Site-Specific Quantification of Protein Ubiquitination on MS2 Fragment Ion Level via Isobaric Peptide Labeling

Proteome-wide
quantitative analysis of protein ubiquitination is
important to gain insight into its various cellular functions. However,
it is still challenging to monitor how ubiquitination at each individual
lysine residue is independently regulated, especially the whereabouts
of peptides containing more than one ubiquitination site. In recent
years, isobaric peptide termini labeling has been considered a promising
strategy in quantitative proteomics, benefiting from its high accuracy
by quantifying with a series of b, y fragment ion pairs. Herein, we
extended the concept of isobaric peptide termini labeling to large-scale
quantitative analysis of protein ubiquitination. A novel MS2 fragment
ion based quantitative approach was developed, allowing the quantification
of ubiquitination at site level via isobaric K-ε-GG peptide
labeling, which combined metabolic labeling, K-ε-GG immunoaffinity
enrichment, and site-selective N-terminus dimethylation. The feasibility
of this proposed strategy was demonstrated through the ubiquitin proteome
analysis of differently labeled MCF-7 cell digests. As a result, 2970
unique K-ε-GG peptides of 1383 proteins containing 2874 ubiquitinated
sites were confidently quantified with high accuracy and sensitivity.
In addition, we demonstrated that quantification on MS2 fragment ion
level makes it possible to precisely quantify each individual ubiquitinated
lysine residue in 39 K-ε-GG peptides bearing two ubiquitination
sites by the use of specific ubiquitinated b, y ion pairs. It is expected
that this proposed approach will serve as a powerful tool to quantify
ubiquitination at the site level, especially for those multiubiquitinated
peptides.