5.3.4. Peptide and Protein Identification

The MaxQuant software (version 1.6.0.16) was employed for peptide/protein identification from the raw MS data.⁴⁸ The raw mass spectral data were searched against the Uniprot Mtb protein database containing 3993 protein sequences (Proteome ID: UP000001584, Organism ID: 83332) concatenated to reverse decoy database and protein sequences for common contaminants. Trypsin [KR].[^P] was specified as a cleavage enzyme with up to two missed cleavages. The “re-quantify” and “match between runs” options were utilized with a retention time alignment window of 3 min. Carbamidomethylation of cysteine residues was specified as a fixed modification and acetylation on protein N-terminal, conversion of N-terminal glutamine and glutamic acid to pyroglutamic acid, and oxidation of methionine were set as the variable modifications.

Both unique and razor peptides were used for the quantification of protein abundance. Peptides with a minimum length of seven amino acids and detected in at least one or more of the replicates were considered for identification. For protein identification, a minimum of two peptides, of which at least one was unique, was required per protein group. All other parameters in MaxQuant were set to default values.