Fig. 1

Experimental approach and computational analysis for the assessment of ubiquitin occupancy and total protein ratios. a Experimental approach: SILAC LC–MS/MS was used to identify changes in the ubiquitinome of SKOV3 ovarian cancer cells in response to 26S proteasome inhibition by MG132. Cells were cultured in either light or heavy (containing isotopically labeled arginine and lysine residues) RPMI 1640 media. Light cells were treated with either DMSO negative control of MG132 26S proteasome inhibitor, while cells grown in heavy media remained in a native, untreated state. Light and heavy lysates were combined in a 1:1 ratio and after trypsin digestion were either fractionated by bRPLC or ubiquitin-enriched and then fractionated, corresponding to the global and ubiquitinome data sets respectively. Peptides in the global and ubiquitin-enriched samples were detected by LC–MS/MS analysis, which successfully distinguished peptides as originating from the treated (light) or native (heavy) samples based on their m/z ratio. b Partially ubiquitinated peptides can exist in one of two forms, ubiquitin occupied or non-ubiquitinated and the percent abundance of both has to equal 100%. Relative ubiquitinated, non-ubiquitinated and protein ratios (Rub, Rnon-ub and Rprotein) were calculated for all partially ubiquitinated peptides in the MG132 treated (State 2) vs. native (State 1) condition. These rations were subsequently used to determine percent ubiquitin occupancy in State 1 (Pub1), which was then utilized for calculating percent ubiquitin occupancy in State 1 (Pub2)