some clarifications needed for SILAC

support
some clarifications needed for SILAC zongtao  2018-11-07
 

hi, skyline team

  1. "filter for document peptides". if i have multiple mzXML files and one mzid file. will this function stop extracting a peptide in mzid file from a specific mzXML file that does not contain identification of this peptide?
  2. "use hign-selectivity extraction". when using orbitrap fusion, what are the differences (parameter criteria) between checking this function and not using it?
  3. "min peptides per protein". what is the definition of peptides here? if a peptide in both heavy and light forms showed their intact form (charge 2 and 3 identified) and Met-oxidized form (charge 2 and 3 identified), will skyline consider this case as one peptide or multiple (how many?)?
  4. "remove duplicate peptide" and "remove repeated peptide". what are the differences between these two?
  5. see attached partial data from report export. for this peptide, the total areas of the first 6 rows are different from the last 6, while the other parameters are all the same. what is your explanation of such scenario?
 
 
Nick Shulman responded:  2018-11-07
1. When you check the "Filter for document peptides" checkbox on the Build Library Dialog, the resulting .blib file (spectral library) will only contain the peptides that are in your Skyline document.

2. When you check the "Use high selectivity extraction" checkbox on the Transition Settings Full Scan, Skyline will sum intensities across an m/z channel that is half as wide. Checking that checkbox is the same as telling Skyline that your resolution is a number that is twice as large. For Thermo data, I believe we actually recommend that you choose "Centroided" as the mass analyzer, since that will use Thermo's centroiding algorithm, which does a better job of separating things in the spectrum that have m/z's that are close to each other.

3. In terms of the "Min peptides per protein" setting, modified forms of the same peptide count as separate peptides, but heavy/light forms of the peptide count as one peptide. Skyline is just counting up the number of items at the second level in the Targets tree.

4. If you hover the mouse over the "Remove repeated" and "Remove duplicate" items, it gives you a tooltip which should explain the difference. "Remove duplicate peptides" removes all instances of peptides that appear more than once. "Remove repeated" leaves the first occurrence of the peptide, but removes all subsequent ones.

5. I don't know the answer, but if you send us your Skyline document we can take a look.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms and spectral libraries.
If that .zip file is less than 50MB, you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url

-- Nick