Computational protocol: Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

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Protocol publication

[…] RAW spectra were submitted to an Andromeda search in MaxQuant (version 1.5.3.30) using the default settings. Label-free quantification and match-between-runs was activated. MS/MS spectra data were searched against the respective reference databases downloaded from the Uniprot website (Sulfolobus acidocaldarius (TaxID 330779): UP000001018_330779.fasta (2,221 entries); Haloferax volcanii DS2 (TaxID 309800): UP000008243_309800.fasta (3987 entries); Sulfolobus solfataricus P2 (TaxID 273057): UP000001974_273057.fasta (2938 entries); Thermotoga maritima (TaxID 243274): UP000008183_243274.fasta (1852 entries)). All searches included a contaminants database (as implemented in MaxQuant, 267 sequences). The contaminants database contains known MS contaminants and was included to estimate the level of contamination. Andromeda searches allowed oxidation of methionine residues (16 Da) and acetylation of the protein N-terminus (42 Da) as dynamic modifications and the static modification of cysteine (57 Da, alkylation with iodoacetamide). Enzyme specificity was set to ‘Trypsin/P'. The instrument type in Andromeda searches was set to Orbitrap and the precursor mass tolerance was set to ±20 p.p.m. (first search) and ±4.5 p.p.m. (main search). The MS/MS match tolerance was set to ±0.5 Da. The peptide spectrum match FDR and the protein FDR were set to 0.01 (based on target-decoy approach). Minimum peptide length was seven amino acids. For protein quantification unique and razor peptides were allowed. Modified peptides were allowed for quantification. The minimum score for modified peptides was 40. […]

Pipeline specifications

Software tools Andromeda, MaxQuant
Application MS-based untargeted proteomics