Shotgun Proteomics of E. coli

Samples were loaded on a NuPAGE Bis-Tris 4%–12% gradient gel (Invitrogen) and the Coomassie stained bands were pooled and in gel digested with trypsin and GluC, respectively, as described elsewhere [62 (link)]. LC-MS analyses of the peptides were done on an EasyLC nano-HPLC (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) as described elsewhere [63 (link)]. MS data were processed using the software suite MaxQuant, version 1.2.2.9 [64 (link)] and searched using Andromeda search engine [65 (link)] against a target-decoy E. coli database containing 4,311 forward protein sequences, the sequences of the tagged and overexpressed proteins and 248 frequently observed protein contaminants. Trypsin or GluC, were set as proteases in which two missed cleavage sites were allowed. Carbamidomethylation of cysteine was set as fixed modification; N-terminal acetylation, methionine oxidation and serine/threonine/tyrosine phosphorylation were set as variable modifications. Initial precursor mass tolerance was set to 6 parts per million (ppm) at the precursor ion and 20 ppm at the fragment ion level. False discovery rates were set to 1% at peptide, phosphorylation site, and protein group level.