Tryptic in-gel digests gained from cross-linked and enriched protein mixture were analyzed, whereas a comparison between protein mixtures incubated with and without biotin-SG-Api88(Y7B) was achieved. Samples were analyzed on a nanoACQUITY UPLC system (Waters Corp., Eschborn, Germany) coupled online to an electrospray ionization quadrupole-time-of-flight mass spectrometer (Synapt G2-Si MS, Waters). The acquired data were processed using PEAKS Studio 10.6 Xpro (Bioinformatics Solutions, Waterloo,
...Canada). Database searches were performed using an E. coli database downloaded from uniprot (March 28, 2020) containing 4392 protein sequences and additionally the sequences of Api88, mCherry, mAzamy, trypsin, and streptavidin, with a false discovery rate of 1% at the peptide level. The data were exported as peptides.pep.xml files and loaded to Skyline (64-bit, version 22.2.0.351, MacCoss Lab Software, Washington, US). A spectral library was built from all identified peptides.
Proteins identified in both replicates were considered enriched based on the peak area ratios obtained in 70S ribosome samples UV-irradiated in the presence or absence (control) of biotin-SG-Api88(Y7B).[Show more]