMAF) technology to profile and quantify succinylated peptides. The unique combination of liquid chromatography, ion mobility in a very long ion path (13 m), and alternate acquisition of MS and MS/MS spectra for all ions entering the mass spectrometer provided comprehensive profiling and accurate quantification of succinylated peptides in complex matrices. This technology enabled confident resolution of succinylated isomeric peptides, that could not be differentiated without high-resolution ion mobility separation and subsequent MS/MS PTM site identification. We investigated the kidney succinylome of Sirtuin-5 (desuccinylase) knockout mice compared to wild-type mice, with over 1000 succinylated peptides identified and quantified. We analyzed the hypersuccinylation of proteins upon Sirtuin-5 deletion, especially of mitochondrial proteins involved in diverse metabolic processes.