Computational protocol: Characterization of the Mycobacterial Acyl-CoA Carboxylase Holo ComplexesReveals Their Functional Expansion into Amino Acid Catabolism

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Protocol publication

[…] All YCC genes from M. smegmatis () were amplified and cloned into a pKW08 mycobacterial tetracycline-inducible vector [,]. The amplicons were cloned upstream of the Tobacco Etch Virus (TEF) cleavage site, followed by an Enhanced Green Fluorescent Protein (eGFP) tag, and downstream of the TetR08 promoter. Cloning, expression, and a single-step protein purification protocol followed by LC-MS/MS analysis was applied as described []. Proteins were identified by searching the data against the M. smegmatis protein database (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). The database was modified to add randomized sequences of all entries as a control of false-positive identifications during analysis. Fragmentation spectra were searched using the Andromeda search engine integrated into the MaxQuant (v1.3.0.5) platform. Computational data analysis was performed using the Perseus tool (v1.3.0.4, Cox J., Max Planck, 2012). Contaminants and random protein identification were excluded. Proteins that were identified by less than two peptides were excluded from the result list. The proteomics data have been submitted to the Proteomicsdb database (https://www.proteomicsdb.org/proteomicsdb/#projects/4170). […]

Pipeline specifications

Software tools Andromeda, MaxQuant, Perseus
Databases ProteomicsDB
Application MS-based untargeted proteomics
Organisms Mycobacterium tuberculosis, Mycobacterium, Corynebacteriales
Diseases Tuberculosis
Chemicals Acyl Coenzyme A, Biotin, Fatty Acids