Peptide Mutations & Homology Searching

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Why not BLAST?

It should be pointed out that the general homology tool such as BLAST is not the best option for searching with de novo sequence tags. It is very common that some fragment ions are missing from a peptide’s MS/MS spectrum, leading to possible de novo sequencing errors. Thus, an appropriate de novo tag homology search should tolerate common de novo sequencing errors such as (AT/TA) and (N/GG). However, being designed for a different purpose, BLAST penalizes those errors too much and may significantly reduce the search sensitivity [2]. Moreover, BLAST will not attempt to reconstruct the real peptide sequence.

Besides the apparent mutation detection and cross-species search function, a very useful application of SPIDER is to use it iteratively to sequence a complete protein (e.g. antibody sequencing). This is achieved by:

  • Using PEAKS’ standard workflow (de novo + PEAKS DB + PEAKS PTM + SPIDER) to search in a homologous database. This will identify a homologous protein.
  • Then in the coverage pane, select tools “copy mutated protein sequence”. This will copy the mutated protein sequence (after applying the confident mutations) to Windows’ clipboard.
  • Invoke another standard search by paste the copied sequence as the protein database.
  • Repeat the above procedure multiple times to gradually improve the sequence quality.

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