Computational protocol: Interdependence of Primary Metabolism and Xenobiotic Mitigation Characterizes the Proteome of Bjerkandera adusta during Wood Decomposition

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Protocol publication

[…] HPLC-MS/MS was carried out using electrospray ionization (ESI) ion trap mass spectrometry. Full experimental details can be found in the supplemental material (information S1). The B. adusta predicted proteome () was downloaded from the JGI website (http://genome.jgi.doe.gov/Bjead1_1/Bjead1_1.home.html) and used to construct a proteomic database for Mascot to search. This gave a database of predicted proteins, the amino acid sequence, and the genetic location, but it did not supply the protein identities. The spectra from each sample were compared against the bespoke Mascot database to identify individual proteins. During the Mascot search, a significance threshold of a P value of <0.05 was applied as part of the search criteria; hence, all proteins identified were included in the analysis. Furthermore, the use of a bespoke proteomic database ensured that false-positive identifications from organisms more abundantly represented within generic protein databases were eliminated. A table showing all the proteins detected by mass spectrometry, their proteome reference number (as relating to the JGI database), Mascot score, and peptide coverage are included in the supplemental material (Table S1). Each amino acid sequence was submitted to NCBI BLASTP (using a BLOSUM62 matrix), using only fungal sequences from the nonredundant protein database, with an E value cutoff of <1e−5. To characterize the proteins with unknown function, the CDD/SPARCLE domain analysis function () on NCBI was used to generate a map of any domains. Domain identifications on CDD had an E value cutoff of <1e−03, but each domain was also manually curated for accuracy. Each domain was then investigated using InterPro () to assign a putative function to the protein. In the case that more than one domain was found, each type of domain was treated as a separate entity and assigned a function. […]

Pipeline specifications

Software tools BLASTP, InterPro
Databases SPARCLE
Application Protein sequence analysis