A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway
Tandem mass-spectrometry identification of −GG signature peptides from ubiquitinated TCRα on Lys-118, Lys-144, and Lys-178. HEK293T cells were transiently transfected with WT-TCRα or vector alone, incubated with MG132, and then lysed. Lysates were subjected to immunoprecipitation with an anti-HA antibody and examined by SDS-PAGE followed by Coomassie staining. Four regions of the gel were excised and trypsinized as shown in (a). Samples were analyzed by tandem mass-spectrometry and the results are summarized in (b). Peptide spectral matches to −GG signature peptides (ggSP) at Lys-118, Lys-144, or Lys-178 in each region of the gel are indicated by red shading. Representative MS/MS spectra for each of the ggSP are shown: (c) Lys-118, (d) Lys-144, and (e) Lys-178. The peaks are labeled with the fragment ion and its charge state. For example, y14-1 is the singly charged version of y14. 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 manual inspection of MS/MS data.
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