Eastman, Samuel (2024), "IssA protein LC/MS/MS"
Description
Protein LC/MS/MS. In-gel digestion of two separate IssA protein bands using trypsin was performed as in Shevchenko et al. Files were searched against E. coli (K12) database downloaded from Uniprot.org with both the full-length IssA and IssA beginning at T157 protein sequences added. Both size bands isolated in the study resulted in identical protein coverage maps. Note: in older versions of this study pre-publication IssA was internally referred to as "Dis1". This may be reflected in some of these files. Data informs Figure S4B. Number_1.raw = upper band in figure S4A Number_2.raw = second band from top in figure S4A.
Files
Steps to reproduce
In-gel digestion of two separate IssA protein bands using trypsin was performed as in Shevchenko et al. Trypsin digested samples were dried completely in a SpeedVac, resuspended with 20 µl of 0.1% formic acid pH 3 in water, and 2 µL (~ 360ng) was injected per run using an Easy-nLC 1200 UPLC system. Samples were loaded directly onto a 45cm long 100um inner diameter nano capillary column packed with 1.9um C18-AQ resin (Dr. Maisch, Germany) mated to metal emitter in-line with an Orbitrap Fusion Lumos (Thermo Scientific, USA). The column temperature was set at 45 °C with a 1-hour gradient method and 360 nL/min flow rate. The mass spectrometer was operated in data dependent mode with the 120,000 resolution MS1 scan (positive mode, profile data type, AGC gain of 4e5, maximum injection time of 54 sec and mass range of 375-1500 m/z) in the Orbitrap followed by HCD fragmentation in ion trap with 35% collision energy. A dynamic exclusion list was invoked to exclude previously sequenced peptides for 60s and maximum cycle time of 3s was used. Peptides were isolated for fragmentation using quadrupole (1.2 m/z isolation window). Ion-trap was operated in Rapid mode. Raw files were searched using Sequest-HT algorithms 73 within the Proteome Discoverer 2.5.0 suite (Thermo Scientific, USA). 10 ppm MS1 and 0.6 Da MS2 mass tolerances were specified. Carbamidomethylation of cysteine was used as fixed modification, oxidation of methionine, deamidation of asparagine and glutamine were specified as dynamic modifications. Pyro glutamate conversion from glutamic acid and glutamine were set as dynamic modifications at peptide N-terminus. Acetylation was specified as dynamic modification at protein N-terminus. Trypsin digestion with maximum of 2 missed cleavages were allowed. Files were searched against E. coli (K12) database downloaded from Uniprot.org with both the full-length IssA and IssA beginning at T157 protein sequences added.