Import Peptide Library from PEAKS Online from TIMS TOF data

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Import Peptide Library from PEAKS Online from TIMS TOF data ed3  2020-10-09
 

Hello,

I have been trying to import a peptide search from PEAKS online into Skyline but I keep getting an error and I was hoping somebody could please help me understand what is going on. The spectrum was acquired with a Bruker TIMS TOF, and when I try to import the pep.XML file in the "Peptide Search" option, I get an error saying "The .pep.xml file is not from one of the recognized sources." I have the IMS-TOF spectrum in .mzXML format in the same folder as the pep.xml file, and I am not sure why I get this error.

Thank you very much for your help.

Best,
Ed

 
 
Nick Shulman responded:  2020-10-09
BiblioSpec only understands certain pepxml because BiblioSpec needs to be able to convert the scores into a false discovery rate so that it knows what to include in the library.

Here is the page which lists all of the formats that BiblioSpec understands:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild

I see that PEAKS DB is in that list. Is that the same as PEAKS Online? If not, it might be that we should add support for it.

If you would like to send us your .pep.xml and .mzXML files you can upload them here:
https://skyline.ms/files.url

-- Nick
 
ed3 responded:  2020-10-14
Hi Nick

I believe that PEAKS Online does have PEAKS DB. The .mzXML file is very large (36 gb). Should I still upload it to the link you sent, or is there somewhere else that I should upload it?

Thank You,
Ed
 
Nick Shulman responded:  2020-10-14
It's no problem for us if you want to upload a file that large.

.mzXML compress very well, so you can save yourself a lot of time by putting it in a .zip file first.

If you would like, you could send us the .raw file instead, and we can create the .mzXML file ourselves (assuming you created that .mzXML using ProteoWizard MSConvert, and let us know if you used any special settings).

I will send you an email in case you want to use something like Google Drive or DropBox.
-- Nick
 
ed3 responded:  2020-10-14
Hi Nick,

I uploaded the mzXML and the pepXML files to https://skyline.ms/files.url.

Thanks,
Ed
 
Nick Shulman responded:  2020-10-14
Thank you for sending those files.

Near the beginning of your .pep.xml file, it says:
search_engine="PEAKS_DB"
If you were to edit that file and change it so that it says:
search_engine="PEAKSDB"
then everything seems to work.

I am going to ask around and see if we can update BiblioSpec so that it can handle "PEAKS_DB" with or without the underscore.
-- Nick
 
ed3 responded:  2020-10-14
Hi Nick,

Thanks for the suggestion. Unfortunately though, I am still getting the error. The error message also states that the error is on line 19 of the pepXML file, so I'm not sure if maybe something in that line is not properly formatted.

Thanks,
Ed
 
Nick Shulman responded:  2020-10-14
Really? It works for me (except that I eventually get an error about not being able to find "082820-CedenoSC-QC-Shams_Slot2-19_1_493").

I am attaching the pep.xml file that I edited.

If you are still getting an error, please attach your pep.xml file.
-- Nick
 
ed3 responded:  2020-10-20
Hi Nick,

Sorry for my delayed response, and thanks for attaching your pepXML file. I tried importing my library with your XML file and it still didn't work; however, I updated the version of skyline that I was using to the current version and that seemed to fix the error and I am able to import my search results.

Now I am having another issue though. When I try to create the library, I am unable to import the peptides with post-translational modifications and I get an error saying:

---------------------------
Skyline
---------------------------
Invalid entries were found in the library 'QC Peptide List'.
35 of the 3900 peptides or molecules were invalid, including:
Q[-17.02655](-17.03)KSQRTQDENPVVH++
Q[-17.02655](-17.03)KSQRTQDENPVVH+++
A[+42.01056](+42.01)EDIKTKIKNYK+++
A[+42.01056](+42.01)EDIKTKIKNY+++
S[+42.01057](+42.01)DKPDMAEIEKFDKSKLKK++++
S[+42.01057](+42.01)DKPDMAEIEKFDKSK+++
S[+42.01057](+42.01)DKPDMAEIEKFDK++
S[+42.01057](+42.01)DKPDMAEIEKFDK+++
S[+42.01057](+42.01)DKPDMAEIEKFD++
S[+42.01057](+42.01)DKPDMAEIEKF++
---------------------------
OK
---------------------------
I was wondering if you knew why this is happening? For your reference, I am doing a peptidomics study, so what I have been doing is I go to the 'Modifications" tab in "Peptide Settings" and select the PTMs that I am interested in. Then I go to the "Library" tab and go to "Build" and I import the pepXML file to try to create the library and I am able to create the library, but the modified peptides are missing and I get the error message.

Thank you,
Ed
 
Nick Shulman responded:  2020-10-20

Ed,

Those peptides seem to be coming from things in your pep.xml file which look like:
<search_hit hit_rank="1" peptide="S(+42.01)DKPDMAEIEKFDKSKLKK" calc_neutral_pep_mass="2278.1877" massdiff="-6.1793626666059485" num_tot_proteins="1" protein="P62329|TYB4_RAT" protein_descr="Thymosin beta-4 OS=Rattus norvegicus OX=10116 GN=Tmsb4x PE=1 SV=2" protein_mw="5052.650259082031"> <modification_info modified_peptide="S(+42.01)DKPDMAEIEKFDKSKLKK"> <mod_aminoacid_mass position="1" mass="129.0425967114258"></mod_aminoacid_mass> </modification_info>
I imagine that BiblioSpec is not expecting the modification to be in parentheses like that, but I am not very familiar with the pep.xml format.

I will ask around and see whether there is something we can do to fix this.

In the meantime, can you send us the other file that is used by the .pep.xml file: "082820-CedenoSC-QC-Shams_Slot2-19_1_493.mzXML"? (Or, sending us the original .d file would probably work too).
-- Nick

 
matt.chambers42 responded:  2020-10-20

Nick is right, the search_hit peptide attribute should not have modifications in it (that's what modified_peptide is for below). This would seem to be a bug with PEAKS DB's latest pepXML exporter. We could theoretically change BiblioSpec to ignore anything in that attribute that isn't an amino acid, but I'm not very comfortable with that idea. It would create a risk of silently accepting bad input.

 
ed3 responded:  2020-10-20

Hi Nick,

I uploaded the other mzXML file to https://skyline.ms/files.url.

Ed

 
ed3 responded:  2020-10-23

Hello,

I tried changing the search_hit peptide attribute to remove the modification, and that seemed to resolve the issue of not importing the modified peptides. However, when I look at the Spectral Library, the retention times do not seem to match with the true retention times that are listed in the pepXML file. Do you have any idea why the retention times are incorrect?

Thank you for taking the time to answer my questions.

Best,
Ed