Issue With Retention Time Selection

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Issue With Retention Time Selection ed3  2020-12-11
 

Hello,

I am trying to import a pepXML file from PEAKS Online into Skyline to generate a peptide library. However, I am having an issue when doing this as for some reason Skyline is not selecting the appropriate retention time for the peptides in the pepXML file. I have attached a picture of this issue to illustrate what I am talking about. In the attached picture, Skyline has selected the retention time to be 0.02 minutes for the selected peptide; however, the actual retention time of this peptide is 15.20 minutes. Is there a way to resolve this issue?

Thank you in advance for your help, and please let me know if any clarification is needed.

Best,
Ed

 
 
Nick Shulman responded:  2020-12-11
Ed,

Can you send us your pepxml and the mzml, mzxml or raw file that goes with it?

Usually things like this are caused by a misunderstanding about whether times are in minutes or seconds, but it sounds like the retention time is off by a factor of 750, so that's probably not the cause this time.

If your files are less than 50MB you can attach them to this support request.
Otherwise, you can upload them here:
https://skyline.ms/files.url

-- Nick
 
ed3 responded:  2020-12-11
Hello Nick,

Both of the mzXML files have already been uploaded from previous question that I had. They are titled "082820-CedenoSC-QC-Shams_Slot2-19_1_493.zip" and "082820-CedenoSC-QC-SNI_Slot2-20_1_495.zip." I have also uploaded the pepXML file titled "peaks_ptm_ED.pep.xml."

Thank You,
Ed
 
Nick Shulman responded:  2020-12-11
In your pepxml file, it says this, it says that the spectrum number is 5038, and the retention time is 911.953 seconds.

    <spectrum_query spectrum="082820-CedenoSC-QC-Shams_Slot2-19_1_493.d" start_scan="5038" end_scan="5038" retention_time_sec="911.953" activation_method="CID, CAD(y and b ions)" precursor_neutral_mass="1112.5248773097992" assumed_charge="2" index="5038">
      <search_result>
        <search_hit hit_rank="1" peptide="AAAPEEETPAK" calc_neutral_pep_mass="1112.5349" massdiff="-5.516811484734001" num_tot_proteins="2" protein="F1LRZ7|F1LRZ7_RAT" protein_descr="Neurofilament heavy polypeptide OS=Rattus norvegicus OX=10116 GN=Nefh PE=1 SV=1" protein_mw="114411.90365908203">

I believe BiblioSpec does not pay attention to the retention time in peptide search results and only cares about the scan number.

Scan number 5038 in "082820-CedenoSC-QC-Shams_Slot2-19_1_493.mzxml" looks like this:
    <scan num="5038" msLevel="1" peaksCount="36" retentionTime="PT1.008S" activationMethod="CID" lowMz="179.0168" highMz="1641.004" totIonCurrent="2498.0">

It looks like the .mzxml file has a very different spectrum numbering scheme from what your peptide search engine was using. Were you maybe searching a slightly different file than this particular mzxml?

There are a lot of empty spectra in your mzxml file such as:
    <scan num="0" msLevel="1" peaksCount="0" retentionTime="PT0.348S" activationMethod="CID" lowMz="0.0" highMz="0.0" totIonCurrent="0.0">
      <peaks precision="32" byteOrder="network" pairOrder="m/z-int"></peaks>
    </scan>
    <scan num="1" msLevel="1" peaksCount="0" retentionTime="PT0.348S" activationMethod="CID" lowMz="0.0" highMz="0.0" totIonCurrent="0.0">
      <peaks precision="32" byteOrder="network" pairOrder="m/z-int"></peaks>
    </scan>

You might find things work better if you use mzml instead of mzxml.

-- Nick