MaxQuant.Live Enables Enhanced Selectivity and Identification of Peptides Modified by Endogenous SUMO and Ubiquitin

Small ubiquitin-like
modifiers (SUMO) 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 the need for genetic
engineering. Here we investigated the applicability of precursor mass
filtering enabled by MaxQuant.Live 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 precursor mass filtering, we achieved a much higher selectivity
of modified peptides, ultimately resulting in up to 30% more SUMO
and ubiquitin sites identified from replicate samples. Real-time exclusion
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
precursor mass filtering strategy to the new Exploris 480 mass spectrometer,
achieving comparable gains in SUMO precursor selectivity and identification
rates. Collectively, precursor 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.