Hi Srikanth,
You can use as many as you want, and in Skyline-daily, you can currently choose non-linear alignment, such as Loess. I am not sure what makes you feel "it takes 25". I have built iRT libraries with around 75 to 100 peptides, including for this paper.

https://www.ncbi.nlm.nih.gov/pubmed/30510204

I would expect that you could more or less follow the iRT tutorial to train your own iRT standards in much the same way the tutorial does:

https://skyline.ms/tutorial_irt.url

But, I will also ask Kaipo who has recently improved this workflow (including adding the Loess regression) to make sure you understand how to do this with your choice of Skyline 20.1 or Skyline-daily.

It is definitely possible and should not be a blocking issue.

Thanks for posting to the Skyline support board.

--Brendan

Hi Srikanth,
Was the tutorial Brendan linked helpful? If you are still having issues, can you provide a screenshot of the error message you are seeing?

Thanks,
Kaipo

Hi Brendan and Kaipo,

Thanks for the response. I had seen the tutorial and is quite informative, but I am still not clear as to how to use your own peptides to create a library. The tutorial shows how to use all the peptides as results and calculate iRTs. I have results as mzid file. When I import into skyline to build the library, I am still not sure as to how to select some of the peptides (my endogenous RTs) and use them as standards rather than the ones provided. Do I need to also load the SWATH runs and the find the peptides of interest?
I want to export the results as a report (OpenSWATH) to analyze with OpenSWATH.

Any help appreciated.

Regards,
Srikanth

Hi Srikanth,
To use a non-predefined set of iRT peptides, you can manually set them in the Edit iRT Calculator dialog.
If the peptides aren't already in your document, you can add them from a spectral library through View -> Spectral Libraries -> Add / Add All. I would recommend having all the iRT peptides in a single peptide group. Then from the Edit iRT Calculator dialog, you can click "Choose Standards..." and select that peptide group to use.
I hope that is helpful, let me know if it works for you.

Thanks,
Kaipo

Hi Kaipo,

Thanks for the information. I am able to add non-predefined set using the method you mentioned.
I am more interested in just creating DDA spectral library and export the library in openswath/peakview format with the iRT columns.
Do you have any tutorial which tells how to ONLY create a spectral library using DDA files (without any use of DIA data) and exporting the report in some format.

Srikanth

Hi Srikanth,
I'm not sure about the details of openswath/peakview formats - can you provide more details on which information/columns you'd like to export from spectral libraries? They are SQLite databases, so any data in a library can be extracted via SQL queries.

Thanks,
Kaipo

Hi Kaipo,

I am interested in these columns:
ˆ PrecursorMz: precursor m/z
ˆ ProductMz: fragment m/z
ˆ Tr recalibrated: retention time
ˆ ProteinName: protein name
ˆ GroupLabel: isotype type
ˆ LibraryIntensity: fragment ion intensity
ˆ PeptideSequence: peptide sequences without modifications
ˆ FullUniModPeptideName: peptide sequences with modifications
ˆ UniprotID: database accession number
ˆ decoy: whether the peptide a decoy or not
ˆ PrecursorCharge: precursor charge
ˆ FragmentType: fragment type
ˆ FragmentCharge: fragment charge
ˆ FragmentSeriesNumber: fragment ion number

Regards,
Srikanth

Hi Kaipo,

Sorry to bother you, but I realized what I really want is to be able to just do the following (using Skyline or CMD):

1. Import search engine results as mzid and build library using BliBuild
2. Filter using BlibFilter
3. And export the result in OpenSwath or Peakview compatible format (columns as mentioned earlier).

Any help would be appreciated.

Regards,
Srikanth

Hi Srikanth,
Unfortunately this workflow is not possible with BiblioSpec because several of these fields (and decoy peptides) are not included in the spectral libraries. If you have familiarity with C++, you may be able to use the ProteoWizard library (what BiblioSpec/Skyline use) to do this: http://proteowizard.sourceforge.net/
I think this part of the documentation would be relevant: http://proteowizard.sourceforge.net/dox/namespacepwiz_1_1identdata.html

Thanks,
Kaipo

Hi Kaipo,

I am not an expert in C++, but I did look at the all the tables and information in the SQL database (bibliospec). I can use the OpenSwath report template attached and export the report with most of the columns except the observed RT. Can you tell me where should I be looking /or adding to this template to to get the observed RT values, instead of the calibrated RT.

Regards,
Srikanth

Hi Srikanth,

Does Peptide, Average Measured Retention Time work for you?

Thanks,
Kaipo

Hi Kaipo,

I get only #N/A for all the targets with Average Measured Retention Time. I just want the observed retention time for the peptides. I could see that in the SQL tables but not once imported.

Srikanth