Number of Output Parameter

[Natalie Heller]

Hi all,

The parameter num_output_lines controls the number of search results hits (peptides) that are reported for each spectrum query.  In my parameter file, I have this set to 100, I would like to have the maximum number of hits reported.  But when I run my .mzML file through Comet, it only reports one hit.  Is there another parameter that I should be changing as well?  I also have num_results set to 100.

Thanks!

Natalie

[Jimmy Eng]

Natalie,

What output format are you looking at and are you using Comet version 2016.01 rev. 3?  The Percolator .pin output only reports the top hit per spectrum query but the rest of the output formats should report multiple peptides per spectrum.  Attaching your comet.params file might help to clarify what's happening.

Jimmy

[Natalie Heller]

Hi Jimmy,

I am using 2016.01 rev. 2, I would like to keep using this older version, for repeatability reasons for the project I am working on.  I have attached the parameter file that I have been using.  Thanks for all your help!

[Jimmy Eng]

Natalie,

2016.01 rev. 2 is fine; the changes in rev. 3 shouldn't be pertinent here.  I did use your parameters file and Comet 2016.01.2 to search a random raw file that I had against a human database just to confirm I was getting up to 100 search hits per spectrum query which I did see.  There are definitely entries where there are less than 100 hits reported because of a combination of (a) there are less than that many peptides in the database within the precursor mass tolerance and (b) the peptides must have a positive, non-zero cross-correlation score to be reported.  Negative cross-correlation scores can occur when you have very bad matches to very poor spectra.

I'm happy to investigate more but would need your fasta file, the mzML file, and a copy of your pep.xml search result.  If you could make those available to me through some file share like Dropbox or OneDrive, I'll hopefully be able to figure out why you're getting only single hits out.

[Natalie Heller]

Does it matter that the database that I'm using only has two proteins in it?

[Jimmy Eng]

If you have just 2 proteins, there's only a small number of tryptic peptides in it.  And for each query that you're searching using a 20 ppm tolerance, you probably will just have 1 peptide at most within that mass tolerance.  I would guess a lot of your spectra have no peptides that fall within the precursor tolerance.  Hope this makes sense.