Saturday, October 12, 2013

Iterative searching in Proteome Discoverer!

Want to boost your peptide IDs in Proteome Discoverer as well as the confidence of those IDs?  Move from your regular search to iterative searching.  I looked it up (it·er·a·tive ( t -r t v, - r- -t v). adj. 1. Characterized by or involving repetition, recurrence, reiteration, or repetitiousness ).

Here is the gist of it.  You take that file and you give it different instances of search engines with different combinations of likely modifications.  For example, if your peptide is one that has acetylation and phosphorylation, you need to have both of those mods in that search, but it is impossible to have every combination (or even very many) in one search engine.  Using multiple engines allows you to search more modifications.  It also allows you to get better confidence on your ID'ed peptides.  For example, if Mascot, Sequest and MSAmanda all give you a peptide spectral match, then it is probably more likely to be true than if only one of the engines give it to you.  So your confidence increases.

How are the results?

On each sample, this iterative (comprehensive search) increased the number of ID'ed peptides.  By a lot.  Yes, at first it seems crazy, but it sure looks like it works.  For full information, look for this on Planet Orbitrap:


  1. Hi Ben,
    I was wondering whether PD takes care of same scan ID identified with different peptide with/without other modifications?
    - Santosh

  2. Santosh,
    Good question. Ultimately, the search engines are going to disagree once in a while, especially when looking at lower res MS or MS/MS spectra. Due to the basic construction of Sequest (and we assume, Mascot as well, but no one really knows), if all things are equal between two spectral matches, the one with the PTM will still end up with a lower score. From a biological perspective it makes sense, as PTMs tend to have a low stoichiometry as compared to their non-modified counterparts.