Ubiquitinated proteome: ready for global?

Abstract

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms.

Fig. 1.

A schematic presentation of three different strategies to isolate ubiquitinated substrates. A, The affinity purification strategy using Ub tag that allows purification under proteome denaturing condition. B, Tandem Ub binding domain for endogenous substrate enrichment. C, Poly-Ub chain specific antibody or ubiquitination signature remnant antibody. D, Three different contaminants of either protein precipitation during incubation, nonspecific binders to the matrix, or specific binding proteins to the affinity bait and poly-Ub conjugates exist during purification process.

Fig. 2.

A comparison between Sequest and Mascot for ubiquitination identification at 1% FDR.

Fig. 3.

Four sample MS/MS spectra misassigned for ubiquitination filtered at 1% FDR using Mascot. Note all the spectra have good ion score (25–120). A, A peptide spectrum with C-terminal Asn (N) results in false-positive ubiquitination assignment as C-terminal lysine. B, Two Cys (C) alkylation (+57.021 Da) leads to false positive ubiquitination identification. C, An ambiguous ubiquitination spectrum where major fragmentation peaks do not match. D, A false postive ubiquitination assignment rejected by precursor mass deviation and low peptide coverage.

Fig. 4.

A–C, MS/MS spectra of K11, K48 and K63 ubiquitination tryptic peptides conjugated with larger remnant (LRGG).