support

Welcome to the Skyline support forum. If you have a question about using Skyline, or if you encounter a problem, you can post your questions here.

It is likely that your question has already been asked and answered.  Please use the search box in the upper right corner of this screen before posting a new question.

Support is provided by the creators of the software, as time allows, though we hope others will share their experience as the user community is now quite large.

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

In order to post to the forum, you'll need to sign-in or if you don't yet have an account sign up. Forgot your password? You can reset it using the "(forgot password)" link on the sign-in page.

You can also follow the Skyline support board through email updates after you sign up.

When you post a question, please include the following information:

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

If you are including text output from a tool, please attach files to your message, rather than pasting in long text.

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that .sky.zip file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
CE optimization on Agilent QQQ
(3 responses) Sarah Michaud 2017-11-06
Hello,

I getting some strange results when optimizing collision energies for an Agilent 6495 QQQ using Skyline. I am generating the transition list for CE optimization using skyline, then using the method to acquire the data, and importing the data back into Skyline. After importing the data, sometimes the transition results look fine and as expected, but other times the pattern in the peak area graph is really unusual (almost random instead of a curve shape), and other times the results are missing completely. When I check the same data file in the Agilent MassHunter software, the data appears to have been acquired correctly and the peak area pattern is normal. I've attached some slides with examples to try and show what I'm seeing.

I think the problem might be that for some transitions the product m/z value is for some reason different in the acquired data file compared to the method generated by Skyline, and when that data is imported back into Skyline the peak areas associated with each CE step gets jumbled.

Thank you for the help!

Sarah
 CEoptimization.pptx 
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Quantifying with surrogate standards
(1 response) Joerg 2020-11-24

Hi,
I am tring to quantify different lipid classes using several surrogate standards. Although I set the respecitve molecules as "surrogate standard" and set the "Normalization Method" to the respective standards for the other molecules, there is no option to normalize the area view to these standards. Nor is it possibl to use the surrogate standards in the Quantification tab of the "Molecule Settings". I am obviously missing something...
Many thanks in advance!
Joerg

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Chromatogram information unavailable after importing mzML files
(3 responses) Andreas 2020-11-24

I am analyzing Lipid data in the Small Molecule Mode of Skyline. I can import the Sciex MRM wiff files. However, importing peak boundaries changed the first sample of the batch only. Therefore, I converted the wiff file to mzML files using Msconvert (ProteoWizard). Unfortunately, after importing the results as mzML some lipids could not be imported with certain samples (Chromatogram information unavailable, see attached files). Are there special parameter for Msconvert required for usage with Skyline?

 LPX_STDs_mzML_ajh.sky.zip  LPX_STDs_mzML_ajh.png 
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Specifying Integration Boundaries
(18 responses) jrenders 2020-11-09

Hi there,
My small molecule analysis methods produce some pretty ugly peaks for a few analytes. Skyline does its best, but I do not expect any integration algorithm could figure out some of my data. What I have been searching for to fix the problem is some place to specify explicit integration boundaries. In reading back on support, I understand that "explicit retention time" and "window" will get me close but these are more "suggestive" to skyline (and in fact do not work for my peaks). Integrating a peak and then rt-clicking and hitting "Apply Peak to All" also seems more suggestive than explicit and does not work. I would like to have skyline integrate all peak area between two specified RT's without any consideration to inflection points or anything else and then apply this setting across the whole batch for that particular molecule. Is that possible at this time? Thanks!

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ppm-level calculation
(2 responses) laura nieder 2020-11-23

Hi, thank’s for the great software and the really nice tutorials. Is there any way to let skyline calculate the ppm-levels of a certain protein (using for example 3 peptides for quantification) in sample A regarding to a housekeeping protein in the same sample?
I always have the same two Proteins in my samples. Protein X with a steady concentration, protein Y with changing levels. Usually we do absolute quantification with an external calibration curve. This time I would like to get the ppm levels of Y regarding the concentration of X within the same sample. Similar to HCP analysis.
Is there a possible way, that Skyline calculates this for me?
Thank you,
L.

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Trouble finding spectrum files - can they be input through a command line interface
(2 responses) jharrison 2020-11-23

Hi Skyline team,

Thank you for making such a useful software. I want to do some ms1 filtering in some data and I have been having some issues when I try to open the .pep.xml file. I keep getting the error message below and I do have the .mzML file in the appropriate folder. I think the issue is that I am using VMware fusion on a mac to run windows and I think it is somehow frustrating your auto search tools. I am wondering if there is a way I can pass the spectrum file into skyline through a command line interface like skyline runner. Or maybe I am doing something else silly that is giving me an issue. I am grateful for any help you can offer.

System.IO.IOException: ERROR: Could not find spectrum file 'NA[.mz5|.mzML|.mzXML|.ms2|.cms2|.bms2|.pms2]' for search results file 'WT_Roos_1.pep.xml' in current directory, ../,../../.
ERROR

Best,
Joe

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Annotation of Light peptide fragments instead of heavy fragments
(3 responses) nyalwijo 2020-11-22

Dear colleagues,
I have PRM data for a SIL peptide. THe MS2 fragmentation data is perfect but Skyline annotates fragments for the Light peptide which are not in the MS2 spectrum instead of the Heavy peptide fragments. How can I correct the issue? Thank you. J

 Skyline_peptide_Capture.PNG 
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Precursor Report: Duplicate lines?
(4 responses) alejandro.cohen 2020-11-19

Skyline people... again requesting your help. Here a curve ball:

I'll try to summarize:

I'm developing a targeted SIM (tSIM) method for 18 steroid hormones using a LC-QExactive and Skyline's Small Molecule interface. For each molecule, I have a light and deuterated standard. ALSO, for each molecule, I'm monitoring both the [M+H] and [M+Na] precursors. Reason being, these samples are ocean water derived, so I'm concerned Na might not be easy to completely removed during sample prep, and signals will likely be split (I'm clearly seeing this in the data I've analyzed so far, even with the standards).

I've followed the following tutorials which were very helpful:

https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_small_quant
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_hi_res_metabolomics

So, I have one question and one concern.

Q: For the calibration curves for any of the molecules, I see that they remain constant regardless whether I select the [M+H] or the [M+Na] (Curves DO change selecting different molecules, of course) . Does that mean the areas of both [M+H] and [M+Na] are summed up to make the final areas? Does this also apply to their Internal standards and the ratios? I'm finding it hard to navigate the tables to see how the data is compounded together.

Concern: in my precursor-Quant table, for each Replicate, I see TWO lines having the same column content (Rep, Rt, Precursor, Molecule, Ion Formula, Precursor Adduct, etc etc) EXCEPT the Area, Area Ratio and Background. Attached a screenshot.

Could you help me out with this? Thanks again for your fabulous work with Skyline.

 Screenshot 2020-11-19 151723_double entry.jpg  Screenshot 2020-11-19 153841.jpg 
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Question about iRT prediction in skyline
(2 responses) 16622080827 2020-11-15

I have used iRT kit(Biognosis)in my experiment and I have also detected all the iRT peptide in my data both in PD software or check mannually.But when I try to import the PD result, it always shows that RT prediction failed.Maybe some peptides are in low intensity.Faced with this problem,what can I do for this condition?

 questions.ppt 
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Small molecule: calibration curves for individual targets
(2 responses) alejandro.cohen 2020-11-18

Hi !

I'm developing a targeted SIM method for 18 steroid hormones using Skylines Small Molecule interface. So far so good.
I have prepared calibration curves for each standard (3 concentration levels) separately and run them on a LC-MS (QExactive). What I mean is that I did NOT pool all standards together in the same tubes, rather, have a separate calibration curve for each standard.

Example:
Replicate 1: Std X at 0.1ng
Replicate 2: Std X at 1ng
Replicate 3: Std X at 10 ng
Replicate 4: Std Y at 0.1ng
Replicate 5: Std Y at 1ng
Replicate 6: Std Y at 10 ng
Replicate 7 :Std Z at 0.1 ng, etc etc

In the Replicate Tables, I have already classified them as Standards and their concentrations as in https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/HiResMetabolomics-20_1.pdf tutorial.

Is there any way to inform Skyline which Molecule each Standard corresponds to? By default, Syline tries to find ALL Molecules in each each Replicate. Manually removing peaks from each Replicate is very time consuming.

All tutorials I have read so far combine all standards into single replicate injections.

Thanks!

Alejandro

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Train Drift Time predictor - "Skyline Drift Time Predictor Training" tutorial
(5 responses) s1899812 2020-11-09

Hello,

I am following the "Skyline Drift Time Predictor Training" tutorial and when I was going to train the drift-time predictor on page 17/26, the Prediction tab under Peptide Settings is only showing me a "Retention time predictor", but not a "Drift time predictor". I have attached an image to show this.

Do I need to do anything to add the Drift time predictor?

The link to the tutorial can be found: https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/DriftTraining-3_1_1.pdf

Thanks
Joan

 Skyline Peptide Settings.png 
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MPPARRReport tool not working
(7 responses) roman koziy 2020-11-16

Hi,

I have constructed a library from DDA SpectrumMill search results in order to evaluate MS1 spectra. I further wanted to export the Skyline results into Agilent MPP software for analysis. However, whenever I try to run the MPPAPRReport tool I get an error message (please see attached). Could you please suggest what could be the problem?

Thank you very much for your help,
Roman.

 MPPARRReport_message.pptx 
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Skyline external tool using SkylineTool not able to import small molecule blib from directory using AddSpectralLibrary command
(3 responses) Chris Ashwood 2020-11-13

Hi Skyline Support Team,

I hope you are doing well.

I am currently trying to fix a new error that I have tried to fix with no avail. Has the new Skyline updates (daily or stable) changed how they handle external tool directories and paths? Or the SkylineTool?

The C# app I am developing loads small molecule spectral libraries using the SkylineTool with the following command:
_toolClient.AddSpectralLibrary("NGlyCat_Library", NGlyCatBlib);

Skyline appears to be unable to find the blib file (as shown in red in the attached image) despite the blib being in the tool directory. To my knowledge, this command has not caused any issues until I updated to the newest Skyline versions. I also get an error (shown below) that the document must be fully loaded so I cannot share the full Skyline document with the Share button.
System.IO.IOException: Settings.PeptideSettings.Libraries: null library
at pwiz.Skyline.SkylineWindow.ShareDocument() in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1185

I rolled back my daily version but it did not seem to fix the issue. And I also tried an older version of my program that worked fine and it also failed.

Any help would be appreciated because I'm going a little crazy trying to find what I changed on my end to cause this issue. I have included the Skyline assay and spectral libraries in the uploaded file (Skyline_SpecLib_error.zip).

Cheers,
Chris

 Imported_SpecLib_Issue.png 
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peptides with mixtures of modified and unmodified residues
(24 responses) Keri 2018-05-09

Hi Brendan,

I have attempted to use the peptide settings > modifications feature to quantify samples that contain both fixed and variable modifications (e.g. some but not all cysteines in a peptide are isotopically labeled (+521 or +527) and all cysteines are carbamidomethylated +57). I input both the structural modification for carbamidomethylation and the variable modifications for the additional isotopic label, but when I apply those settings, I see multiple modifications, including the sums of multiple modifications (e.g. 1022, 1028, which are not correct). Can Skyline handle samples with mixed modifications and, if so, how?

Thank you

Keri

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Variable MRM time windows
(1 response) benoit fatou 2020-11-13

Hi Skyline Team,

I was wondering if there is any way in Skyline to generate variable MRM time window for each peptide when exporting the method or transition list.
I am asking you because some of my peptides have a broad peak width so if I set a very short time window, I would crop them out. On the other hand, making a short time window for the peptides with very small peak width might increase their sensitivity because we decreased the number of concurrent transition pairs.

Let me know if you need more information or if you have any question.
Thanks for your help,
All the best,
Benoit

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Silicone isotope pattern: is it possible to add it?
(2 responses) marcel reimann 2020-11-13

Hi Skyline Team,

I´m using Skyline in a kind of "off-label-use" for my GPC-MS. For that purpose it would be great to add also the silicone isotope pattern. Is there a way to add this to the skyline "automated isotop calculation"?
Adding it to the transition list import would be a nerve wrecking work.

Thank you in advance,

best Marcel

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MS1-Filtering small molecules: Threshold - "empty samples"
(1 response) marcel reimann 2020-11-13

Hi Skyline Team,

I´m using Skyline in a kind of "off-label-use" for my GPC-MS.
Sometimes it happens, that I dont have a Signal at all when using the MS1-Filtering. No noise, no error, just nothing. Using the same transition list on the next sample (more concentrate) it works.
Could it be that there is a "hidden" threshold? I couldn´t find anything, would be great to know where it is.
Could be also the case that I have to much precursors with one name. For example I have 47 precursors with 3 charge states each. Is this too much for skyline? In some cases I got "signal" when I removed some precursors.

Thank you in advance!
best Marcel

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spectral library from Peaks Online pepXML
(4 responses) Tharan Srikumar 2020-11-11

Hi Skyline team,

I'm trying to use exports from Peaks Online v1.4 to create a spectral library in Skyline daily (yesterday's release). I've exported pepXML, MzIdenML and a mgf. If I rename the pepXML file from the original .xml to pepXML, Skyline begins to read the file, but throws an error looking for the spectra file. It seems like Skyline is not looking for the mgf file but rather several other formats (see screenshot attached). I've also tried to rename the mgf file to the same filename Skyline is requesting with no success either.

Best,

Tharan

 Screenshot 2020-11-11 123329.jpg 
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typing sequence name
(2 responses) gabriele philipps 2020-11-09

Hello,

when I paste an entire amino acid sequence into the left pane in order to identify peptides according to my peptide settings, I am always struggling with filling in the sequence name, i.e. replacing the "sequence 1" name with my protein name. By some strange reason the name flips back to "sequence 1" after some seconds and I have to type my protein name a second time an then it will remain.
Is there any trick to keep already the first entry?

Thanks a lot for your help!
Gabriele

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Set Pause Time and Dwell Time
(6 responses) benoit fatou 2020-10-27

Dear Skyline Team,

I am working on the LCMS 8060 from Shimadzu and I was wondering if it is possible to set the Pause Time and the Dwell Time when exporting the methods from Skyline.
Thanks for your help,
Best,
Benoit

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peptide prediction tool (stability and signal strength)
(1 response) gabriele philipps 2020-11-09

Hi,

I have a rather general question which is not necessarily related to the Skyline software:
is there a software that can predict which peptides are better or worse suited for targeted proteomics (with respect to stability, retention time and above all signal intensity of the fragments which to my knowledge correlates with their ability to ionize)?
At the moment I know I should avoid cysteine and methionine and N-terminal glutamine and asparagine. But a tool which gives an overview on the peptide properties (when pasted in ) together with a judgement on the suitablility would be very helpful.

I hope you can help. Thanks for every suggestion.
Best regards,

Gabriele

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predict peptide from the first amino
(1 response) spklongz 2020-11-09

Hi,

I want to predict peptide starting from the first amino acid (N-term) of a protein. How to set parameters of Skyline?

I varied Skyline parameters, while it failed to do that.

 skyline.PNG 
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Spectral Library Creation
(1 response) pg23 2020-11-06

Hi!

I would like to create a spectral libray for a targeted method set-up similar to the one that is included in the Targeted Method Edit Tutorial (from Yeast).
I have been searching for potential spectra at PRIDE, but I fail to download the right format to upload in the library (Peptide Settings). Where can I find suitable files for spectral library creation?

Thank you

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Error 0x800F0954 when installing Skyline
(1 response) alvaro sanchez 2020-11-06

I could not install Skyline because of that error.
It says:
The following feature could not be installed:
.NET Framework 3.5

Any solutions?

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Drif time predictor
(7 responses) mariangela valletta 2020-10-30

Hi,
I have skyline 20.2 64 bit on my Windows 10. To get familiar with skyline and ion mobility data , l followed the tutorial 'Skyline Ion Mobility Spectrum Filtering' and I downloaded zip file. I read that with new version of skyline IMS filtering settings moved to Transition Settings - Ion Mobility. My problems is: 'Use Results' button is not clickable. Why?

Thank you

 Drif time Predictor.pptx 
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Why Can't Peptide Search Be Imported Without Full-scan MS1 Filtering?
(3 responses) philip remes 2020-11-03

I'm importing search results into Skyline from a DIA chromatogram building experiment. After the search results are imported, and Skyline asks to Configure the Full-Scan Settings to Import the files, I want to select 'None' for the MS1 Filtering. I thought part of the advantage of DIA is that the parent ion may not be visible in the MS1 Full Scan or may have a high probability of having interferences, so why require that it be processed? There may be no MS1 scans, or they may be of such low resolution that they are not helpful. I realize I can then cancel and import the data the usual way after setting up Transitions. But I wondered if this MS1 filtering requirement could be waved for DIA and PRM peptide search imports.

Thanks
Philip

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Need Skyline 20.1.0.31 32bit (f09d9bed5)
(2 responses) wnc4 2020-11-04

Hi, I am working with a collaborator and they are using an older version of skyline. I would like to import the current transitions everyone else is using but I do not have the same software version. I have the newer software version. Where can I download the older version from?

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Error on Importing after installing a UV/VIS detector
(1 response) chris brown-2 2020-11-02

Hello -

I received an error message when attempting to import data to Skyline 20.1.0.31.

Up until I configured a UV/Vis detector on the instrument I had been able to import data just fine. After installing that detector, Skyline is unable to import the .raw files. If the work around is just to de-configure the UV/Vis detector, that is fine.

I am attaching the error message that is returned in the import box below.

Thanks, CJB


At 8:15 AM:
Failed importing results file 'D:\Studies\Lumos-DailyQC\Data\100ppb-HypersilGold_20201031014802.raw'.
error reading spectrum controllerType=4 controllerNumber=1 scan=1

Inner exceptions:
Exception type: System.Reflection.TargetInvocationException
Error message: error reading spectrum controllerType=4 controllerNumber=1 scan=1
error reading spectrum controllerType=4 controllerNumber=1 scan=1
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1909
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.NextSpectrum() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 915
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 258
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 238
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 575
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 381
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

Exception type: System.Exception
Error message: error reading spectrum controllerType=4 controllerNumber=1 scan=1
error reading spectrum controllerType=4 controllerNumber=1 scan=1
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1125
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.Read() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 952
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.<RunAsync>b__33_0() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 809

Exception type: System.Exception
Error message: [RawFileThreadImpl::getTrailerExtraValue(SPS Masses Continued:)] GetTrailerExtraValue
[RawFileThreadImpl::getTrailerExtraValue(SPS Masses Continued:)] GetTrailerExtraValue
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1056
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetMetaDataValue[TVal](Int32 scanIndex, Func2 getValue, Func2 isUsableValue, Func2 returnValue, DetailLevel& detailLevel) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1116 at pwiz.ProteowizardWrapper.MsDataFileImpl.GetMetaDataValue[TVal](Int32 scanIndex, Func2 getValue, Func2 isUsableValue, Func2 returnValue, DetailLevel& detailLevel) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1123
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetMsLevel(Int32 scanIndex) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1128
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.LookaheadContext.GetMsLevel(Int32 index) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1359
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1036

view request
Import Library Assay for small molecules process?
alejandro.cohen 2020-10-29

HI Skyline people

I'm developing a PRM (Targetted MS/MS) method on Skyline from data acquired on LC-QExactive setup on the latest Skyline daily (20.2.1)

I have already:

1- Generated the molecule list and imported it into Skyline (Edit>Insert>TransitionList). I have so far the Precursors but NOT selected the Fragments.
2- I already acquired DDA files on all my standards (I have a single .raw file for each standard with good MSMS spectra for each one)

I'd like now to add the 'transitions' (Fragments) to the PRM method on skyline. I've looked at tutorials and webinars, and I cant find one that specifically explains the process to Import Assay Library for PRM. I'm hoping this could be done automatically from the DDA experiments on the standards (.raw files) instead of manually inserting the PRM transitions through the Edit>Insert>TransitionList option.

I notice the Import Assay Library requires a .csv,.tsv file as input. Could you take me through the generals steps from going from the .raw files (DDA of my stds) to the Import Assay Library for small molecules and which programs to use at every step? I can handle the rest of the steps such as importing the results of the PRM acquisition, etc etc.

Thanks again

Alejandro

Thanks!

Alejandro

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Calibration curve data export
(2 responses) Zac 2020-10-28

I want to reproduce the calibration curve data in graphpad prism, when i export the data i see two sets of data one 'Standard' and one 'Calibration Curve' each with columns 'Analyte concentration' and 'Normalized Peak Area'. What i am confused about is that there are more rows of data for the 'Calibration Curve'. 68 rows for Standard and 100 rows for the Calibration curve. Based on the number of calibration points and replicates i expect 68 rows of data. Should there be extra for the Calibration Curve, are means for replicates included? Thanks for all your great help and support.

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wiff2 file from Sciex Echo MS fails to import to skyline
(4 responses) michelle robinson 2020-10-26

Hi Brendan,
I'm unable to import MRM data generated on the Sciex Echo MS into skyline (wiff2 file format). I am using Skyline daily. The error message says, "[Experiment2ImpI::ctor()] Object reference not set to an instance of an object"
Thanks for your assistance,
Michelle

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Issue with Skyline transition list export
(1 response) Shalini Aggarwal 2020-10-27
Dear Skyline team,

I am facing an issue with the export of the transition list from skyline daily software. It is showing the below shown error. I cross-check each step but still unable to figure out the issue. My colleagues tried the same thing on their PC and it worked. Kindly guide me with the error rectification.

thank you in anticipation,
Shalini
 image.png 
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Peaks of light and heavy peptides have unequal peak boundaries
(9 responses) fcsigloch 2020-10-22

Hi!

In an MRM experiment, I came across the problem that Skyline picks non-coeluting peaks for the light endogenous peptide and the heavy labelled reference peptide (screenshot attached). I never observed this behaviour in PRM data, where I am used to perfectly matching peak boundaries.
I could not determine a specific setting that caused this behaviour. Can I somehow force Skyline to choose the same peak boundaries for light and heavy trace?

Also, I tried to train a default or mProphet model to improve the peak picking. Training the model worked, but when I want to apply it to the data, I get the following error message:

---------------------------
Skyline
---------------------------
Failed attempting to reintegrate peaks.
The index 6 must be between 0 and 2
---------------------------
OK More Info
---------------------------
System.Reflection.TargetInvocationException: The index 6 must be between 0 and 2 ---> System.Reflection.TargetInvocationException: The index 6 must be between 0 and 2 ---> System.IndexOutOfRangeException: The index 6 must be between 0 and 2
   bei pwiz.Skyline.Model.Results.ChromatogramInfo.GetPeak(Int32 peakIndex) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:Zeile 2773.
   bei pwiz.Skyline.Model.TransitionGroupDocNode.UpdateResults(SrmSettings settingsNew, SrmSettingsDiff diff, PeptideDocNode nodePep, TransitionGroupDocNode nodePrevious) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:Zeile 1487.
   bei pwiz.Skyline.Model.TransitionGroupDocNode.ChangeSettings(SrmSettings settingsNew, PeptideDocNode nodePep, ExplicitMods mods, SrmSettingsDiff diff) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:Zeile 1116.
   bei pwiz.Skyline.Model.PeptideDocNode.ChangeSettings(SrmSettings settingsNew, SrmSettingsDiff diff, Boolean recurse) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:Zeile 979.
   bei pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass142_5.<ChangeSettingsInternal>b__7(Int32 i) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:Zeile 1080.
   bei pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:Zeile 186.
   --- Ende der internen Ausnahmestapelüberwachung ---
   bei pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 1944.
   bei pwiz.Skyline.Util.ParallelEx.LoopWithExceptionHandling(Action loop, Action`1 catchClause) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 2246.
   bei pwiz.Skyline.Util.ParallelEx.For(Int32 fromInclusive, Int32 toExclusive, Action`1 body, Action`1 catchClause, Nullable`1 maxThreads) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 2190.
   bei pwiz.Skyline.Model.SrmDocument.ChangeSettingsInternal(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:Zeile 1068.
   bei pwiz.Skyline.Model.MProphetResultsHandler.ChangePeaks(IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\MProphetResultsHandler.cs:Zeile 172.
   bei pwiz.Skyline.EditUI.ReintegrateDlg.<>c__DisplayClass12_1.<OkDialog>b__1(IProgressMonitor pm) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:Zeile 133.
   bei pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 254.
   --- Ende der internen Ausnahmestapelüberwachung ---
   bei pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 1944.
   bei pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 202.
   bei pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 140.
   bei pwiz.Skyline.EditUI.ReintegrateDlg.OkDialog() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:Zeile 135.
---------------------------

Thanks for your help!

 unequalRTs.png 
view request
MRM triggered MSMS
(4 responses) heyang 2020-02-04

Dear Skyline team,

I want to compare MRM triggered MS2 with library spectra. Could I do this in skyline? if yes, how?

thanks,

Heyi

view request
Precursors per sample injection thinks it is transitions per sample injection
(2 responses) philip remes 2020-04-04

Hi Brendan,

This is a repost of issue 723. Sorry I didn't know to first post to Support and not Issues.

I've attached the skyline file. To reproduce what I saw,

  1. File->Export->Isolation List
  2. Instrument Type -> Thermo Fusion. Interestingly, if I use the first Instrument in the list, Agilent QTOF, and enter 216 in max precursors, the number of methods is correctly set to 9
  3. For Fusion, if 216 is set to max precursors, the number of methods is 138.

Thanks
Philip

 200404_export_list_debug.sky.zip 
view request
MS1 filtering: is manual XIC window mass tolerances possible?
(5 responses) alejandro.cohen 2020-10-23

Hi Skyline,

I'm using Skyline for a targeted metabolomics project on an QExactive (running at 70K resolution @ 200m/z according to Thermo). I inserted this value in the Transition Settings> Full-Scan>MS1- filtering>Resolving Power tab.

My standards are all showing at the expected Rt and with mass errors <2ppm. However, Skyline is also labeling other peaks between + - 2 to 9ppms. Is there any way to manually reduce the XIC mass windows, or set mass error thresholds to reduce the 'labeling clutter' in the chromatogram windows? Or do I just have to increase/fake the Resolving Power value on the Transition Settings pane.

Thanks again for you continuous support, Skyline rocks!

Alejandro

view request
Import Peptide Library from PEAKS Online from TIMS TOF data
(12 responses) 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

view request
Problem library creation for cross-linking
(1 response) tatianibl 2020-10-22
Hi,

I am trying to build a cross-linking library using an output created by proxl from plink 2.0. However, I am having a problem. Follow bellow the error message.

Thanks,
All the best.
Tatiani.

---------------------------
Skyline-daily
---------------------------
ERROR: No spectra were found for the new library.

Command-line: C:\Users\tatiz\AppData\Local\Apps\2.0\49BTDOGX.MMZ\O1B3EWR8.PN0\skyl..tion_e4141a2a22107248_0014.0002_e354297bfe67025e\BlibBuild -s -A -H -o -c 0.95 -i teste -S "C:\Users\tatiz\AppData\Local\Temp\tmpA7B.tmp" "C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input\teste.redundant.blib"
Working directory: C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\tatiz\AppData\Local\Apps\2.0\49BTDOGX.MMZ\O1B3EWR8.PN0\skyl..tion_e4141a2a22107248_0014.0002_e354297bfe67025e\BlibBuild -s -A -H -o -c 0.95 -i teste -S "C:\Users\tatiz\AppData\Local\Temp\tmpA7B.tmp" "C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input\teste.redundant.blib"
Working directory: C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156
view request
Confusion with Skyline and Proteome Discoverer
(3 responses) Mark Athanason 2020-10-20

Hi all,

First off thanks for providing the support that you do, hope I don't bother you all too much.

For right now I'm fine using the proteome discover software, but ultimately I want to perform DIA quant on similar data using skyline. Within PD, I perform a search using sequest and peptide validation with percolator with an FDR of 1%. Using the "import DDA search" wizard in the most recent version of skyline daily, I use the .msf file with a cut off score of 0.99. What I get in skyline doesn't seem to match what I see in PD at all. For example PD says I have 411 protein groups, but in skyline when I check remove repeat and duplicate peptides, I'm left with 222. Shouldn't these be the same since percolator is filtering for high confidence peptides? additionally, for some peptides PD does not correlate an MS1 trace where skyline does ... in my opinion incorrectly.

I'm uploading the .msf file, "Ibu1_DDA_1.raw" "Ibu1_DDA_2.raw" and "Ibu_DDA_3.raw" to https://skyline.ms/files.url

Any help would be greatly appreciated!
Mark

view request
Some peptides show incomplete peaks but in raw data they are okay
(17 responses) yingzhu1992 2020-10-05

Hi Skyline team,

I imported my result into the Skyline, some peptides show incomplete peaks, I checked from the raw data, they look fine. Can you please check and explain it for me and is there anything I can do to solve this?

And I also upload the raw data.

 20201001 H+L all transition.sky.zip  Capture.PNG  Raw data.PNG 
view request
HMDB-msp file import problem
(7 responses) Joerg 2020-10-14

Hi,
we tried to import the HMDB for small molecule quantification into Skyline. We downloaded the msp-file from Oliver Fiehn´s website. Skyline import part of the database, but just "forgets" the majority of the data. Is there any way to change the msp-file in a way that Skyline recognizes all entries? Or is import of the "original" xml-file from the HMDB website possible?
Many thanks in advance!
Jörg

view request
Storage location for custom PTMs
(2 responses) dkueltz 2020-10-07

Hi Nick, Brendan,
I have generated quite a few custom PTMS for one of my skyline documents (peptide settings/ modifications/ add) and would like to have them available for future documents. Where are these added PTMs stored? With only the Skyline document I entered them for or somewhere central?
Thanks,
Dietmar

view request
installation problem
(1 response) niyatti 2020-10-17

Hi, I tried to install Skyline (64 bit) on 64 bit Dell home basic desktop with Windows 7 through google chrome. The install.log is attached. Any idea what is wrong?

 install.log 
view request
Problem building spectral library
(3 responses) itv005 2020-10-15

Hi,

I am a farily new user of Skyline, and I am struggling to build my spectral library. Aside from some of the tutorials, my training is very limited. The source of my spectral library is a search from Proteome Discoverer 2.4 that I exported to a pep.xml file. I also have a used MSconvert to generate mzml files from my raw files.

I suspect that my issue is not due to any of the settings I made when setting up skyline, therefore I have not included those in this request. As seen in the attachment, I get an error saying the wrong directory is used, but I cannot seem to find out how it chooses this directory, or how to change it. I have put the skyline file, the raw files, and the converted spectrum files all in the same folder. The only thing missing, as I see it, is the program itself. However, I struggle to see the logic if this is the missing link, as this would be impractical for further projects.

I used the 20.2 version of Skyline and Windows 10 Home. The data I have is a non-targeted HPLC-MS/MS of CSF. I wish to use this library in a PRM project which is ready to be analyzed.

Thanks in advance! Let me know if I should add more info.
Ingrid

 skyline_error.PNG 
view request
Problems with import of peak boundaries in smallmolecule mode for .wiff data
(4 responses) Max 2020-08-24

Hi,

i am trying to import peak boundaries for a number of data sets based on .wiff data. We have set up some small molecule transitions for small molecules and peptides (due to qunatifiaction porpuses). I have used the allready discussed workarounds for small molecules (see https://skyline.ms/issues/home/issues/details.view?issueId=671) which is perfectly working for data from e.g. waters were a single filne represents a single injection. However, in the case of AbSciex 5500 the data is stored in a single .wiff file. I added therefore the sample name (alias Sample identifier) column to the peak boundaries list as stated in the FAQ for importing peak boundaries (see here: https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/ImportingIntegrationBoundaries-2_6.pdf). Nevertheless, skyline gives me an error message back stating that the sample names are not fitting to the sample names in the raw file . We have not changed the names of the single sample upon importing in Skyline thus i am looking for help to resolve this. I know i am missing something but i can not figure it out....

Looking forward to hear from you guys!

Max

PS: I have example files ready but can only send them confidentally.

view request
Skyline using questions
(5 responses) chaoxue 2020-10-05
  1. Can skyline show the same precursor and same product, but with different transition settings, such as CE and RF lens?
  2. Can skyline show different monoisotopic transitions, such as M+1, M+2?
view request
Reduction in the number of true peptide identification as the number of DIA raw files increases in the spectral library search
(13 responses) Chinmaya k 2020-09-28

Hello,

I have carried out a Spectral Library search for one of our DIA data generated with a 25m/z isolation window from 400-1000 m/z range. The spectral library search of single DIA.Raw file is able to identify 8000+ peptides with Q-Value < 0.01 from mProphet model. However, when multiple replicates of the same sample (Which includes the DIA file used for the first search) were searched against the same spectral library keeping all other parameters as the same is resulting in a drastically less number of peptides (<5000) with Q-value < 0.01 from mProphet model. And this goes on as the number of files increases.

What might be the reason for this? Why the number of true peptide identifications is decreasing as the number of DIA files are increased?

Prior to applying the mProphet model, the peptides RT was calibrated using Pierce iRT standard spiked in the sample. The screenshots of parameters used for RT prediction and calculation are attached below for your references.

Please let me whether any parameters have to be changed and if so, why? How does it will effect the DIA Spectral library search and statistical validation?

--
Chinmaya

 Peptide_Setting.PNG  Edit_iRT_Calculator.PNG  Edit_Retention_Time_Predictor.PNG 
view request
Identifying a significant protein in Skyline but it's not found in raw files.
(1 response) renad almahdali 2020-10-15

Dear Skyline team,

First, thank you loads for this amazing efficient software!
I am now working on constructing human proteomics profile from PBMC samples using Sciex TripleTOF 5600 and DDA method. I happened to come across a significant (highly expressed) protein in Skyline but when I wanted to check it in the proteins list exported from ProteinPilot raw file I didn't find it. My question is, is it possible to find a protein in Skyline software only but not from the original files?? I even tried to look it up by its protein name and accession including it synonyms.

Waiting for your support guys,
Thanks.
Renad Abdulrahman

view request
Unable to retrieve application files. Files corrupt in deployment. Skyline-64_20_2_0_286 unplugged version
(1 response) h a ebhardt 2020-10-15

Hi Brendan & team members of the Skyline team.

I tried installing Skyline-64_20_2_0_286 unplugged.

I first downloaded the zip file,
unpacked it,
ran setup.exe as administrator

Then: the setup.exe still accessed the internet and after downloading 3/4 of the program, the "Unable to retrieve application files. Files corrupt in deployment" occurred.

I attached a screenshot of the error plus the txt of Details...

There is a two problems here: first, the error itself.

Second, and more important, my IT dep't does not allow me to use programs which access the internet. Hence, the unplugged version. Is it possible to get an unplugged version which does not require the internet for installation?

Maybe the error is related to the fact that programs here are not allowed to access the outside world.

Kind regards,
Holger Alexander Ebhardt.

 skyline202error.jpg  26BB66Q3error.txt 
view request
fail to recognize spectral library from NIST
(3 responses) 851813663 2020-10-13

Dear nick
I have downloaded the Rat's spectral library from NIST, it was in msp format, and when I wanted to use the skyline to build the library, I found that the software could not recognize this format, and I was very eager for your help.
Thank you sincerely

 picture1.PNG 
view request
manual selection and change of ion mobility range for prm-PASEF
(2 responses) RBl 2020-10-14

Dear Skyline Team,
thank you very much for your constant efforts and all that you provide to the mass spec community! The software developed very much over past few years when i did not use it a lot!

I have started using Skyline in combination with the TimsTOF Pro recently. I needed to create a prm-PASEF method also with the ion mobility dimension. I noticed that ion mobility range that is showed in the Skyline was in many cases on the edge of the mobility peak/ion cloud and thereby a big proportion of the peak was not included. In some cases the ion mobility range in Skyline was in the middle of the mobility peak/ion cloud but edges were missing. I prepared some pictures so you can get the impression of what I am talking about. Please see the file attached.
I tried to set the ion mobility ranges in Skyline but I failed. Is there an option available for manual selection of ion mobility range or is it on me and I can not find it?
If there is no option for manual selection of ion mobility range, would it be possible to get something similar as manual chromatographic peak selection for Ion mobility peak/ion cloud in the future? And in the more distant future something like automatic peak peaking for ion mobility dimension if that is possible at all?

May you need more information from me, please do not hesitate to contact me.

Thank you very much for your efforts and your answer.
Best regards,
Renata

 ion mobility in skyline.pdf 
view request
Transition settings ion mobility "use spectral library mobility values when present" cannot be unchecked
(1 response) pierre-olivier schmit 2020-10-14

While unchecking the corresponding radio button and choosing "OK" : the radio button is automatically checked again if we re-open the transition settings ion mobility tab.

view request
ERROR: No Spectra were found for the new library
(1 response) lake n paul 2020-10-13

All,

Excited to see the DDA search implemented into Skyline. However, I tried unsuccessfully to run a search. The search goes to completion however I don't get any spectra for the detected peptides. The XIC for the peptides are present but no MS/MS spectra. An error (see attached) is given.

Help!

Lake

 ERROR.png 
view request
Skyline CMD help
(7 responses) smanda 2020-08-13

Hi Brendon/Kaipo,

I am trying to build a spectral library all using command line as we are trying to automate some of the steps at our lab. To this, I am unable to figure out, how can I choose peptides standards for RT calibration (after I import the library). My current steps are:

  1. SkylineCmd.exe --in=Empty.sky --out=New_library.sky --import-search-file=searchresults.mzid --import-fasta=20190717_Uniprot_concat_decoys.fasta
    This step creates a nr library and adds the peptides as targets.
    I do not specify RT peptides here.
    I then import a report format

  2. SkylineCmd.exe" --report-add=skyline.skyr

  3. SkylineCmd.exe --in=New_library.sky --report-name=OpenSWATH --report-file=test_report.tsv --report-format=tsv

I see that the exported reported as several NAs in the iRT column. We have around 40 endogenous peptides that are added as iRT standards in all experiments, can you please let me know, how to I add them at RT peptides and export the report with iRT values.

Regards,
Srikanth

view request
Some bugs shown in software
(4 responses) fenglinshen yzu 2020-10-13

Some peptides can't show the intact peak when we analysed MRM HR results using skyline which was the lastest released. Only three peptides were analysed with a 15 min gredient and 7.5 min-window was set. We also found the same scan data imported to the latest released skyline software showed only three transitions which was set as "full scan", and when the data was imported to earlier version, all of the transitions were shown. The details are shown in the attachments.

 新建 Microsoft PowerPoint 演示文稿.pptx 
view request
Unable to see any fragment ions in Skyline
(2 responses) clgunth2 2020-10-13

Hello - I created a test transition list with ions I can see in the raw mass spectra and imported into Skyline. When I imported the data, only the precursor mass is identified with the green circle next to the precursor name, even though the ions in my transition list were extracted from the raw file. The fragment ions don't have a red circle for being not detected but rather say "Chromatogram not available" if clicked on. I'm not sure if it's just not reading my data correctly or not. Again, these fragment ions were chosen directly from the raw spectra to test the workflow in Skyline, so they are present in the raw data.

My data file contains only MS2 scans for one precursor. My goal is to be able to filter through a much longer list of theoretical fragments for one complex structure. I'm following similar to what I would do if I was filtering through a transition list with MS1 data except the data file contains solely MS2 scans.

Thanks,
Crystal

 MS2_Test.sky.zip  SkylineError.PNG 
view request
Skyline 20.2 refuses to install
(5 responses) Tomas Vaisar 2020-10-13

For some reason, the manual install of 20.2 does not want to go - after Microsoft Store message - clicking Install Anyway does not do anything. It does not matter whether I run setup.exe normally or as Administrator?
My Windows 10 problem or Skyline setup.exe problem?

Thanks,

Tomas

view request
MS Amanda search result files?
(3 responses) dkueltz 2020-10-12

Hi Nick,
Are the msamanda search results stored somewhere in a format that can be imported into Scaffold or other viewers? It would be useful to have those search result files for comparison with other search engines.
Thanks,
Dietmar

view request
Customise reports
(3 responses) giulia lambiase 2020-10-09

Hello,

I happen to analyse lots of data and would like to automate the data analysis process. Is it possible to customise Skyline reports to make them run scripts for you?

Thanks,
Julia

view request
Question New Protein Abundance Report Feature
(4 responses) roman sakson 2020-10-12

Dear Skyline team,

thank you for providing us with the possibility to get numbers representing protein abundance per replicate directly from Skyline, which is also supposed to be part of the next main Skyline release. I intend to use this option rather frequently in my research and would like to understand it a bit better. I am mainly interested in the normalization case against heavy (each light signal must have a heavy counterpart to be considered).

In the release notes, you state that this protein abundance value is the same that the Group Comparison (GC) framework uses. I thought that, as default, on protein level GC sums up all transition areas that belong to the same protein (also across different peptides) that are quantitative and have a heavy counterpart and then normalizes this sum to the sum of heavy signals. This is slightly different compared with calculating normalized ratios transition by transition and then averaging those ratios, since strong signals dominate the sums of values, as previously described on several occasions. However, the explanation in the reporting window for the "Protein Abundance" feature now states that "The Protein Abundance is calculated by taking the average of the normalized areas of all of the Transitions under the Protein." This now sounds to me exactly as the latter option to do it and the question is, whether protein abundance numbers really are exactly what the GC framework uses?

Thanks a lot,

Roman

view request
Collision Energy Optimization for Precursors?
(2 responses) philip remes 2020-10-09

I've done the tutorial the does collision energy optimization by exporting transition lists: https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/SmallMoleculeMethodDevCEOpt.pdf
For small molecules, it would be useful to do the same thing for PRM experiments. But the Optimizing Dropdown box is disabled for File->Export->Isolation list, even though I see it for the Export->Transition List option. Is this a feature that could be added?

Thanks
Philip

view request
No chromatograms or spectra are reported for peptides with modified cys or met
(12 responses) caminha marcelle 2020-10-07

Hi,
I'm a new Skyline (20.1) user and I'm having difficulties analyzing peptides with cysteine carbamidomethylation and methionine oxidation. The spectra of peptides with cysteine only show up when I select carbamidomethylation as a variable modification, thus reporting them as from m/z without the expected mass differences; no chromatogram is shown in any settings. As for the peptide with methionine oxidation (as a variable modification), neither the spectra nor the chromatogram is reported. However, in both cases, the peptides are detected by the Comet search tool integrated into the PatternLab after either DDA or PRM method acquision. Could you, please, explain me how to solve this? Any advice is greatly appreciated.
Best regards,
Marcelle

view request
Crosslink search from Mascot to create library for Skyline
(8 responses) sandberg 2020-08-05

Hi,
Is it possible to use crosslinking results from the Mascot search engine to create a library to search the files for crosslinked peptides in Skyline?

Best,
Magdalena

view request
Optimizing FAIMS on new Thermo instruments
(4 responses) Martijn van Duijn 2019-09-18

Hi,

Our institution acquired a shining new Orbitrap Eclipse with a FAIMS source module. The FAIMS module need optimization for targeted analysis of our analytes, in order to obtain the best compensation voltage for transmission of the ion of interest.

It would be great if Skyline could facilitate this optimization step, similar to the optimization of collision energies that is already done. In fact, Skyline already seems to include a very similar optimization feature "Compensation Voltage', which seems to be intended for use with the SciEx Differential Mobility Separation. Can this feature be adapted to be used with Thermo FAIMS Compensation Voltages, or is this something that is intended for implementation in a future release of Skyline?

Regards,

Martijn van Duijn

view request
Peak removal from multi-injection replicate
(7 responses) Julienlab 2020-10-06

Hi,

I am analyzing PRM data collected as a multi-injection replicate (2 injections per sample) however for ~10% of my targets, skyline is quantifying the same peptide in both injections when it should only be quantified in one.

I have attached a pdf document showing an example of this where the first page is the correctly identified peak from the second injection as found in all four samples. The second page shows the chromatogram from injection 1 which should not identify this peptide, however it is still being quantified. The dotp scores in these cases are low and appear to be a combination of the two injections instead of only injection 2.

Is there any way to remove single injections from one peptide so the dotp score only reflects one injection?

Thank you,
Bridgette

 PRM-screenshots-False.pdf 
view request
Crashes for export of empty target lists for prm-PASEF
(1 response) jens decker 2020-10-05

We have observed crashes in case of most likely empty target lists. A version of the prmscheduler.dll which should be immune is in the build process. Nevertheless it would be good if Skyline will give a warning and stop the preparations for the export if there are no targets, which seems to be easy to get if certain flags are not set correctly for new users: Get aware of the fact that something is wrong.

view request
A request for support
(1 response) wangcg1119 2020-10-07
As far as I concern, Skyline is the best choice for MS data analysis. Quality data can be obtained based on Orbitrap which is equipped with FAIMS module. We wonder whether Skyline can be employed for procession on PRM data which is generated from Orbitrap Lumos equipped with FAIMS module. If so, please kindly provide relative tutorial to support our research. 
view request
Issue opening any files in Skyline-Daily
(1 response) wlstutts 2020-10-06

I just upgraded Skyline-daily today and now when I click "open file" or the folder icon to open a file, the program freezes. At this point I am unable to open any previously created Skyline documents.

view request
"Failed importing results...The calculator ... requires all of its standared peptides in order to determine a regression
(2 responses) Mark Athanason 2020-10-05

Hello,

I'm getting the following error, seen below. I'm simply trying to build a spectral library to perform DIA quant with 2 DDA runs. I've never seen this error before. I'm using the most current version of skyline daily. For RT standards, I selected automatic because when I select biognosys 10 or 11 it doesn't find any standards, for some reason. When selecting automatic, it finds 10 standards with a r^2 = 1. Going down the wizard is fine, then when it's importing the DDA data it cancels and throws this error:

At 11:25 AM:
Failed importing results file 'E:\Mark\spheroid\firstPlate\DMSO_DDA_1.mzXML'.
The calculator DMSO1 requires all of its standard peptides in order to determine a regression.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'E:\Mark\spheroid\firstPlate\DMSO_DDA_1.mzXML'.
The calculator DMSO1 requires all of its standard peptides in order to determine a regression. ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator DMSO1 requires all of its standard peptides in order to determine a regression.
at pwiz.Skyline.Model.Irt.RCalcIrt.ChooseRegressionPeptides(IEnumerable`1 peptides, Int32& minCount) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Irt\RCalcIrt.cs:line 152
at pwiz.Skyline.Model.Results.ChromCacheBuilder.RetentionTimePredictor.CreateConversion() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 838
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 404
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

The search was performed with comet with .RAW files converted to .mzXML with default settings. The output file is a pep.xml. I think the error is occurring when it's trying to automatically train the mProphet model. In the search settings I performed a concatenated decoy search, with skyline being told to make 1 reverse decoy to train mProphet.
Any help would be appreciated,
Mark

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Exportation of peaks
(3 responses) bousahba imene 2020-10-05

Hello Skyteam :
i need to export the peaks of all my compounds in a report, I checked all the boxes in the editing list of the reports but i couldn't find it .. Is it possible ? if yes, how can i do ?

Thank you very much

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MS Amanda error
(4 responses) dkueltz 2020-10-03
Hi,
I am getting the following error message after specifying the PTMS (just CAM and MetOx) for a DDA search with MS Amanda within the latest build of Skyline daily. The error pops up when clicking next after specifying PTMs. I am not sure why the algorithm is looking for heavy isotope mods since I did not specify any of these in the peptide settings or ms amanda ptm options. I did report the error using the otion in the error popup and checked to include the Skyline document (since it was small) but the error report did not seem to work (at least there was no indication that anything happened). Let me know if you need a DDA raw data file for debugging. I am using bruker .d data and can upload a BSA QC file that I used to generate this error.
Thanks,
Dietmar

Skyline version: 20.1.9.268-f970ef4c5 (64-bit)
Installation ID: 62026530-da44-466f-b8a5-1c67fe40d053
Exception type: IndexOutOfRangeException
Error message: Modification type heavy not found.

--------------------

System.IndexOutOfRangeException: Modification type heavy not found.
   at pwiz.Skyline.Model.DocSettings.PeptideModifications.ChangeModifications(IsotopeLabelType labelType, IList`1 prop) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\DocSettings\PeptideSettings.cs:line 1473
   at pwiz.Skyline.FileUI.PeptideSearch.MatchModificationsControl.AddCheckedModifications(SrmDocument document) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\MatchModificationsControl.cs:line 179
   at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.UpdateModificationSettings() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 0
   at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.NextPage() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 523
   at System.Windows.Forms.Control.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnMouseUp(MouseEventArgs mevent)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ButtonBase.WndProc(Message& m)
   at System.Windows.Forms.Button.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
   at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
   at System.Windows.Forms.Control.WndProcException(Exception e)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.SkylineWindow.ShowImportPeptideSearchDlg(Nullable`1 workflowType) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 3006
   at pwiz.Skyline.SkylineWindow.importPeptideSearchMenuItem_Click(Object sender, EventArgs e) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2988
   at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
   at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
   at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ToolStrip.WndProc(Message& m)
   at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at pwiz.Skyline.Program.Main(String[] args) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Program.cs:line 306
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Error - Skyline failing to download
(1 response) claire tonry 2020-10-03

Hi,

I keep getting the error log when I attempt to download Skyline:

The following properties have been set:
Property: [AdminUser] = true {boolean}
Property: [InstallMode] = HomeSite {string}
Property: [ProcessorArchitecture] = AMD64 {string}
Property: [VersionNT] = 6.2.0 {version}
Running checks for package 'Windows Installer 3.1', phase BuildList
The following properties have been set for package 'Windows Installer 3.1':
Running checks for command 'WindowsInstaller3_1\WindowsInstaller-KB893803-v2-x86.exe'
Result of running operator 'VersionGreaterThanOrEqualTo' on property 'VersionMsi' and value '3.1': true
Result of checks for command 'WindowsInstaller3_1\WindowsInstaller-KB893803-v2-x86.exe' is 'Bypass'
'Windows Installer 3.1' RunCheck result: No Install Needed
Running checks for package 'Microsoft .NET Framework 4 (x86 and x64)', phase BuildList
Reading value 'Version' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v4\Full'
Read string value '4.5.51641'
Setting value '4.5.51641 {string}' for property 'DotNet40Full_TargetVersion'
The following properties have been set for package 'Microsoft .NET Framework 4 (x86 and x64)':
Property: [DotNet40Full_TargetVersion] = 4.5.51641 {string}
Running checks for command 'DotNetFX40\dotNetFx40_Full_x86_x64.exe'
Result of running operator 'ValueEqualTo' on property 'InstallMode' and value 'HomeSite': true
Result of checks for command 'DotNetFX40\dotNetFx40_Full_x86_x64.exe' is 'Bypass'
Running checks for command 'DotNetFX40\dotNetFx40_Full_setup.exe'
Result of running operator 'ValueNotEqualTo' on property 'InstallMode' and value 'HomeSite': false
Result of running operator 'VersionGreaterThanOrEqualTo' on property 'DotNet40Full_TargetVersion' and value '4.0.30129': true
Result of checks for command 'DotNetFX40\dotNetFx40_Full_setup.exe' is 'Bypass'
'Microsoft .NET Framework 4 (x86 and x64)' RunCheck result: No Install Needed
Running checks for package '.NET Framework 3.5 SP1', phase BuildList
Reading value 'SP' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v3.5'
Read integer value 1
Setting value '1 {int}' for property 'DotNet35SP'
The following properties have been set for package '.NET Framework 3.5 SP1':
Property: [DotNet35SP] = 1 {int}
Running checks for command 'DotNetFX35SP1\dotNetFx35setup.exe'
Result of running operator 'ValueGreaterThanEqualTo' on property 'DotNet35SP' and value '1': true
Result of checks for command 'DotNetFX35SP1\dotNetFx35setup.exe' is 'Bypass'
'.NET Framework 3.5 SP1' RunCheck result: No Install Needed
Launching Application.
Application appears to be an application manifest

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Error Import Skyline Daily SureQuant data
(4 responses) romeally 2020-10-02

I am developing a SureQuant assay using Skyline Daily and for the most part it is working fine, however sometimes randomly my .raw data files will not import. I can open them in Qualbrowser and they seem fine but they seem to randomly stop importing at certain scan numbers. Sometimes it fails early and sometimes it fails late in the chromatogram. I have attached the error message I get upon import. This was acquired on a Lumos instrument with software version 3.3 and the latest Skyline Daily using the triggered acquisition tab checked. Any suggestions? Appreciated!

 ImportError.docx 
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Failed importing results from mzML file
(2 responses) michal zawadzki 2020-10-02

Dear Skyline Team,

I am trying to upload an mzML file converted from a Thermo .raw file with MSConvert into a Skyline document where I have already imported a chromatogram library generated by EncyclopeDIA. I do not think the file is corrupted as I am able to use it in EncyclopeDIA without any issues.

I use Skyline (64bit) 20.1.0.155 (a0e7323e3) and MSConvert (Version: 3.0.20219-6a8ecb8e3 (developer build)).

Below is a full text of the error I am getting:

At 12:11:
Failed importing results file 'C:\Users\u525581\Desktop\Skyline EX4163G_DIA_maize_embryos_310720\20200731_EX4163C_001_01.mzML'.
[IO::HandlerBinaryDataArray] Unknown binary data type.

Inner exceptions:
Exception type: System.Exception
Error message: [IO::HandlerBinaryDataArray] Unknown binary data type.
[IO::HandlerBinaryDataArray] Unknown binary data type.
at pwiz.CLI.msdata.ChromatogramList.chromatogram(Int32 index, Boolean getBinaryData)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetChromatogram(Int32 chromIndex, String& id, Single[]& timeArray, Single[]& intensityArray) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 616
at pwiz.Skyline.Model.Results.GlobalChromatogramExtractor.GetChromatogram(Int32 index, Single[]& times, Single[]& intensities) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 172
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 599
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 236
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 439
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

Would you be able to advice how to solve this problem, please?

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Broken compatibility with Shimadzu LCMS 8050 after replacing Shimadzu DataReader with IoModule for MRM files
(3 responses) Pawel 2020-09-30
Hi Skyline Team!

Thanks for your continuous work on the software. Just wanted to make you aware that the most recent update 20.1.9.268 broke the compatibility with Shimadzu LCMS 8050. The following error appears:

"Failed importing results file 'C:\LabSolutions\Data\Chalani\20200929_QC(100x)_021.lcd'.
[ShimadzuReader::getSpectrum] GetMSSpectrumByScan: E_INVALIDARG"

Old version of Skyline still works but I am unable to use Skyline for importing Shimadzu data on any computer on which I updated the software to the above version. Would you mind having a look into this problem?

Regards,
Pawel
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Reprocessing of data obtained by neutral loss on a QQQ using skyline
(2 responses) julien faugere 2020-10-01

Hello,

I did analysis by neutral loss on QQQ on a lipid sample to observe triglycerides. Can we use skyline to reprocess this type of data ? I have often used skyline for reprocessing MRM data but never for neutral loss.

Thank you for your answer.

Kind regards,

Julien Faugere

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Retention time recognized in sec format
(1 response) Khang Huynh 2020-09-29

Hi Brendan and Team,

I have just updated to the latest version of Skyline-daily today. When I imported the LC-MS/MS data files, the retention times on the chromatograms were in "second" format instead of in "minute" as usual (photo attached). I have tried to look into the Settings but could not figure out how to change it back to the "minute" format. Could you please help?

Operating system: Windows 10.
Small molecules for Quantification.
Raw data files: Shimadzu .lcd

Thanks,
Khang Huynh

 Screenshot 2020-09-29 172658.jpg 
view request
Correct spectrum selection for non-redundant library
(1 response) Juan C. Rojas E. 2020-09-30

Dear support,

Attached you can find a few slides displaying my issue.

In multiple instances I have observed that the spectrum selected for the non-redundant library is suboptimal compared to some other spectra available (Slide 1 and 2). For DDA files the problem is easily circumvented by working with the redundant library, but for library generation for DIA, MRM, and/or PRM methods could lead to comparisons to suboptimal representative library spectrum.

The .mzXML files (exported from PEAKS) for data acquired in resolution mode are kept in resolution mode format when exported (Slide 3). Maybe this is the reason for the mismatch due to better random matching (i.e. in mass tolerance consideration) to some of the "split" peaks of some spectra compared to others even if the absolute abundance is lower.

Is it possible to manually exchange the best representative spectrum for the non-redundant library?

If not, could a peak picking step be included in the library building procedure? Or should I just perform it with MSConvert externally?

As always, thank you for your time and support.
Sincerely,
JC

 Library_msms_spectrum_selection.pptx 
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Broad mass range for peak extraction
(4 responses) harald schoeny 2020-09-18

Dear Skyline Team,

I have lipid data measured on an HILIC- Orbitrap QExactive HF with an all ion fragmentation (AIF) approach. I figured it out to extract single masses with the help of the forum but I would like to extract a broader mass range (m/z 530-650 ) withing one peak. It should be the sum of all lipid species within one lipid class. Unfortunately, I couldn't find a way to do that in Skyline. I have tried to set the MS/MS filtering to Isolation scheme/Use results data isolation targets/ Isolation width/fixed/120 mz but nothing really happened. Is there another workaround for that?

Thank you in advance.
Best, Harald

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Problem in building of spectral library
(1 response) Verma AK 2020-09-29

Dear Skyline team,

Recently I used Skyline to build a spectral library from .mgf files. However, I want to convert .mgf file to blib format, because skyline do not accept .mgf file. If a direct conversion is not possible, perhaps there is a way to convert the library to an intermediate format that you are aware of?
Moreover, it is very helpful to me. If you assist me to building of spectral library from my data generated from Thermo Scientific Orbitrap Fusion MS platform.

Best regards,
Arivnd

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Missing light product ions in SureQuant experiment
(3 responses) carmen.gonzaleztejedo 2020-09-28
Dear Team, We performed a SureQuant experiment in an Orbitrap Exploris 480. When we load the data into the Skyline, we can see the XIC for the light and heavy precursors and the XICs for the heavy fragments. However, we cannot see the fragments of the light precursors (see attached). I have checked all the peptide and transition settings but I cannot figure out what the issue is. I would really appreciate if you can help me with that. Thanks a lot in advance. Regards, Carmen.
 SkylineSupport_OE480_SureQuant.pptx 
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Custom iRT peptides
(3 responses) benoit fatou 2020-09-24

Dear Skyline Team,

I was wondering if it is possible to create custom iRT peptides for RT adjustment in MRM experiments. In fact, I am working on plasma samples and I would like to use some endogenous peptides from the serum albumin in my samples.
Thanks for your help,
Best,
Benoit

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DIA 3-plex SILAC
(1 response) trinidad martincampos 2020-09-27

Hello all,

First of all, thank you very much for the effort you put into Skyline for all of us ;)

I took a look at the post with "DIA SILAC" but my problem is still not solved... I have a 3-plex SILAC (light, medium and heavy) experiment that I am going to analyse with DIA in an orbitrap fusion.
Any hint about how could I do that? What happens with a peptide whose light and medium (for example)tags are in different windows? Any possible workflow to do this?

Thank you very much!!!
Trini

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How did skyline read the OpenSWATH results?
(6 responses) andyzcq 2020-09-19

I followed the tutorial, and found there is no merged.osw file in OpenSWATH results.

I have used OpenSWATH (V. 2.5) to analyze my DIA data.

So how did Skyline read OpenSWATH (V. 2.5) results?

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Oxonium ions for MRM glycan analysis
(3 responses) giulia lambiase 2020-09-24

Hello,
I am writing an MRM method for monitoring glycans in mAbs. I need to add oxonium ions in my transition list but cannot work out how to do it in Skyline. Could you please support me with this?
Thanks,
Julia

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Skyline-daily Program Won't Open
(2 responses) ehubb004 2020-09-22

Yesterday, for no clear reason, the skyline-daily program (the most current version) stopped opening on my computer. When I try to open it, my computer briefly registers that it's loading, but nothing actually happens, and task manager never registers Skyline activity. I've tried restarting my computer, as well as completely reinstalling skyline-daily and even redownloading the installer program, but nothing has affected this issue.

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Retention time selection
(6 responses) ojulien 2020-09-17

Hi,

We imported 4 .raw files into skyline, and used a bibliospec file to import all the sequences into skyline.

We then created an inclusion list for all the peptides of interest (~700) for PRM.

However, the RT in the inclusion list are all off if I select "average RT", since the peptides selected were not found in all datasets. The other option is to use a single dataset out of 4, but then the RT will be off for the peptides that were not found in that specific dataset.

The best way would be to use the RT from the dataset that matches the spectral library. Is there a way to do this, am I missing something? If not, is it possible to add this as an option in future release?

This would be great, as of right now, we are adjusting the RT window manually for each peptides in each datasets...

OJ

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Waters Method Export Failure (Masslynx 4.2 SCN986)
(1 response) Will Thompson 2020-09-23

Hi Brian et al

We have recently upgraded two of our Waters TQ-S systems to Windows 10, and purchased licenses for Masslynx 4.2 SCN986 to do this upgrade. When trying to export *.exp methods for the instrument, we are now getting errors that the method cannot be exported. I am pasting in the long text of the method below. Do you have any hints from this text on whether or not it is a masslynx version issue, or something else? We have confirmed the method export is functional on MassLynx 4.2 SCN1007, which runs on the TQ-XS. I have asked Waters if there is a more recent upgrade for the TQ-S, or if we can run SCN1007 on the TQ-S. Maybe there are other questions you guys can ask your Waters contacts?

This would mean that any Waters TQ-S users who upgrade to Windows 10 will not be able to export methods from Skyline any more... :(

Cheers,

Will

Error text below


System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

Command-line: Method\Waters\BuildWatersMethod -w 2 -s -m "C:\VerifyETemplate.exp"
Working directory: D:\5633
Standard input:
D:\5633~SKEB17.tmp
D:\5633\5633_TQS_v1.exp
protein.name,peptide.seq,precursor.mz,precursor.retT,product.m_z,collision_energy,cone_voltage,peptide_unmod.seq,ion_name,library_rank
1,C0.[M+],162.1,0.56,85.1,18,32,C0,Ion [85.100549/85.100549],-1
2,d3-C0.[M+],165.2,0.56,85.1,18,32,d3-C0,Ion [85.100549/85.100549],-1
3,C2.[M+],204.1,0.9,85.1,18,26,C2,Ion [85.100549/85.100549],-1
4,d3-C2.[M+],207.1,0.9,85.1,18,26,d3-C2,Ion [85.100549/85.100549],-1
13,C3.[M+],218.1,1.73,85.1,18,28,C3,Ion [85.100549/85.100549],-1
14,d3-C3.[M+],221.1,1.73,85.1,18,28,d3-C3,Ion [85.100549/85.100549],-1
15,iC4.[M+],232.1,2.19,85.1,18,28,iC4,Ion [85.100549/85.100549],-1
16,C4.[M+],232.1,2.29,85.1,18,28,C4,Ion [85.100549/85.100549],-1
12,d3-C4.[M+],235.2,2.27,85.1,18,28,d3-C4,Ion [85.100549/85.100549],-1
21,C5:1.[M+],244.1,2.66,85.1,20,32,C5:1,Ion [85.100549/85.100549],-1
22,C4:1-M.[M+],244.1,2.71,85.1,20,32,C4:1-M,Ion [85.100549/85.100549],-1
23,C5-P.[M+],246.2,2.69,85.1,18,30,C5-P,Ion [85.100549/85.100549],-1
24,C4-2M.[M+],246.2,2.81,85.1,18,30,C4-2M,Ion [85.100549/85.100549],-1
25,iC5.[M+],246.2,2.89,85.1,18,30,iC5,Ion [85.100549/85.100549],-1
26,C5.[M+],246.2,3.01,85.1,18,30,C5,Ion [85.100549/85.100549],-1
5,C3-DC.[M+],248.1,0.8,85.1,21,30,C3-DC,Ion [85.100549/85.100549],-1
6,C4-OH.[M+],248.1,1.25,85.1,21,30,C4-OH,Ion [85.100549/85.100549],-1
19,d9-C5.[M+],255.2,2.86,85.1,18,30,d9-C5,Ion [85.100549/85.100549],-1
20,C6.[M+],260.2,3.56,85.1,18,30,C6,Ion [85.100549/85.100549],-1
7,C4-DC.[M+],262.2,1.2,85.1,21,32,C4-DC,Ion [85.100549/85.100549],-1
8,C3-DC-M.[M+],262.2,1.5,85.1,21,32,C3-DC-M,Ion [85.100549/85.100549],-1
9,C5OH-I.[M+],262.2,1.78,85.1,21,32,C5OH-I,Ion [85.100549/85.100549],-1
29,d3-C6.[M+],263.2,3.55,85.1,18,30,d3-C6,Ion [85.100549/85.100549],-1
10,C5-DC.[M+],276.1,1.71,85.1,20,32,C5-DC,Ion [85.100549/85.100549],-1
11,C6-OH.[M+],276.1,2.53,85.1,20,32,C6-OH,Ion [85.100549/85.100549],-1
31,C8:1.[M+],286.2,4.16,85.1,22,34,C8:1,Ion [85.100549/85.100549],-1
32,C8.[M+],288.2,4.28,85.1,22,34,C8,Ion [85.100549/85.100549],-1
17,C6-DC.[M+],290.2,2.01,85.1,20,34,C6-DC,Ion [85.100549/85.100549],-1
18,C5-M-DC.[M+],290.2,2.08,85.1,20,34,C5-M-DC,Ion [85.100549/85.100549],-1
33,d3-C8.[M+],291.2,4.27,85.1,22,34,d3-C8,Ion [85.100549/85.100549],-1
28,C10:1.[M+],314.2,4.76,85.1,22,37,C10:1,Ion [85.100549/85.100549],-1
34,C10.[M+],316.2,4.86,85.1,22,38,C10,Ion [85.100549/85.100549],-1
27,C8-DC.[M+],318.2,2.94,85.1,22,35,C8-DC,Ion [85.100549/85.100549],-1
35,d3-C10.[M+],318.9,4.85,85.1,22,38,d3-C10,Ion [85.100549/85.100549],-1
37,C12:1.[M+],342.3,5.29,85.1,24,40,C12:1,Ion [85.100549/85.100549],-1
39,C12.[M+],344.2,5.37,85.1,22,38,C12,Ion [85.100549/85.100549],-1
30,C10-DC.[M+],346.2,3.62,85.1,22,36,C10-DC,Ion [85.100549/85.100549],-1
40,d3-C12.[M+],347.2,5.37,85.1,22,38,d3-C12,Ion [85.100549/85.100549],-1
36,C12-OH.[M+],360.3,4.87,85.1,23,38,C12-OH,Ion [85.100549/85.100549],-1
38,C14:2.[M+],368.3,5.33,85.1,23,39,C14:2,Ion [85.100549/85.100549],-1
42,C14:1.[M+],370.2,5.76,85.1,23,39,C14:1,Ion [85.100549/85.100549],-1
43,C14.[M+],372.2,5.85,85.1,24,40,C14,Ion [85.100549/85.100549],-1
44,d3-C14.[M+],375,5.85,85.1,24,40,d3-C14,Ion [85.100549/85.100549],-1
49,cis-9-C16:1.[M+],398.3,5.98,85.1,25,42,cis-9-C16:1,Ion [85.100549/85.100549],-1
50,trans-2-C16:1.[M+],398.3,6.2,85.1,25,42,trans-2-C16:1,Ion [85.100549/85.100549],-1
51,C16.[M+],400.4,6.27,85.1,24,44,C16,Ion [85.100549/85.100549],-1
52,d3-C16.[M+],403.4,6.27,85.1,24,44,d3-C16,Ion [85.100549/85.100549],-1
41,C16:1-OH.[M+],414.3,5.51,85.1,26,44,C16:1-OH,Ion [85.100549/85.100549],-1
45,C16-OH.[M+],416.3,5.85,85.1,26,44,C16-OH,Ion [85.100549/85.100549],-1
48,C18:2.[M+],424.4,6.09,85.1,26,45,C18:2,Ion [85.100549/85.100549],-1
54,C18:1.[M+],426.4,6.34,85.1,27,45,C18:1,Ion [85.100549/85.100549],-1
55,C18.[M+],428.4,6.62,85.1,28,50,C18,Ion [85.100549/85.100549],-1
56,d3-C18.[M+],431.4,6.61,85.1,28,50,d3-C18,Ion [85.100549/85.100549],-1
46,C18:1-OH.[M+],442.4,5.97,85.1,28,46,C18:1-OH,Ion [85.100549/85.100549],-1
53,C18-OH.[M+],444.3,6.27,85.1,28,46,C18-OH,Ion [85.100549/85.100549],-1
47,C20:4.[M+],448.3,6.08,85.1,28,48,C20:4,Ion [85.100549/85.100549],-1
---> System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

Command-line: Method\Waters\BuildWatersMethod -w 2 -s -m "C:\VerifyETemplate.exp"
Working directory: D:\5633
Standard input:
D:\5633~SKEB17.tmp
D:\5633\5633_TQS_v1.exp
protein.name,peptide.seq,precursor.mz,precursor.retT,product.m_z,collision_energy,cone_voltage,peptide_unmod.seq,ion_name,library_rank
1,C0.[M+],162.1,0.56,85.1,18,32,C0,Ion [85.100549/85.100549],-1
2,d3-C0.[M+],165.2,0.56,85.1,18,32,d3-C0,Ion [85.100549/85.100549],-1
3,C2.[M+],204.1,0.9,85.1,18,26,C2,Ion [85.100549/85.100549],-1
4,d3-C2.[M+],207.1,0.9,85.1,18,26,d3-C2,Ion [85.100549/85.100549],-1
13,C3.[M+],218.1,1.73,85.1,18,28,C3,Ion [85.100549/85.100549],-1
14,d3-C3.[M+],221.1,1.73,85.1,18,28,d3-C3,Ion [85.100549/85.100549],-1
15,iC4.[M+],232.1,2.19,85.1,18,28,iC4,Ion [85.100549/85.100549],-1
16,C4.[M+],232.1,2.29,85.1,18,28,C4,Ion [85.100549/85.100549],-1
12,d3-C4.[M+],235.2,2.27,85.1,18,28,d3-C4,Ion [85.100549/85.100549],-1
21,C5:1.[M+],244.1,2.66,85.1,20,32,C5:1,Ion [85.100549/85.100549],-1
22,C4:1-M.[M+],244.1,2.71,85.1,20,32,C4:1-M,Ion [85.100549/85.100549],-1
23,C5-P.[M+],246.2,2.69,85.1,18,30,C5-P,Ion [85.100549/85.100549],-1
24,C4-2M.[M+],246.2,2.81,85.1,18,30,C4-2M,Ion [85.100549/85.100549],-1
25,iC5.[M+],246.2,2.89,85.1,18,30,iC5,Ion [85.100549/85.100549],-1
26,C5.[M+],246.2,3.01,85.1,18,30,C5,Ion [85.100549/85.100549],-1
5,C3-DC.[M+],248.1,0.8,85.1,21,30,C3-DC,Ion [85.100549/85.100549],-1
6,C4-OH.[M+],248.1,1.25,85.1,21,30,C4-OH,Ion [85.100549/85.100549],-1
19,d9-C5.[M+],255.2,2.86,85.1,18,30,d9-C5,Ion [85.100549/85.100549],-1
20,C6.[M+],260.2,3.56,85.1,18,30,C6,Ion [85.100549/85.100549],-1
7,C4-DC.[M+],262.2,1.2,85.1,21,32,C4-DC,Ion [85.100549/85.100549],-1
8,C3-DC-M.[M+],262.2,1.5,85.1,21,32,C3-DC-M,Ion [85.100549/85.100549],-1
9,C5OH-I.[M+],262.2,1.78,85.1,21,32,C5OH-I,Ion [85.100549/85.100549],-1
29,d3-C6.[M+],263.2,3.55,85.1,18,30,d3-C6,Ion [85.100549/85.100549],-1
10,C5-DC.[M+],276.1,1.71,85.1,20,32,C5-DC,Ion [85.100549/85.100549],-1
11,C6-OH.[M+],276.1,2.53,85.1,20,32,C6-OH,Ion [85.100549/85.100549],-1
31,C8:1.[M+],286.2,4.16,85.1,22,34,C8:1,Ion [85.100549/85.100549],-1
32,C8.[M+],288.2,4.28,85.1,22,34,C8,Ion [85.100549/85.100549],-1
17,C6-DC.[M+],290.2,2.01,85.1,20,34,C6-DC,Ion [85.100549/85.100549],-1
18,C5-M-DC.[M+],290.2,2.08,85.1,20,34,C5-M-DC,Ion [85.100549/85.100549],-1
33,d3-C8.[M+],291.2,4.27,85.1,22,34,d3-C8,Ion [85.100549/85.100549],-1
28,C10:1.[M+],314.2,4.76,85.1,22,37,C10:1,Ion [85.100549/85.100549],-1
34,C10.[M+],316.2,4.86,85.1,22,38,C10,Ion [85.100549/85.100549],-1
27,C8-DC.[M+],318.2,2.94,85.1,22,35,C8-DC,Ion [85.100549/85.100549],-1
35,d3-C10.[M+],318.9,4.85,85.1,22,38,d3-C10,Ion [85.100549/85.100549],-1
37,C12:1.[M+],342.3,5.29,85.1,24,40,C12:1,Ion [85.100549/85.100549],-1
39,C12.[M+],344.2,5.37,85.1,22,38,C12,Ion [85.100549/85.100549],-1
30,C10-DC.[M+],346.2,3.62,85.1,22,36,C10-DC,Ion [85.100549/85.100549],-1
40,d3-C12.[M+],347.2,5.37,85.1,22,38,d3-C12,Ion [85.100549/85.100549],-1
36,C12-OH.[M+],360.3,4.87,85.1,23,38,C12-OH,Ion [85.100549/85.100549],-1
38,C14:2.[M+],368.3,5.33,85.1,23,39,C14:2,Ion [85.100549/85.100549],-1
42,C14:1.[M+],370.2,5.76,85.1,23,39,C14:1,Ion [85.100549/85.100549],-1
43,C14.[M+],372.2,5.85,85.1,24,40,C14,Ion [85.100549/85.100549],-1
44,d3-C14.[M+],375,5.85,85.1,24,40,d3-C14,Ion [85.100549/85.100549],-1
49,cis-9-C16:1.[M+],398.3,5.98,85.1,25,42,cis-9-C16:1,Ion [85.100549/85.100549],-1
50,trans-2-C16:1.[M+],398.3,6.2,85.1,25,42,trans-2-C16:1,Ion [85.100549/85.100549],-1
51,C16.[M+],400.4,6.27,85.1,24,44,C16,Ion [85.100549/85.100549],-1
52,d3-C16.[M+],403.4,6.27,85.1,24,44,d3-C16,Ion [85.100549/85.100549],-1
41,C16:1-OH.[M+],414.3,5.51,85.1,26,44,C16:1-OH,Ion [85.100549/85.100549],-1
45,C16-OH.[M+],416.3,5.85,85.1,26,44,C16-OH,Ion [85.100549/85.100549],-1
48,C18:2.[M+],424.4,6.09,85.1,26,45,C18:2,Ion [85.100549/85.100549],-1
54,C18:1.[M+],426.4,6.34,85.1,27,45,C18:1,Ion [85.100549/85.100549],-1
55,C18.[M+],428.4,6.62,85.1,28,50,C18,Ion [85.100549/85.100549],-1
56,d3-C18.[M+],431.4,6.61,85.1,28,50,d3-C18,Ion [85.100549/85.100549],-1
46,C18:1-OH.[M+],442.4,5.97,85.1,28,46,C18:1-OH,Ion [85.100549/85.100549],-1
53,C18-OH.[M+],444.3,6.27,85.1,28,46,C18-OH,Ion [85.100549/85.100549],-1
47,C20:4.[M+],448.3,6.08,85.1,28,48,C20:4,Ion [85.100549/85.100549],-1

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List1 argv, String fileName, String templateName, Dictionary2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 4162
at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 3959
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254 --- End of inner exception stack trace --- at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1942 at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140 at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action1 performExport) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2095

view request
Export method to MassLynx 4.2 version
(6 responses) danielacgranato 2018-11-27

Dear,

I was wondering if the new version of Skyline (v.4.2) allows to export SRM methods to MassLynx version 4.2. Last year I was unable to do so and I received a response that Waters was working on making it possible. As a solution to the problem I have been exporting the method to a computer not connected to the equipment (Xevo TQ-XS) and to an older version of MassLynx (v.4.1). Do you know if it is possible now to export directly to MassLynx 4.2?

Thank you very much. Best, Daniela

view request
Cannot Roundtrip Small Molecule Transition List
(3 responses) damien ready 2020-09-17

Greetings,
We are looking into using Skyline for some of our small molecule processing, and I ran into a rough edge I thought could be improved around managing transition lists. If I graphically build a small molecule transition list in Skyline and export it, Skyline fails to import the list into a new document.

If you follow the below, I was able to manipulate the exported document into a format that Skyline would import, but the workflow is cumbersome for something that feels like it should be natively handled by the GUI.

If you do intend to investigate the issue, I should be clear that my actual interest is in automating the creation of Skyline documents + transition lists through the Skyline Commandline runner. This work through the GUI was just to establish POC.

Skyline Daily 20.1.9.234
To reproduce:

  • Start Skyline
  • Select Small Molecule workflow
  • Edit->Insert-Transition List
    • Molecule List Name: aspirin
    • Precursor Name: aspirin
    • Precursor Formula: C9H8O4
    • Precursor Adduct: [M+H]
    • Precursor m/z: 181.0495348
    • Precursor Charge: 1
  • Insert molecule
  • File->Export->Transition List
  • File->New
  • File->Import->Transition List
    Results in following error message:
System.IO.InvalidDataException: Failed to find peptide column., line 1. ---> System.IO.InvalidDataException: Failed to find peptide column., line 1. ---> pwiz.Skyline.Model.LineColNumberedIoException: Failed to find peptide column., line 1.
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, ColumnIndices indices, IDictionary`2 dictNameSeq, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 485
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 423
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 427
   at pwiz.Skyline.Model.SrmDocument.ImportMassList(MassListInputs inputs, IProgressMonitor progressMonitor, IdentityPath to, IdentityPath& firstAdded, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList, List`1& peptideGroups) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1454
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass1079_0.<ImportMassList>b__2(IProgressMonitor longWaitBroker) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1925
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.SkylineWindow.ImportMassList(MassListInputs inputs, String description, Boolean assayLibrary) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1923
   at pwiz.Skyline.SkylineWindow.ImportMassList(String fileName) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1902

Consulting the Help on the "Transition List" window, I attempted to hand-edit the exported CSV to include the columns (MoleculeGroup, PrecursorName, ProductName, PrecursorFormula, ProductFormula, PrecursorMz, ProductMz, PrecursorCharge, ProductCharge, PrecursorAdduct, ProductAdduct, PrecursorRT, PrecursorRTWindow, PrecursorCE, PrecursorDT, HighEnergyDTOffset, PrecursorIM, HighEnergyIMOffset, IMUnits, PrecursorCCS, SLens, ConeVoltage, CompensationVoltage, DeclusteringPotential, Note, LabelType, InChiKey, CAS, HMDB, InChi, SMILES, KEGG, ProductNeutralLoss), added the required data, but this too failed to import. Lastly, I added the suggested columns, filled in the extra data, and deleted the originally exported columns - this worked.

Attached:

  • small_molecule_ts_export.csv - failed to import - Skyline small molecule built and exported transition list
  • small_molecule_ts_export_extracolumns.csv - failed to import - Skyline small molecule export + manually added suggested columns
  • small_molecule_ts_export_extracolumns_only.csv - successful import - Skyline small molecule export + manually added suggested columns only

Thanks for your attention. Happy to give any more details that you require.

 small_molecule_ts_export.csv  small_molecule_ts_export_extracolumns.csv  small_molecule_ts_export_extracolumns_only.csv 
view request
Comments need to be deleted
(5 responses) mattkarasu 2020-09-11

When exporting an MS Method for our Waters Xevo TQ from Skyline it seems to be creating an .exp file with comments added to every line of the MRM table. Oddly, there seem to be two different comments though? One mentioning Quanpedia and the other mentioning VerifyESkylinLibrary.

It is also worth pointing out that the output .exp file has these comments present even when opened as a raw text file, so I know it is not something our MS File Editor is adding or misinterpreting.

view request
Extracting MS1 peak area for peptides of known mass from DDA/bbCID experiments
(3 responses) Mus 2020-09-14
Hi folks, I have 6 datafiles from DDA runs on a Bruker QTOF. I collected MS/MS fragmentation data in bbCID mode (similar to all ion fragmentation), - only to assign MS1 peaks confidently. Each of the 6 samples are varying but defined mixtures of the following, in a sea of trypsin digested BSA: - 35 synthetically made LIGHT peptides (with K or R C-terminus to mimic a trypsin digest) - 35 synthetically made HEAVY (deuterium labelled) peptides (with K or R C-terminus to mimic a trypsin digest) The peptide sequences are known and I have precurser masses for +2/+3 charge states of all the 70 peptides. The samples contain varying ratio's of LIGHT:HEAVY peptides and I am trying to determine these ratio's by extracting peak area for MS1 peaks for each of 70 peptides. What's the best way to do this in Skyline? I have tried MS1 filtering workflow without success. Thanks in advanvce.
view request
support in Shimadzu LabSOlutions LCMS 5.99 SP2
(4 responses) Stephane MOREAU 2020-08-28

Hi,

it seems that Skyline cannot read data coming from the latest version of SHimadzu LabSolutions LCMS 5.99 SP2 ?

sincerely

view request
Prosit Server Unavailibility
(3 responses) Chinmaya k 2020-09-14

Hello,

I am not able to connect to the Prosit server (131.159.152.7:8500) through Skyline. What is the issue?

--
Chinmaya

 Skyline_Prosit_Server_Unavailability.PNG 
view request
error building Prosit library
(1 response) bin fang 2020-08-28
pwiz.Skyline.Model.Prosit.PrositException: Status(StatusCode=Unknown, Detail="CUDNN_STATUS_BAD_PARAM
in external/org_tensorflow/tensorflow/stream_executor/cuda/cuda_dnn.cc(1249): 'cudnnSetTensorNdDescriptor( tensor_desc.get(), data_type, sizeof(dims) / sizeof(dims[0]), dims, strides)'
     [[{{node encoder1/CudnnRNN}} = CudnnRNN[T=DT_FLOAT, direction="unidirectional", dropout=0, input_mode="linear_input", is_training=true, rnn_mode="gru", seed=87654321, seed2=0, _device="/job:localhost/replica:0/task:0/device:GPU:0"](encoder1/transpose, encoder1/ExpandDims_1, encoder1/Const, encoder1/concat)]]
     [[{{node out/Reshape/_55}} = _Recv[client_terminated=false, recv_device="/job:localhost/replica:0/task:0/device:CPU:0", send_device="/job:localhost/replica:0/task:0/device:GPU:0", send_device_incarnation=1, tensor_name="edge_605_out/Reshape", tensor_type=DT_FLOAT, _device="/job:localhost/replica:0/task:0/device:CPU:0"]()]]") ---> Grpc.Core.RpcException: Status(StatusCode=Unknown, Detail="CUDNN_STATUS_BAD_PARAM
in external/org_tensorflow/tensorflow/stream_executor/cuda/cuda_dnn.cc(1249): 'cudnnSetTensorNdDescriptor( tensor_desc.get(), data_type, sizeof(dims) / sizeof(dims[0]), dims, strides)'
     [[{{node encoder1/CudnnRNN}} = CudnnRNN[T=DT_FLOAT, direction="unidirectional", dropout=0, input_mode="linear_input", is_training=true, rnn_mode="gru", seed=87654321, seed2=0, _device="/job:localhost/replica:0/task:0/device:GPU:0"](encoder1/transpose, encoder1/ExpandDims_1, encoder1/Const, encoder1/concat)]]
     [[{{node out/Reshape/_55}} = _Recv[client_terminated=false, recv_device="/job:localhost/replica:0/task:0/device:CPU:0", send_device="/job:localhost/replica:0/task:0/device:GPU:0", send_device_incarnation=1, tensor_name="edge_605_out/Reshape", tensor_type=DT_FLOAT, _device="/job:localhost/replica:0/task:0/device:CPU:0"]()]]")
   at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   at System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   at Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   at Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   at Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 96
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 86
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 230
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 234
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 314
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 113
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 69
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Cannot fill in Protein Description field in 'Edit>Insert>Peptides'
(2 responses) shaoen 2020-09-09

Hi,

I would like to fill all three fields (peptide sequence, protein name, protein description) when adding a list of peptides but it seems I can't enter anything in the protein description field. Data in the first two columns work fine. What am I missing?

Skyline version: Skyline-Daily 20.1.9.234

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System.IO.IOException: ERROR: Error parsing modifications file: modifications.xml(line 5662): Missing required attribute 'composition'.
(2 responses) m kuppusamy 2020-09-09

I am trying to load some DDA data on skyline using the import DDA peptide search option. I get an error "System.IO.IOException: ERROR: Error parsing modifications file: modifications.xml(line 5662): Missing required attribute 'composition'." when I try to load the msms.txt file from the Maxquant search. I already have modifications.xml file from maxquant in the same directory as well. Could you please tell me what could be the problem here?

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issue when importing peptide search from a MaxQuant msms.txt file - ignored modifications
(2 responses) marie locard-paulet21061 2020-09-08

Hello,

I would like to use Skyline to generate a spectral library compatible with a library-based DIA search in DIA-NN.
My issue is that when I import an output from MaxQuant (1.6.14) using the "import peptide search" pipeline (msms.txt file in the same folder as the raw files, the mqpar.xml and the modifications.xml containing the list of variable modifications -I added the Ammonium and Ammonia loss in the bottom-), the only modifications that I find in the output are oxidation (M) and carbamydomethylation (C). So several modifications are missing.
I am pretty sure that I miss something but I cannot figure out what.

The idea is to then export a custom report from Skyline that would be in a format that I can make compatible with a DIA-NN input library. This bit works.

I work with Skyline (64-bit) 20.1.0.155 (a0e7323e3).
I have uploaded the MaxQuant outputs I work with on the skyline upload site, with the name "Ticket_MLocardPaulet.zip".
Please let me know if you need more information.
Thank you for your help.
Kind regards,
Marie.

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Bibliospec for spectral library searching
(2 responses) david morgenstern 2020-09-06

Hi,

I'm considering moving to using spectral library searching for glycoprotoemics due to the prohibitive search times (especially for large datasets). apart from the old manuscript - is there any tutorial I can use for it? there are multiple questions I've got regarding the use of the software:

  1. how does FDR performed after the search?
  2. how does it deal with very small database (50 entries) for FDR calculations?
  3. how does the scoring algorithm (as well as the annotator) deal with c/z B/Y spectra from glycopeptides and EThcD/sHCD?
  4. can it look for modifications on top of what comes out of the library?
  5. Can you convert the .blib libraries to .msp?
  6. can it deal with PRM data for library preparation? PRM with separate MS1?

I'd appreciate any resource you can point out to me.

Thanks!
David.

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bibliospec error
(4 responses) dkueltz 2020-09-05

Hi Nick, Brendan,
I have used the integrated bibliospec for many years and never had a problem but now for some files the error below pops up when trying to build a new spectral library. I am using pep.xml + corresponding mzxml files generated in PEAKS Xplus for building libraries. DDA data from two consecutive runs that look similar in terms of peptide and protein coverage and file size give different results. One file works fine for library generation but the other throws this error. I tried this with Skyline v19, v20, and the latest daily. I also tried windows admin and regular user accounts - same result. There is no disk space issue either. I can upload an example pep.xml + mzxml file pair that works and another one that does not work if that would help troubleshoot the issue - let me know.
Thanks,
Dietmar
System.IO.IOException: Error occurred running process.

Command-line: C:\Users\dkueltz\AppData\Local\Apps\2.0\CHO9N7RC.HQC\PCZXTA8K.NOQ\skyl..tion_e4141a2a22107248_0013.0001_e50c2c68eab478d7\BlibBuild -s -A -H -o -c 0.95 -i t -S "C:\Users\dkueltz\AppData\Local\Temp\tmpA47.tmp" "D:\Spectral-Libraries\t.redundant.blib"
Working directory: D:\LR0013_Oremo-Kidney\PEAKS_mzID-mzXml_Export\PEAKS10P__LR0013_Oremo-Kidney_gradual2weeks_PEAKS PTM_42
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 186
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144

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