Hi Fengchao,

This doesn't look like the OpenSWATH files we have seen in the past. Normally we expect these columns:

• filename
• RT
• FullPeptideName
• Charge
• m/z
• decoy
• aggr_Peak_Area
• aggr_Fragment_Annotation
• m_score

and optionally these ones:

• ProteinName
• leftWidth
• rightWidth

Additionally, to build spectral libraries, Skyline requires results files that have some type of probability scores (e.g. q-values) and a way to map results to a spectrum file (e.g. filename and scan number). If you have that information, it should be possible to generate a generic ssl file (https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec input and output file formats) that can be used to build a library.

Thanks,
Kaipo

Hi Kaipo,

Thanks for your reply.

I am trying another approach to import this library file through File -> Import -> Assay Library. Unfortunately, I get another error says: Precursor m/z 300.8289 does not match the closest possible value 295.4972 (delta = 5.3317), peptide MALLHSGR. Check the Modification tab in the Peptide Settings, the m/z types on the Prediction tab, or the m/z match tolerance on the Instrument tab of the Transition Settings.

The corresponding row is

It seems that Skyline cannot recognize the Unimod notation (UniMod:35).

Could you please tell me how to solve this issue?

Thanks,

Fengchao

Hi Fengchao,

First you will need to ensure that the necessary modifications are added (through Settings > Peptide Settings > Modifications, as the error mentions).

Then, normally you would be able to use "[UniMod:##]" format but it looks like there is a bug preventing this from working. As a workaround for the time being you can use a text editor to find and replace the UniMod strings with the modification masses, e.g.:

• oxidation: replace "(UniMod:35)" with "[+16.0]"
• carbamidomethylation: replace "(UniMod:4)" with "[+57.0]"

And retry the import after the replacements have been made.

Thanks,
Kaipo

Hi Kaipo,

Thanks for your prompt reply.

Your suggestions work for those modifications now. But I have new errors:
Precursor m/z 351.1602 does not match the closest possible value 350.4883 (delta = 0.6719), peptide EREMAEMR. Check the Modification tab in the Peptide Settings, the m/z types on the Prediction tab, or the m/z match tolerance on the Instrument tab of the Transition Settings.

The row is like

The m/z difference (351.1602 vs 350.4883 ) is from isotope errors (we have isotope error 0/1/2 during the search). Could you please provide any suggestions or solutions to this issue?

Another question I would like to ask is that do you have any suggestion regarding the library format that can be natively supported by Skyline? The file I am using is from EasyPQP without any file name or scan name information because those are consensus spectra.

Thanks,

Fengchao

Hi Kaipo,

I figured out the isotope error issue. But I have another one (sorry that I have so many questions):
Failed to explain all transitions for SVVLMSHLGRPDGVPMPDK m/z 513.5158 with a single set of modifications

The row is

The b14^2 ion do have 724.8876 Th. I am not sure what causes this error.

Thank in advance for your help.

Best,

Fengchao

Hi Fengchao,

I don't see that line in the tsv - is it from the same one you uploaded earlier?

Thanks,
Kaipo

Hi Kaipi,

I am sorry for the confusion. Yes, it is from another library. I put it alone with Skyline files in the attachment.

Thanks again for your time and help,

Fengchao

Hi Fengchao,
Skyline is having trouble with certain sequences that have two modifications on the same amino acid, e.g.:
[+42.010600]C[+57.021464]DAFVGTWK
which Skyline interprets as
C[+42.010600][+57.021464]DAFVGTWK
There are 35 lines like this in the uploaded file - you can replace these instances with a single modification that combines the formula of both modifications (something like C[+99.032064]DAFVGTWK and create a corresponding modification in Skyline).

There does also appear to be some sequences missing the carbamidomethyl modification, which will result in errors like this:
Precursor m/z 372.4297 does not match the closest possible value 358.1743 (delta = 14.2554), peptide CTAGTLHNLSHHR. Check the Modification tab in the Peptide Settings, the m/z types on the Prediction tab, or the m/z match tolerance on the Instrument tab of the Transition Settings.
After adding a +57 mass shift to the cysteines the errors went away. Hope that helps.

Thanks,
Kaipo