Reliable identification of protein-protein interactions by crosslinking mass spectrometry_AP-MS

Quantitative proteomics dataset on soluble, affinity-enriched proteins from SPA-pulldowns in E. coli K12, on rpoB-SPA / nusG-SPA / yacL-SPA. This data was used to identify proteins for enrichment analysis and subsequent crosslinking mass spectrometry search (DB creation). Cleared E. coli lysate was added to anti-FLAG M2 beads to isolate binders to rpoB (RNA polymerase), nusG or yacL (RNA polymerase binders. Elution was accomplished via TEV protease cleavage. A mock K12 E.coli lysate ETV elution was used to assess protein enrichment in the obtained (specific) eluates. The proteins in each fraction were precipitated using acetone, derivatized and proteolysed in-solution. Each sample was acquired as injection triplicate via LC-MS on Q Exactive HF and the raw data searched with MaxQuant with LFQ and iBAQ quantitation enabled. Further processing was done using Perseus.