Re: Tips on the use of SEQUEST now available
Pat,
Thanks for your post! With regard to your point #1. I can redirect SEQUEST to databases stored on C: or D: drive and not in an Xcalibur directory. The search works but it looks like the reference links (to the database) in the HTML document still refer to the SEQUEST database install directory. That looks like a bug, maybe a "feature".
Point #2 is a good point. These indexed databases take up a lot of space. If you are searching small databases, you may find the speed increase of TurboSEQUEST not worth the hassle, especially since you can't do differential modifications with Turbo indexed for speed. I usually reserve my TurboSEQUEST searches for broad searches of the non-redundant databases.
I would probably index the databases using average mass. Likewise, when performing a search, I usually like to set the parent ion mass to average and the fragment to monoisotopic in the "ions and modifications" parameter box. The parent masses are determined from the normal mass spectra. Centroiding of the unresolved charge states will make the experimental mass of the peptide come out somewhere between the monoisotopic and average mass. It will be closer to average for higher charge state ions. However, the fragment ions are mostly singly charged, so monoisotopic is a better setting for fragment ions. I would set the peptide mass tolerance to 2-3 mass units in the parameters tab (the box at the very top). The default setting is 1, which is too low. You want to open up the mass tolerance on the front end of SEQUEST (which is what the peptide tolerance does), so as not to exclude something that is barely outside the mass tolerance. Sequest will figure it out on the back end.
I get 4 hits on human myoglobin with the trypmyo01 dataset when I search it against the human.fasta.bin file. Maybe the reason you are not seeing any hits is that your peptide mass tolerance is set too low? Let me know if this helps.
Mark
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