Automated On-antibody TMT Labeling for Ubiquitin Profiling in Multiomics Sample Analyses

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. UbiFast is a highly sensitive method for multiplexed ubiquitylation profiling in which K-ɛ-GG peptides are enriched using antibodies specific to ubiquitin remnants (K-ɛ-GG) followed by on-antibody labeling with TMT reagents. Here, we present methods for automation of the UbiFast method using anti-K-ε-GG antibody covalently linked to magnetic beads and a magnetic particle processer. The workflow enables processing of up to 96 samples in parallel and greatly reduces sample processing times and variability. We have also incorporated automated UbiFast as part of a serial enrichment strategy that enables efficient and sample-sparing analysis of limited biological and clinical samples. Optimized FAIMS MS methods for the analysis of Ub samples will also be described.