Isobaric Labeling Enables 10-Plex Quantitative Analysis of Ubiquitylated Peptides: A diagnostic Ion to Improve Identification and quantification

摘要

We describe a 10-plex isobaric labeling method to derivatize ubiquitylated (K-GG) peptides after immuno-enrichment and identify a diagnostic fragment ion that can be used to trigger high resolution SPS-MS~3 scans. Utilizing a di-glycine remnant specific antibody cross-linked to agarose beads, we find the reproducibility of multiple K-GG immunoprecipitations (IPs) to be excellent, enabling the labeling of multiple IPs after immuno-enrichment. Additionally, isobaric labeling of KGG peptides enables online filtering based on a specific K-GG-TMT diagnostic ion and precursor charge state to increase the depth of MS/MS analysis and trigger accurate SPS-MS~3 scans. We use this method to simultaneously interrogate the changes in the ubiquitylome of HTC116 cells in biological quintuplicate after treatment with bortezomib.