A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway
Figure 5
Tandem mass-spectrometry identification of −GG signature on active site serine based on peptide spectral matches from multiple charge states. In vitro E2 charging assays were performed on wildtype UbcH5c or C85S-mutant UbcH5c where the catalytic cysteine has been mutated to a serine residue to stabilize its interaction with bound ubiquitin. Ubiquitin-UbcH5c thioester (wildtype) and ester linked ubiquitin-C85S-UbcH5c (CS) were separated by SDS-PAGE and stained with Coomassie Blue (a). Regions denoted in red were excised from the gel, digested, and analyzed by LC-MS/MS. Peptide spectral matches for serine linked ggSP were identified for UbcH5c wild type (b) and for C85S-UbcH5c (c). Representative MS/MS spectra for several charge states are shown: (right panel) and (left panel). b- and y-fragment ions are denoted by blue and red, respectively. Green squares denote precursor ions, multiply charged fragment ions or predictable neutral loss ions accounted for during the manual inspection of MS/MS data.
