Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis

Carbon metabolism in Mycobacterium smegmatis was analysed using reverse-phase chromatography coupled to orbitrap high-resolution mass spectrometer using trypsin digestion. MS/MS files were searched against the Mycobacterium smegmatis strain MC2-155 database Release 1 - July 2010 ) by MaxQuant software (version 1.3.0.5). Mass spectra were searched with an initial mass tolerance of 7 ppm in MS mode and 0.5 Da in MS/MS mode. Up to two missed cleavages were allowed. Carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (Protein N-term) and Phospho (S, T, Y) were considered as variable modifications. Minimum required peptide length was set to seven amino acids and at least two (unique + razor) peptides were required for protein identification. A cut-off was fixed at 1% FDR at the peptide and protein level. Reverse and contaminants sequences were removed and proteins with a Posterior Error Probability (PEP) lower than 0.1 were accepted for further data treatment. Protein quantification was performed with razor and unique peptides, using only unmodified, oxidated (M) and acetylated (Protein N-term) peptides. A minimum of two ratio counts was required to quantify proteins.