PEAKS Multi-Round Search


Video Dialogue

Multi-Round search is a very helpful feature found in PEAKS. It is designed to help you identify spectra unmatched by an initial database search; however they matched promising de novo sequences. We call these results de novo only spectra. These can be found in any identification result by clicking on the ‘de novo only’ tab. This tab contains spectra that could not be matched by PEAKS DB, PEAKS PTM, or SPIDER. However, using de novo sequencing these spectra can still have excellent peptide spectrum matches. This makes us question why these good peptides are missed by database search.

Multi-Round search gives us the opportunity to answer that question. It takes the de novo only spectra and separates them from the scans already matched using a database search algorithm. It allows you to search just those spectra with different parameters. So, you can choose a new database, different ptms, different enzyme cleavage rules, the possibilities are endless.

For example, take this spectrum from a human antibody sample. Searching it against Swissprot we are able to identify a peptide sequence but not very confidently. The peptide does not come from an antibody protein, it is a poor peptide spectrum match, and it has a low score below our 1% false discovery threshold. The de novo peptide spectrum match is much better. It explains the majority of the high intensity peaks with major fragment ions. Using Multi-Round search, this spectrum will be carried forward to the next round. We searched the de novo only spectra identified in the original Swissprot search against NCBI. With our example spectrum, an exact match to the peptide sequenced by de novo was found in an antibody protein. Here is a summary of the hits we were able to find with this spectrum. Searching Swissprot we identified an unexpected protein with a low score, likely a false positive. Now with the new Multi-Round search we achieve ideal results, where we identified a peptide from an antibody protein with a high score and no mutations.

One of the best applications of this new search type is filtering out contaminant proteins. For example, you can download a contaminant database like cRAP shown here. First do a search with the contaminant database. Here you can see that we were able to identify a few contaminants in this dataset. These scans will be removed from the Multi-Round search. Then when you search your target database, this will give you a list of protein identifications without contaminants. Another good benefit of Multi-Round search is limiting your search space. By filtering out identified spectra, you can limit your search space in order to identify difficult targets. For example you can identify endogenous peptides in a digested sample. First run a search using the enzyme you used to digest the sample. Then run a Multi-Round search with no enzyme to find endogenous peptides.

To actually set up a Multi-Round search, all you have to do is select an existing database search result and click the Multi-Round search button. This will bring up a new search parameters pane where you can select any new parameters you wish to search the de novo only results with. Keep in mind; the de novo only results are controlled by the filters you set in the summary view of the initial search. Scans with identification results below the database search filters, and above the de novo ALC filters will be included in the de novo only results. So, check to make sure you are satisfied with those settings before starting your Multi-Round search.

That’s all you need to know to get started with Multi-Round search in PEAKS. Thanks for listening!