A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway
KR-TCRα peptide VAGFNLLMTLR (aa 254–264) is modified. HEK293T cells were incubated in either heavy or light SILAC media for five days and then transiently transfected with KR-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. (a) Gel pieces containing heavy, unmodified TCRα were combined with gel pieces containing light SILAC labeled, ubiquitin-modified KR-TCRα. Heavy and light proteins were digested with trypsin and analyzed by LC-MS/MS. (b) Seven narrow range full-MS spectra for representative KR-TCRα peptides are shown. Data were collected in high-resolution on an LTQ-Orbitrap, with light ions shown in red and heavy ions in blue. (c) HEK293T cells were incubated in either heavy or light SILAC media for five days, transfected with KR-TCRα or KR-TCRα-T262A, and the same experimental procedure was followed as in Table 1. The relative abundance of six peptides are quantified and represented in the bar graph. Numbers represent the area under the curve for the peptide in the high molecular weight region (corresponding to modified TCRα) divided by the peak height of the same peptide in the low molecular weight region (corresponding to unmodified TCRα).
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