Mass Spectrometry-Based Protein Identification

Samples from immunoprecipitates were reduced, alkylated, and loaded onto SDS-polyacrylamide gel electrophoresis gels to remove LC-MS incompatible reagents. Gel plugs were excised, destained, and subjected to proteolytic digestion with trypsin and resulting peptides extracted and desalted, as previously described^{26 (link)}. Aliquots of the peptides were analyzed with LC-MS using a 60 min gradient on an EASY nLC 1000 coupled to a ThermoFisher Scientific Orbitrap Elite Hybrid Ion Trap Mass Spectrometer. The data were searched against a UniProt human database using Sequest within Proteome Discoverer. The results were filtered to only include proteins identified by at least two peptides. Protein quantitation was preformed using the ratios of PSMs in DAP5, eIF4GI, and eIF4GII AP over the PSMs in the control AP. In addition, the data were analyzed using the SAINT algorithm^{27 (link)}, including experiments 52–54 from the crapome.org database^{28 (link)} as additional controls.