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How to prepare a protein database

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Sarah Haynes edited this page Oct 26, 2020 · 4 revisions

Proteomics pipelines and toolkits like Philosopher rely on properly formatted protein sequence databases to correctly identify peptides. Here are some tips on how to prepare a protein database for your experiment.

If you do not have a protein sequence database: --id

Run Philosopher from the command line to download one from UniProt by executing the following two commands:

philosopher workspace --init
philosopher database --reviewed --contam --id UP000005640

This will generate a human UniProt/SwissProt (i.e. reviewed sequences only) database, with common contaminants and decoys added (with a default decoy prefix rev_). If you would like to use the full UniProt, remove the --reviewed tag.

For mouse, for example, use the proteome ID UP000000589. To find the proteome ID for other organisms, search within the UniProt proteomes.

If you have your own database, without decoys and contaminants: --custom

Add decoys and contaminants and format it for FragPipe using the following commands:

philosopher workspace --init 
philosopher database --custom <file_name> --contam

If you have your own database, with decoys and contaminants: --annotate

Reformat it for FragPipe using the following commands:

philosopher workspace --init 
philosopher database --annotate <file_name> --prefix <prefix>

Header Formatting

If you need to run the --custom or the --annotate command, you may manually inspect the formatted files to ensure it will be compatible with Philosopher, it should follow one of these formats (see example for each):

  • UniProt: >sp|P02489|CRYAA_HUMAN Alpha-crystallin A chain OS=Homo sapiens OX=9606 GN=CRYAA PE=1 SV=2

  • NCBI: >NP_000385.1 alpha-crystallin A chain isoform 1 [Homo sapiens]

  • ENSEMBL: >ENSP00000291554.2 pep chromosome:GRCh38:21:43169008:43172805:1 gene:ENSG00000160202.7 transcript:ENST00000291554.6 gene_biotype:protein_coding transcript_biotype:protein_coding gene_symbol:CRYAA description:crystallin alpha A [Source:HGNC Symbol;Acc:HGNC:2388]

  • or generic: >P02489

If you are adding you own decoys, they also need to follow a specific formatting; sequences need to be formatted as a whole protein string in FASTA file with a decoy (e.g. rev_ or DECOY_) added at the beginning.

Examples of compatible decoy formats:

  • >rev_tr|J3KNE0|J3KNE0_HUMAN
  • >DECOY_tr|J3KNE0|J3KNE0_HUMAN

Examples of incompatible decoy formats:

  • >tr_REVERSED|J3KNE0|J3KNE0_HUMAN
  • >tr|fake_J3KNE0|J3KNE0_HUMAN RanBP2-like
  • >tr|J3KNE0_DECOY|J3KNE0_HUMAN
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