The ubiquitin (Ub) modification pathway is a highly regulated, transient and reversible event that is conserved amongst eukaryotes. The covalent modification of cellular substrates with Ub plays a principal regulatory role in many cellular processes, such as proteasome-mediated degradation1, 2
, protein sorting3
. Ubiquitination occurs via the carboxyl terminus of the Ub glycine, which forms an isopeptide bond primarily with the ε-amino group of lysine residues on targeted substrates. This event is catalyzed by a cascade of enzymes that include Ub activating enzyme (E1), Ub-conjugating enzymes (E2s) and Ub-ligases (E3s)1, 6, 7
. The substrates can either be mono-ubiquitinated (mono-Ub) or poly-ubiquitinated (poly-Ub) at a single or multiple Lys sites. Polyubiquitin chains are assembled when additional Ub molecules are conjugated to any of the seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) or even N-terminal amine group of pre-existing Ub molecules8–10
. Conversely, deubiquitination enzymes (DUBs) remove Ub from modified substrates to further contribute to dynamic ubiquitination process11, 12
. Importantly, dysregulation of ubiquitination has profound impact on cellular functions and is involved in the pathogenesis of many diseases, including cancer and neurodegenerative disorders13, 14
, and the inhibition of ubiquitin-proteasome system has been demonstrated to be a successful strategy to treat multiple myeloma. Thus, methodologies that assist in the global analysis of Ub-conjugates are essential for the characterization of pathways that are regulated by ubiquitination.
Recent advances in the development of mass spectrometry (MS)-based technologies have allowed for the detection and quantification of hundreds to thousands of proteins with accuracy and sensitivity16–18
. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is typically used to analyze protein mixtures for large-scale proteomic applications and has become the preferred method for the analysis of ubiquitinated proteome19–22
. However, because ubiquitinated conjugates are present at a low steady-state level, due in part to proteasome-mediated degradation and highly active deubiquitination enzymes in cells, it is difficult to identify Ub-conjugates without prior enrichment. To this end, various affinity approaches have been used to isolate Ub-conjugates, including Ub-antibodies23, 24
, Ub-binding proteins25, 26
, and epitope-tagged ubiquitin derivatives (e.g. FLAG, HA-tag, myc
-tag, His-tag and biotin)8, 27–30
. Co-purification of unmodified proteins is controlled using extensive wash and/or stringent denaturing conditions (e.g.
8 M urea), but many are still identified in enriched Ub samples. During nickel affinity chromatography, the contaminants are usually found to be endogenous His-rich or highly abundant proteins8
. Additional efforts have also been made to reduce non-specific binding by introducing two-step affinity purification schemes28
. The presence of protein contaminants is often exacerbated when employing non-tagging affinity strategies (e.g.
Ub antibodies or Ub-binding proteins) under native conditions23
. Thus, it is critical to distinguish Ub-conjugates from false-positive contaminants before subsequent functional studies31
The common method for validating Ub-conjugates in large-scale proteomic analyses is the direct mapping of ubiquitination sites by MS/MS. Trypsin digestion of Ub-conjugates generates a di-glycine remnant (-GG, a monoisotopic mass of 114.043 Da) on modified lysine residues, producing unique MS/MS spectra that can be matched by database-searching algorithms8, 32, 33
. One technical challenge is that complete mapping of modification sites requires almost 100% coverage of proteins/peptides “sequenced” by MS/MS. Thus, in large-scale analysis from yeast, only a small fraction of GG-sites can be mapped to peptides, matching to less than 10% of the proteins identified21
. Therefore, secondary strategies are necessary to complement Ub site mapping to improve validation of large datasets.
Western blot analysis of immunoprecipitated Ub-conjugates is commonly used to confirm Ub-conjugates independently8
. Two principles are utilized in the method: (i) ubiquitination causes dramatic increase in apparent molecular weight (MW) in Western blot, as Ub-conjugates display an increase of approximately 8 kDa after mono-ubiquitination and an even larger increase after poly-Ub events; (ii) ubiquitination often generates heterogeneous modified substrates that display as a ladder on the Western blot. However, this type of analysis becomes expensive and impractical for large-scale studies in which thousands of Ub-conjugate candidates are identified.
Herein we describe a robust method for large-scale validation of protein ubiquitination based on virtual Western blots reconstituted from MS data. MW information of every protein identified was extracted after 1D SDS gel and LC-MS/MS (1D geLC-MS/MS). To evaluate false discovery rate of the method, two geLC-MS/MS analyses were performed before and after Ub affinity purification. Multiple statistical analyses were implemented to improve the approach. Finally, we found that only ~30% of identified proteins in the Ub-conjugate samples survived the MW filtering, even though they were purified in the presence of 8M urea, suggesting that false discovery rate in previously published datasets of ubiquitinated proteome may be underestimated.