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Peptide match setting

Posted: Mon Jul 14, 2014 10:36 pm
by Biomarker
Hi,

Can anybody please shade some light on the setting of Q Exactive about peptide match on or preferred mode??? Does it affect the number of identifications?

cheers..
Biomarker

Posted: Tue Jul 15, 2014 12:08 am
by gadsouza
What it does I am not sure. But I have compared acquisition using a digest of Jurkat. What I can see is that when that option is off, the instrument does a lot more MS2 scans, (with a 4 hour gradient number went from 60,000 MS2 scans acquired to 85,000 or so).

However, those addtional 25,000 MS2 only increased the number of PSMs identified by 1,000 or so. So at least in my setup the gain in identification was marginal because probably all the additional MS/MS had low quality. I hadn't test if I loaded more sample that could result on a higher number of identified PSMs.

Posted: Fri Jul 25, 2014 4:54 am
by botte
Dear Biomarker,

the explanation according to our Thermo trainers: the Peptide Match option looks at the isotopic pattern observed in MS and compares them with the expected isotopic pattern for a peptide of that mass. Peptides are a very homogeneous compound class, so the isotopic pattern more or less scales with the molecular weight and is easy to predict.

If the option is set to ON, the SW will only fragment MS precursors that show a good match to a theoretical isotopic pattern. If it is set to PREFERRED, it will first take the peptide-like precursor pattern for MS, but then fill up the available Top N capacity with other precursors as well that match the other DDA criteria.

We tested both settings back and forth after installation of our Q Exactive, and got significantly better results with PREFERRED. Reason is probably that the observed isotopic patterns in real data do not always have the necessary statistics or purity to match to theory. How big the difference is will depend on the complexity and abundance of the samples though, and yes, it does also increase the number of "useless" MS/MS spectra as well.

Hope this helps?

Christof

Posted: Mon Jul 28, 2014 5:26 am
by Biomarker
Clear now. Thanks Christof for the information...