Computational protocol: Unravelling the Structural and Molecular Basis Responsible for the Anti-Biofilm Activity of Zosteric Acid

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Protocol publication

[…] Excised bands and spots were subjected to trypsin digestion as previously described [] and the trypsin digested peptides were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) using the same HPLC and LTQ Orbitrap mass spectrometer previously reported in “Matrix hydrolysis and analysis of hydrolysates”. Reverse-phase chromatography was performed on the same column and at the same flow rate but under different chromatographic conditions: linear gradient 1.6 to 44% acetonitrile in water with 0.1% formic acid over 40 min and total LC-run of 61 min. Mass spectra were collected in positive ion and data dependent scan mode (MS scan at 60000 of resolution in the Orbitrap, mass range 300–2000 m/z, and MS/MS scan on the three most intense peaks in the linear ion trap). Selected peptide charge states were isolated with a width of m/z 2, and were activated for 30 msec using 35% normalized collision energy and an activation q of 0.25. Protein identification was achieved using the embedded ion accounting algorithm (Sequest HT) of the software Proteome Discoverer (version 1.4, Thermo Fisher Scientific) after searching a UniProtKB/Swiss-Prot Protein Knowledgebase [release 2013_12 of 11-Dec-13; taxonomical restriction: Escherichia coli (strain K12), 4431 sequence entries]. The search parameters were 10 ppm tolerance for precursor ions and 0.6 Da for product ions, 1 missed cleavage, carbamydomethylation of cysteine as fixed modification, the oxidation of methionine as variable modification and on a decoy database search calculated false discovery rate under 5%. […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Databases UniProt UniProtKB
Application MS-based untargeted proteomics
Organisms Escherichia coli
Chemicals Hydrogen, NAD