MaxQuant.Live enables enhanced selectivity and identification of peptides modified by endogenous SUMO and ubiquitin

ABSTRACT: Small ubiquitin-like modifiers (SUMOs) and ubiquitin are frequent post-translational modifications of proteins that play pivotal roles in all cellular processes. We previously reported mass spectrometry-based proteomics methods that enable profiling of lysines modified by endogenous SUMO or ubiquitin in an unbiased manner, without requiring genetic engineering. Here we investigated the applicability of precursor mass filtering enabled by MaxQuant.Live (MQL) to our SUMO and ubiquitin proteomics workflows, which efficiently avoided sequencing of precursors too small to be modified but otherwise indistinguishable by mass-to-charge ratio. Using peptide mass filtering, we achieved much higher precursor selectivity, ultimately resulting in up to 30% more SUMO and ubiquitin sites identified from replicate samples. Real-time ‘untargeting’ of unmodified peptides by MQL resulted in 90% SUMO-modified precursor selectivity from a 25% pure sample, demonstrating great applicability for digging deeper into ubiquitin-like modificomes. We adapted the mass filtering strategy to the new Exploris 480 mass spectrometer, achieving comparable gains in SUMO precursor selectivity and identification rates. Collectively, mass filtering via MQL significantly increased identification rates of SUMO- and ubiquitin-modified peptides from the exact same samples, without the requirement for prior knowledge or spectral libraries.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Ivo Hendriks  

LAB HEAD: Michael Lund Nielsen

PROVIDER: PXD022367 | Pride | 2021-02-05

REPOSITORIES: Pride