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
mzTab Output from Skyline
(3 responses) mhadisur 2022-01-12

Hi,

I want to upload the PRM data (manually curated) that I analyzed in Skyline into the MassIVE repository. The MassIVE repository requires the result files, such as mzIdentML and mzTab. How do I export the Skyline results in mzTab format compatible with MassIVE?

Thank you for the help!

Marco Hadisurya

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SQL Failure Error with Peptide Library
(4 responses) ed3 2022-01-15

Hello,

I have been having trouble making a peptide library when I import my pep.XML file. Please see the attached picture to see the error message I receive. I am not sure if this is an issue with the pep.XML file, or the mzXML file that I am using, but any help with this error would be greatly appreciated.

Thank You,
Ed

 skyline error.PNG 
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Selection of transitions that diminish precursor dotp
(1 response) dkueltz 2022-01-14

Hi Nick, Brendan,
I was wondering if there is an automated way for identifying transitions that are interfering with the expected fragment (product) ion intensity pattern and do not conform to the pattern represented in the corresponding library spectrum. Sometimes there is a transitions that greatly "derails" the precursor dotp and when I delete those "interferences" then the dotp value increases greatly, sometimes from 0.5 or so to 0.99 if the interfering transition is high intensity in the samples but very low in library spectra or vice versa. Is there any way to automatically identify those "interfering" transitions in the document view grid? It would be nice to select them all in that grid and then delete them at once rather than having to manually check and delete them 1 by 1 for each precursor.
Thanks,
Dietmar
BTW: thanks for fixing the array dimension error in the latest Skyline daily release - much appreciated!

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RT selection from multiple DDA file, matching highest dotP peptide
(3 responses) af1234 2022-01-12

I have approx ~500 heavy peptides which I want to use to design a PRM-pasef method. I ran them individually in batches of 100 in DDA-pasef and searched all the files with Fragpipe. I then imported all of them into Skyline but, unfortunately, similarly to this https://skyline.ms/announcements/home/support/thread.view?entityId=9a85d70f-db25-1038-8050-e465a3939b46&_docid=thread%3A9a85d70f-db25-1038-8050-e465a3939b46 I want to schedule the PRM using the peptides which are the highest dotP (as each peptide is only in one sample out of 5). It is pretty clear to see which one is correct (see attached), but having to manually change the RT for 500 peptides to match the correct one (and being able to use average RT across the replicates) will take a really long time. I was wondering if there is any update on how to do this.

Thanks!

 Screen Shot 2022-01-12 at 12.10.39 PM.png 
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Merging two documents
(3 responses) Michael Cundell 2022-01-12

I am having trouble merging two documents in Skyline (64-bit) 20.1.0.31 (f09d9bed5) which is probably a result of how I created the two documents:

  1. I had a document with all peptides/transitions of interest in it.
  2. I then imported the raw files.
  3. I then copied the three files .sky, .sky.view and .skyd and shared those with a colleague.
  4. We then both adjusted peak integration boundaries and removed some of the light peptide channels by right clicking chromatograms and choosing 'remove peak' for specific replicates. Generally we kept all heavy label channels, but sometimes we removed these peaks also.
    My colleague started from the top, i started from the bottom.
  5. When we met halfway we both stopped.
  6. we both copied the documents to a separate folder (in case of issues) and each deleted peptides whose peak boundaries were not adjusted in our respective documents.

From this point, is there a way to merge these two documents (referred to as A and B below)?

I tried file > import > Document and select "merge with existing results by replicate name". When i merge A with B, the missing peptides in A are added from B to the targets pane, but the adjusted peak boundaries and removed light labels are not brought over into A from B. Instead, I just get the original skyline peak boundaries created at import in step 2 above (I presume this is because they still reside in skyd in document A). If I do "add new replicates" instead all peak boundaries and selections are brought over from B into A fine, but this just duplicates the replicates (which are identical in both documents).

I also tried exporting the peak boundaries from A and B, then concatenated those lists together in excel and reimported with file > import > peak boundaries. This gave the right windows, but all peaks had both heavy and light labels turned on (it didnt take into account we would like to preserve where we have removed a light label, to keep that light label off/removed from the specific replicates).

Probably the simplest solution is to just keep the documents separate and export the peptide results from each file and merge the two output CSVs. But it would be good to know if I am missing something.

Thank you for your help and a great tool.

Best wishes,

Michael

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Error skyline 21.2
(2 responses) solene bertho 2022-01-13
Hello, I am trying to update my version of Skyline, but the certificate is no valid. Do you know this error and do you know what to do? Thank's
 Erreur instal skyline.txt 
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Using IM predictor on DIA PASEF
(16 responses) paga 2021-12-30

Hi again Skyline team, I come with a new inquiry.

I've been going over the 'Skyline Ion Mobility Spectrum Filtering' tutorial to try and implement the IM dimension into my DIA-PASEF experiment, hoping it could lower the noise and therefore help on the identification of our 30 Heavy labeled peptides and their non-labeled counterparts. I realize that the tutorial I'm referring to is not directly showing this kind of application, but in theory, it should work as well.

So far, my attempts have only worsened identification when compared to a spectral library-based predictor, which can be seen in the picture attached, where I totally lose the signal on an otherwise confidently-identified peptide. Based on this, I come with the following inquiry. What is the nature of the Q_2014_0522_ASMS2014 library? Is this a file acquired in DDA acquisition mode, while BSA_Frag_100nM_18May15_Fir_15-04-02.d and Yeast_0pt1ug_BSA_100nM_18May15_Fir_15-04-01.d files are samples acquired in DIA mode?

If this is the case, then the difficulties I'm experiencing may be because the DIA file I'm using to train the IM prediction is not a pure sample (it contains Hela extract) and therefore selects the wrong drift time range.

Once again, thanks for your help.

 Screenshot 2021-12-30 at 12.22.28.png 
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File Loading Error
(5 responses) germolus 2021-10-17

Hi there. Hoping this will be a quick issue.

I've encountered this error a couple of times now, where I go to open my most recently saved file and there's some error. In this case...

  • Positive mode: "Failure opening ........\pos_07Oct.sky. The file contains an error on line 16933 at column 9."
  • Negative mode: "Failure opening ........\neg_07Oct.sky. The file contains an error on line 10862 at column 9."

Now usually I'm pretty good about doing a "Save As'" with the current date so if something gets ganked up I can go back to the prior date. I didn't do that last week, and now the file's broken and I'd like this to not happen again.

I'll upload the files shortly. Thank you so much!

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#N/A values for Normalised Area in Calibration curve grid
(1 response) sa825 2022-01-10

Hi Skyline,

Happy New Year!

I am viewing some results and I keep getting #N/A values in for Normalised Area in the Calibration curve grid.

I don't understand why? as Skyline can clearly see all 3 transitions (As shown in the image attached) but I keep getting that error?

The image shows 3 replicates of a peptide called : "GWVTDGFSSLK" and you can see all 3 transitions of the peptide detected at each replicate but as highlighted in blue, replicates 1 (31L_1) and 2 (31L_2) are showing #N/A values.

Is there a way I can fix this or is there something I am doing wrong?

Thanks,
Shimon

 Screenshot 2022-01-11 at 02.10.13.png 
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log4j issues?
(1 response) anders honore 2022-01-07

Hi
Sorry to display my complete ignorance regarding the technical foundation for Skyline. Me and colleagues are happy users (small molecules and proteins) - and curious to know whether the current log4j threat has any relevance for Skyline?

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iRT peptides/protein as global standard
(2 responses) Juan C. Rojas E. 2022-01-06

Hi all,

I spiked a protein to my sample before performing the digestion. Then I used the peptides of this protein to define the custom iRT peptides.

The main purpose of the protein spike was to control variance of the digestion procedure so I would like to set it as a global standard. However, when I tried to set the peptides of this protein as a global standard the option is greyed out (image attached). What is the reason behind this? Besides removing the iRT predictor (and with it the annotated iRT peptides) what else could I do to consider these peptides as global standards?

Would you suggest I use all the peptides of the protein or just a few intense ones? If the second, how many?

As always, thank you for your help and time.
Sincerely,
Juan C.

PS: I am using Skyline 21.2.0.369

 iRT_standard_example.png 
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merging documents for library construction
(2 responses) Will Thompson 2022-01-03

Hi Brendan

Following up on our conversation before the holidays about a 'work around' method for building a small molecule library from files which are sparsely populated with analytes. I have attached two skyline files, each containing a 'common' set of iMT standards and a 'unique' set of library molecules. The skyline documents have also each been curated such that the correct peak is integrated for the unique set of molecules, in two raw files (two injections) for each set of standards. Whenever I use the "File/Import/Document" method, it correctly combines the target lists, but Skyline is still integrating the 'unique' molecules in the raw files from the other document. What I want it to do is to merge the molecules which are the same, and leave the unique molecules empty (essentially the same as if I right clicked on the chromatograms and selected 'remove peak')...those molecules are absent in those runs, and if there are integrations performed in those runs it will not be able to build the indexed retention time regression properly. I have tried all combinations of import/Document that I can think of, to no avail. Would you be willing to give it a shot?

Thanks

Will

 P36_IROA_HS4863_112421_Plate1_RowA.sky.zip  P36_IROA_HS4863_112421_Plate1_RowB.sky.zip 
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Flash player question for videos
(1 response) bconisko 2022-01-02

Newbie here.

I tried to watch the video #3 on the home page and got this error:

"...please upgrade your version of the free Flash Player by downloading here."

I clicked on the 'download here" link, got directed to Adobe, and was told updates are no longer available.

What should I do?

Thanks!

Bruce

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Wishlist: Indicator for transitions that are diagnostic of PTMs
dkueltz 2022-01-02

Hi Nick, Brendan et al.,

Wishlist item: It would be nice to have an identifier for transitions that are diagnostic of fragment ions containing a specific PTM, i.e. some sort of flag or column that can be sorted and filtered in the document view grids and indicates those transitions that are diagnostic for a PTM (i.e. all y ions greater than the position of the PTM in question from the c terminus and all b ions greater than the position of the PTM in question from the N terminus, etc.). Having such an identifier would help a lot in automating the process of finding diagnostic transitions for PTMs and save a lot of time by not having to do this manually. Perhaps there is already a way to do this and I am just missing it.

Thanks for considering this wishlist request,
Dietmar

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PTM scoring
dkueltz 2022-01-02

Hi Nick, Brendan et al.,

The following is a wishlist item I have for future implementation in Skyline re: PTM scoring. Right now the scoring of PTM peptides is based on the ratio of modified versus unmodified peptides but that ratio is based on the peptide sequence rather than the AA position. This works fine if one does not have any missed cleavages or semi-cleaved peptides but in reality there is always a certain amount of missed cleavages and semi-cleavages in complex samples or histone extracts collected from cells or tissues. It would be more accurate to base the ratio of modified versus unmodified peptide on the AA position in addition to the type of modification such that peptides of different starting and ending AAs (semi- or missed cleavages) that contain the PTM AA will be considered for calculating the overall peptide ratio.

So the PTM ratio would be the ratio of the sum of transition intensities for all peptides that have a specific PTM in a specific AA position over the sum of transition intensities for all peptides that include that AA position (whether modified or not). This ratio would not require normalization as it is intrinsic to each sample and it would allow relative comparisons of PTM ratios from samples collected under different biological contexts. My lab is currently calculating these ratios by exporting raw transition areas from SKyline and generating a corresponding excel template but generating this template manually takes a very long time. I think this would be a very nice function to include in Skyline that should be of interest to anyone working on PTM quantitation.

I am attaching a screenshot that illustrates that the ModifiedAreaProportion is currently calculated only for exactly the same peptide. However it should be calculated for any peptide that includes the modified AA even if the peptides differ in start/ end/ length. Otherwise PTM ratios will be inaccurate. For example in the screenshot the peptide KNYKVGDNADVQIKM (unmodified) is shown as having a 100% value even though there are other peptides (of different start or end AA) that are modified at AAs included in that peptide. So basing these ratios on identical peptide seqs rather than identical AA position gives misleading results re: peptide PTM ratio.

Thanks for considering this wishlist request,
Dietmar

 ModifiedAreaProportion.png 
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Having an error when importing FragPipe DIA interact.pep.xml files
(2 responses) fcyu 2021-12-31

When I search DIA mzML files using FragPipe, and import the interact-*_rankX.pep.xml files to Skyline, there is an error:

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

Command-line: C:\Users\yufe\AppData\Local\Apps\2.0\181WWENP.RYH\PWGEADGY.2C2\skyl..tion_e4141a2a22107248_0015.0001_9a4bb3d7cf8a899e\BlibBuild -s -A -H -o -c 0.95 -i 1 -S "C:\Users\yufe\AppData\Local\Temp\tmp9D35.tmp" "F:\msfraggerdia\1.redundant.blib"
Working directory: F:\msfraggerdia
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\yufe\AppData\Local\Apps\2.0\181WWENP.RYH\PWGEADGY.2C2\skyl..tion_e4141a2a22107248_0015.0001_9a4bb3d7cf8a899e\BlibBuild -s -A -H -o -c 0.95 -i 1 -S "C:\Users\yufe\AppData\Local\Temp\tmp9D35.tmp" "F:\msfraggerdia\1.redundant.blib"
Working directory: F:\msfraggerdia
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 149
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
---------------------------

When I search the raw files using FragPipe, and import the interact-*_rankX.pep.xml files to Skyline, everything works well.

The difference between using mzML files and raw files is that, with raw files, FragPipe will generate _uncalibrated.mgf files which will be used by Skyline during importing. Also please note that the there is a _rankX suffix in the interact-*_rankX.pep.xml file.

If you want some data to reproduce it, here (https://www.dropbox.com/sh/qc3z8ive4vh5wt7/AABnlEQvqNjysOG2bixl-2wwa?dl=1) it is.

Thank in advance for your help,

Fengchao

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There are peptides with probability than the threshold in the blib library
(6 responses) fcyu 2021-12-30

It is initialized by a FragPipe user. To save some typing time, please read the threads here (https://github.com/Nesvilab/FragPipe/issues/570#issuecomment-1003194356).

Thanks,

Fengchao

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How can I connect to Spectrum Mill Server and what is my IP address
xzhmuxrl 2021-12-23
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Protein identification through MRM transitions generated from triple quad
(4 responses) prajita 2021-12-22

I wanted to know if I have an MRM data of peptides, will I be able to identify the proteins based on that information using Skyline. If yes, is there a tutorial for that? Any help will be appreciated. Thank you.

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Sehr geehrte SkyLine Mitarbeiter
(1 response) alexandria balac 2021-12-22

Ich bräuchte bitte eine Unterstützung,

ich habe bereits ausgesuchte Proteine und wollte einen theoretischen Verdau mit Chymptrypsin(bovine) im SkyLine durchführen.

Wie sollte ich da vorgehen?

Viele Grüße
Alexandria Balac

 Dok1.docx 
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Target Proteomics Course Downloads
jxg918 2021-12-21

Hey everyone,

I'm new to the fascinating world of proteomics and using skyline!
I found the targeted proteomic tutorial page here - the videos are very good, I'm surprised (and thankful) this is freely available!

However, the link towards the tutorial files (and related data) seems to be broken (for both the 2016 and 2018 ETHZ course).
(the "go there" links on this page: https://skyline.ms/wiki/home/software/Skyline/page.view?name=srm_course )
Could someone please share them with me or provide me a link where I can access these?
That would be massively helpful, thank you!

Kind regards and best wishes
Jan

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Exporting Analyzed Results
(1 response) ianO 2021-12-21

Hi!

After I uploaded my raw data files into Skyline and analyzed the results (like changing manually peak integration boundaries, removing peak, etc.), I was wondering if it was possible to somehow export everything so that I can, e.g., open it on another computer and see what was done? One issue I came across by just opening the skyline file on another computer is the path for the raw data files.

Does anyone have any suggestions on how I can do this? Ideally, I want to avoid having to manually check the results again when I open the skyline file on another computer.

Thanks in advance!
Ian

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What are those numbers in the legend of the 3D spectrum?
(1 response) ho-tak lau 2021-12-20

Hi Skyline Team,

I am still new to IMS and trying to learn about the data using Skyline.
What do those numbers in the 3D spectrum legend mean? I have a 3D spectrum captured from Webinar 19. The 89407, 5024, 282 and 16. And there are always 4 different colors?

Thanks in advance.
Ho-Tak

 web19.PNG 
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Dotp vs Rdotp confusion
(2 responses) jfoe 2019-01-25

Dear skyline team,

I have read up on dotp vs rdotp.
For some reason though, the rdotp is almost always much higher in my assays.

I have attached a picture of my issue.
The top row is for the analyte peptide.
The bottom row for the heavy reference.
The Library entries in both rows should be the same.

Why is the rdotp so much better than the dotp for the analyte?
This doesn't make sense to me because the reference seems to be basically identical to the library.

This is my understanding so far:

dotp - between product peak areas and the corresponding intensities in a library spectrum
rdotp - between analyte peak areas and reference standard peak areas (e.g. light to heavy)

What I would conclude then is that if for any given peptide and its heavy reference peptide:
Both peptides use the same library entry for the dotp comparison.
If the dotp of heavy standard to library is 1, then dotp = rdotp.

Your help would be much appreciated!

 rdotp_issue.png 
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Can skyline do manual integration if no peak detected
(3 responses) xue shi 2021-12-16

Dear Skyline team,
Can skyline integrate baseline if no peak detected?
Please see attachment, at retention time 3.1, there is no peak detected in this sample. Can skyline integrate the baseline or does it offer manual integration? we need a number instead 0 for other data process purpose.
Thank you,
Xue Shi

 skyline baseline .PNG 
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Errors happened when Skyline (21.1.9.348) builds the spectral library using the speclib generated by DIA-NN.
(7 responses) andyzcq 2021-12-14
I used the latest version of skyline to build the spectral library based on the speclib file generated by DIA-NN, as the version3 speclib has been supported by Skyline (21.1.9.348) according to the update log.

Errors happened, and the errors are shown below.

"---------------------------
Skyline-daily
---------------------------
ERROR: could not find precursorId '(UniMod:1)SDS(UniMod:21)GEQNYGERES(UniMod:21)R2' in speclib; is 'DDA_library_DeepLearning-first-pass.tsv' the correct report TSV file?
ERROR:
ERROR: reading file DDA_library_DeepLearning-lib.tsv.speclib

Command-line: C:\Users\zcq\AppData\Local\Apps\2.0\15HT56TR.XZ7\XGZRPD5Q.HLT\skyl..tion_e4141a2a22107248_0015.0001_4fbcbec802624e1e\BlibBuild -s -A -H -o -c 0 -i 1 -S "C:\Users\zcq\AppData\Local\Temp\tmpDE3A.tmp" "D:\project\SCASP_PTM\20210923_HeLa_polyIC_CaTiO3\skyline\1.redundant.blib"
Working directory: D:\project\SCASP_PTM\20210923_HeLa_polyIC_CaTiO3\DIA-NN
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: could not find precursorId '(UniMod:1)SDS(UniMod:21)GEQNYGERES(UniMod:21)R2' in speclib; is 'DDA_library_DeepLearning-first-pass.tsv' the correct report TSV file?
ERROR:
ERROR: reading file DDA_library_DeepLearning-lib.tsv.speclib

Command-line: C:\Users\zcq\AppData\Local\Apps\2.0\15HT56TR.XZ7\XGZRPD5Q.HLT\skyl..tion_e4141a2a22107248_0015.0001_4fbcbec802624e1e\BlibBuild -s -A -H -o -c 0 -i 1 -S "C:\Users\zcq\AppData\Local\Temp\tmpDE3A.tmp" "D:\project\SCASP_PTM\20210923_HeLa_polyIC_CaTiO3\skyline\1.redundant.blib"
Working directory: D:\project\SCASP_PTM\20210923_HeLa_polyIC_CaTiO3\DIA-NN
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 149
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
---------------------------
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Missing Values in DIA data analysis
(8 responses) klemens froehlich 2021-12-09

Dear Skyline developer team,

We are currently in the review process of a manuscript where we compare multiple different DIA data analysis suites /spectral library generation methods and data analysis strategies using a large scale benchmark dataset (92 samples with human background / Ecoli spike-in).

We have one condition where no Ecoli proteins are present.

Following the tutorial:
"Analysis of DIA/SWATH data in Skyline" we exported our data on protein level and found that for most Ecoli proteins values are reported in the samples where no Ecoli is present.

My question is this: Which additional filtering options would you recommend to avoid such background quantifications? They are lower than the intensity values of the spike-in conditions so at this point we would just advise users of Skyline to be aware of this "background" reporting of protein intensities.

Best, Klemens

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CCS measured vs. CCS explicit (or library)
(3 responses) tim causon 2021-04-09

Skyline is doing a great job with filtering LC-IM-MS data, but we are looking for some improved reporting options.

  1. When we have a known set of target molecules to search for, we create a simple Skyline transition list for small molecules with: RT, Explicit CCS, molecular formula, and ion species.
  2. In CCS-calibrated files, the target CCS is converted to a target Drift Time, which does a great job filtering the results in the measurement files as we want it to.

However, the reporting options only allow the "Explicit CCS" to be reported (that is our "target value"). Other options available for reporting are identical or are the "target drift time" and not the "measured CCS". Note: "Measured drift time" is not as valuable as we may import data files with different CCS calibration functions applied.

Would it be possible to include an option to report the "Measured CCS" (i.e. calculated from the measured drift peak apex)? This would allow "CCS Error" to be reported in a similar way to "Mass Error PPM".

view request
DIA-NN .speclib support
(38 responses) Tobi 2020-05-27

Dear Skyline Team,

could you please consider implementing support for DIA-NNs .speclib spectral libraries? Its a highly convenient tool for predicted libraries and much faster than Prosit.

https://github.com/vdemichev/DiaNN

With best regards,
tobi

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Questions about skyline read the ion mobility values of the spectral library generated by DIA-NN.
(4 responses) andyzcq 2021-11-27

I noticed that Skyline has supported the speclib format generated by DIA-NN.
I used the "peptide"-"library"-"edit"-"add", found skyline can not recognize the *.speclib format. I modified "speclib" to "clib", which can be recognized by Skyline. But Skyline kept loading the file for a long time (about several hours), and the speclib file is only 7M.
Then I used the "file"-"import"-"assay library" to load the lib.tsv file generated by DIA-NN, which is successful. I assigned the ion mobility column as "explicit ion mobility" in the import wizard. However, there are no "ion mobility" values in the spectral library explorer.

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Settings questions arising from Webinar 21: PASEF
(2 responses) paga 2021-12-13

I've been going through the recently uploaded tutorial on DIA-PASEF, and I have the following doubts I would love to solve.

1.- I understand why they select only precursors with charges 2 and 3, but what I don't understand is why they decide not to look at the precursors (MS1 spectra). They decide to avoid using MS1-filtering, and decided to only use MS/MS filtering with a Centroid Precursor mass analyzer. Why would be a good idea to dispense on valuable precursor information which is used to determine transition quality (idotp)?

2.- On the same line, why is Centroid better than TOF for PASEF?

3.- When selecting MS/MS filtering with TOF Product mass analyzer, the default resolving power is 30.000. On the PASEF tutoriual, the chocen Centroid option was paird with a value of 30 for resolving power. What is the source of this huge difference? Is there a way I can check the resolving power of my DIA files in case I wasn't the one performing them?

Thanks a lot for the support, Skyline team!

view request
Information on critical Log4j 2.x vulnerability
(1 response) taperez 2021-12-13

We have been informed that a critical vulnerability in Java application, Log4j 2.x was reported recently. Just want to check with you if Skyline software runs on Log4j 2.x or uses it for its application.

Let us know.

Thank you,
Tatiana Perez

 IA-2021-005_ Widespread Exploitation of a Critical Apache Utility Vulnerability Likely in the Near-Term.pdf  NYC21-0306 APACHE LOG4J 0-DAY VULNERABILITY.pdf  Restricted_[AgencyName]_Log4J CVE-2021-44228.xlsx 
view request
Some questions for diaPASEF data analysis about library and settings
(4 responses) lourh 2021-12-06

Dear Skyline team,

I'm using Skyline to search diaPASEF data with a tsv assay file, and have some questions in this process. I'd be appreciate if you can help me with them.
DIA data were acquired on timsTOF pro2 with 4 diaPASEF windows in each frame and repeated MS2 series twice after one MS1 frame. And the assay library was built on ddaFASEF data with FragPipe and filtered with OpenSwathAssayGenerator.

My questions are as follows

  1. What's the best time to use "Integrate All"? Previously I directly exported search result after reintegrate with mProphet scoring, while the quantification looked weird. But when I clicked "Integrate All" before exporting (still after reintegrate), the exported result looked good. Is that fine to use this function as the last step? or I think this might be set at will?

  2. Should I do some filters before exporting? Currently I usually use Refine -> Advanced, and set these values after reintegrate and before exporting

    • Min peptides per protein: 1
    • Min transitions per precursor: 3
    • Remove peptides missing library match
    • Q value cutoff 0.01
    • Detected in 1 replicate
    • Should I also restrict "Min peak found ratio" and "Max peptide peak rank", or others? In fact, I'm using a benchmark dataset, so better quantification without ID loss will be good
  3. The exporting in my cases is a little slow. I think I chose some normal columns, for peptide sequence, RT, prec charge, Total area, qvalue, with no fragment level data (skyr is attached). Do you have any idea about it

  4. It seems Skyline would able to not rely on protein information in searching and refinement steps, this it true? Would this means I can get same results if no fasta file assigned and keep the library have no proteins (the proteins in tsv file would be dropped if I build library from tsv file with Peptide settings -> Library -> Build)

  5. About iRT and IM in blib built from tsv file. Now I found two ways to import a tsv assay file, one by import -> add assay file, another by Peptide settings -> Library -> Build.

    • The first one cannot recognize ion mobility column but can import iRT in library correcly (and an IrtLibrary table will be generated in blib).
    • The latter one can recognize ion mobility while the proteins were dropped and iRT values would be transformed to a very small scale (seems -1 to 3) even if I select iRT standard peptides as Biognosys 11 or set it as None, and in this case the RT difference score for mProphet cannot be checked.
    • Is there a suitable solution for importing a tsv library? or do you think the followings make sense to use: first build blib with import -> add assay file; then open blib and add ion mobility values to tables "RefSpectra" and "RetentionTimes" for each precursor; also create table "IonMobilityTypes" to store drift time or reduced IM or compensation like library built from Peptide settings.
  6. For ion mobility settings, is that enough to check "Use spectral library IM when present" and set resolving power to 30 as tutorial shown? or use fixed window to 0.06 (maybe this means 1/k0) like OpenSwath preferred.

  7. What's the best practice to aggregate precursor quantities to protein level? Is top3 average enough, or some other suggestions?

The Skyline version is 21.1.0.278

Sorry for so many questions. Really thanks for your time.

Best regards,
Ronghui

 PepQuant.skyr 
view request
Error on Bruker Impact data import
(1 response) pierre-olivier schmit 2021-12-13

good morning, I am gettin gthe following error message while trying to import Bruker Impact data (NB : timsTOF Pro data are imported correctly)

At 09:49:
Failed importing results file 'G:\Data\2021\CSM_49_MICROPEP_VIP-HESI-ELUTE-IMPACT II-10083_CM\Data\QUALIFICATION\Reference_500ugml_AutoMSMS_T0_33_1_122.xml'.
[ReaderFail] don't know how to read G:\Data\2021\CSM_49_MICROPEP_VIP-HESI-ELUTE-IMPACT II-10083_CM\Data\QUALIFICATION\Reference_500ugml_AutoMSMS_T0_33_1_122.xml

with best regards,

Pierre-Olivier

view request
Error Importing Data from Exactive Plus EMR
(2 responses) mellors 2021-12-10

Hi,
I tried importing data files from our Exactive Plus EMR (Thermo Orbi), and something is not working. It goes through the motions without throwing any errors, but it's not actually fitting the data. I loaded a data file from a similar method run on our Exploris 240 and that loaded properly. I've attached the skyline file along with one of the data files from the Exactive EMR that won't load properly. All of the other files from the Exactive EMR that I tried to load (about 40) behaved the same way.

Thanks,
Scott

 Oligos_Std_Skyline_UltraTrace.sky.zip  20211209_HRB000240_P22_ID262_UltraTraceBGE_42.raw 
view request
skyline is not able identify with my iRT peptides
(2 responses) Sangram 2021-12-07

Hello people,

I am trying to perform a (pretty straightforward) DIA analysis with 32 fractions of a cell line proteome. I performed the spectra search using PeptideShaker and yielded the results in mzid format. Now in the "Add iRT peptide" window all samples shows failed. I would like to know what implications I might have down the analysis. This is because I noticed whenever this happens and I would export a spectral library in openSWATH format, the values from RetentionTimeCalibrationScore is missing !

How can I stop this from happening ?

 regression_error.png 
view request
pepxml-mzXML import from PEAKS no longer works
(4 responses) dkueltz 2021-12-08
Hi,
My lab has used Skyline for many years to import pepxml (and corresponding mzXML) generated by PEAKS and it always worked well until very recently when I get the error pasted below. Importing pepxml + mzML from MSfragger still works well. I wonder whether the pepxml + mzXML import function in Skyline has changed? Could you please look at the error below and let me know how to fix it such that my lab can continue to use our pipeline of generating spec libs from PEAKS DDA data?
Thanks much,
Dietmar

---------------------------
Skyline
---------------------------
ERROR: peptides.pep.xml(line 22656): [Serializer_mzXML::translateSourceFileTypeToNativeIdFormat] unknown file type
ERROR:

Command-line: C:\Users\dkueltz\AppData\Local\Apps\2.0\8JR1V559.JCC\KOCQGHPK.M36\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i test6 -S "C:\Users\dkueltz\AppData\Local\Temp\tmpB6D.tmp" "D:\Spectral-Libraries\test6.redundant.blib"
Working directory: F:\EAM0021-24_Oremo_V8-AmBic_60min\PEAKS-to-Skyline\PEAKS10Pro_Oremo_V8-AmBic_Histones_PEAKS PTM_35
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: peptides.pep.xml(line 22656): [Serializer_mzXML::translateSourceFileTypeToNativeIdFormat] unknown file type
ERROR:

Command-line: C:\Users\dkueltz\AppData\Local\Apps\2.0\8JR1V559.JCC\KOCQGHPK.M36\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i test6 -S "C:\Users\dkueltz\AppData\Local\Temp\tmpB6D.tmp" "D:\Spectral-Libraries\test6.redundant.blib"
Working directory: F:\EAM0021-24_Oremo_V8-AmBic_60min\PEAKS-to-Skyline\PEAKS10Pro_Oremo_V8-AmBic_Histones_PEAKS PTM_35
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 142
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
---------------------------
view request
Issue on library in Peptide setting
(1 response) hogeun kwak 2021-12-08

Dear Skyline team,

Hello, my name is Hogeun.
I am testing the prm-PASEF using skyline-daily(21.1.9.335).
We took our PASEF data including Ion Mobility using timsTOF Pro 2.
And the characterized DDA file was extracted from PEAKS (Xpro).
I found the attached message when I input the .pep.xml (including IM data from PEAKS) in library option in Peptide setting .
Do you know the reason? Could you give me the solution if you know that?

best regards,

Hogeun Kwak

 Error.png 
view request
how skyline do the peak picking
(1 response) xue shi 2021-12-07

Dear Skyline team,
I am using skyline to process Agilent QQQ MRM data now. However, could you please let me know how skyline integrate and calculate the peak area
Let's see for this transition, it has different retention time. Do you just add all the area with same transition 104.07- 58.196 together or you did some fittings(gaussian,EMG) for the peak picking?
PrecursorMZ ProductMZ
104.07 58.196

9.4305
9.44131667
9.44131667
9.44131667
9.45213333
9.45213333
9.45213333
9.46295
9.46295
9.46295
9.47375
9.47376667
9.47376667
9.48456667
9.48456667
9.48456667
9.49538333
9.49538333
9.49538333
9.5062
9.5062
9.5062
9.51701667
9.51701667
9.51701667
9.52783333
9.52783333
9.52783333
9.53865
9.53865
9.53865
9.54946667
9.43026667
9.44108333
9.44108333
9.44108333
9.4519
9.4519
9.4519
9.46271667
9.46271667
9.46271667
9.47353333
9.47353333
9.47353333
9.48435
9.48435
9.48435
9.49516667
9.49516667
9.49516667
9.50598333
9.50598333
9.50598333
9.5168
9.5168
9.5168
9.52761667
9.52761667
9.52761667
9.53843333
9.53843333
9.53843333
9.54923333
9.54923333
9.54923333
9.56005
Thank you a lot

 0714_48 mix_50uM_02.d.zip 
view request
Skyline missing integration limits for some random species
(4 responses) j3 menzel 2021-12-07
Hi,

first of all thanks for providing such a great software and support!

I have encountered a problem that is recurring and I can't find a solution for it.

Basically, in molecule mode, DIA, for transition lists with a few hundred entries, some are missing integration limits entirely and I can't get a reported integration for these species or adjust integration limits.

If you know, how integration limits can be inserted in such cases manually or how Skyline may be forced to always enter some integration limits, even if they seem or may be "wrong" that would be great !

Thanks heaps,

Philipp
 Skyline missing integration limits.pdf 
view request
"error reading spectrum" of .mzML files from OpenChrom, original data files are Agilent MassHunter .D files which skylines cannot import
(5 responses) Allison Haase 2021-12-07

Dear Skylines team,

My lab has been using Skylines MS for a variety of purposes, specifically for small molecule analysis using GCMS and GC MS/MS. We have had success with importing and analyzing MS data from Thermo .raw files, but have run into an issue with data from an Agilent instrument from an outside lab. The data is provided in Agilent MassHunter .D files. Skyline does not recognize this file type, so we have attempted to convert the data files using OpenChrom to .mzML and .mzXML formats. The conversion process works, and Skyline can then see the data files for importing. However, we consistently get an importing error of "error reading spectrum". I have attached the full text of the error below, as well as the data files involved, both in .D and .mzML formats.

I would be appreciative of any help that you could give, even if it is just a recommendation for a file converter. My lab lacks Agilent MassHunter software, so I cannot convert it using the native system.

At 10:47 AM:
Failed importing results file 'C:\Users\Allison\Desktop\ExportOC\TMS2047.mzML'.
error reading spectrum
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\Allison\Desktop\ExportOC\TMS2047.mzML'.
error reading spectrum ---> System.Reflection.TargetInvocationException: error reading spectrum ---> System.Exception: error reading spectrum ---> System.Exception: [IO::HandlerBinaryDataArray] At position 843: encoded lengths differ.
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1155
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetSpectrum(Int32 spectrumIndex) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 893
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1075
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1126
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.Read() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 953
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.<RunAsync>b__33_0() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 810
--- End of inner exception stack trace ---
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.NextSpectrum() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 916
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 259
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 239
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 592
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 384
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

 TMS2047.D.zip  TMS2047.mzML 
view request
Sample Group Labeling Swap incorrectin -daily
(2 responses) Will Thompson 2021-12-06

Seems to be a pretty bad bug in 21.1.9.335 (current daily) where it swaps the labels on the bar chart/replicate intensity chart when you use the Sample Group function. I've tried with a couple different documents and behavior is this:

  1. Add Sample Group to the Document Settings (Value List)
  2. Assign replicates to two different groups in the document grid / replicates
  3. In the replicate intensity pane, right click and choose group by/sample group

Behavior: Skyline then gathers the replicates and labels them with the group name. The problem is, it swaps the group name from what is actually assigned in the document grid.

Will

view request
Modifications on Selenocysteine in MaxQuant data for Skyline
(9 responses) s m hacker 2021-11-09

Dear Skyline Team!
My group is working on a variant of isoTOP-ABPP (MS1 quantification) to study the target engagement of covalent inhibitors using chemoproteomics. I have now tested Skyline for Quantification of MSFragger search results and it works great. We have to use a workaround that uses selenocysteine as placeholder for a modified residue, which we have established before for MaxQuant searches/quantification (https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201912075), but than it works out very well. In principle, this strategy should also be transferable to quantify MaxQuant search results in Skyline. Nevertheless, when I want to load the MaxQuant data with a modification (IA heavy isoDTB U2) on selenocysteine into Skyline, I get the following error message (screenshot attached):

System.IO.IOException: ERROR: No matching mod for IA heavy isoDTB U2 in sequence AAANIGLEPEEVAIITGIGU(IA heavy isoDTB U2)SGR (line 2). Make sure you have provided the correct modifications[.local].xml file.

Command-line: C:\Users\AKHacker3\AppData\Local\Apps\2.0\1AYPALAY.4DC\R6XMQK52.Z7A\skyl..tion_e4141a2a22107248_0015.0001_e8c6979bec685b9e\BlibBuild -s -A -H -o -c 0 -i 181207_P05_P06 -S "C:\Users\AKHacker3\AppData\Local\Temp\tmpEE5D.tmp" "C:\current_analysis\SMH\013_181207_P05_P06\MQ_2_0_3_0_mod_U_CAM_var2\skyline2\181207_P05_P06.redundant.blib"
Working directory: C:\current_analysis\SMH\013_181207_P05_P06\MQ_2_0_3_0_mod_U_CAM_var2\skyline2
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 142
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 157

The IA heavy isoDTB U2 modification on U is defined in the modifications.local.xml (attached), which is put into the same folder. I think, this might be related to the U creating a problem.
Might this be something someone on your end could look into? I have uploaded the full data, I wanted to use, here:
https://gigamove.rwth-aachen.de/en/download/1842568fa824b8253e650dc6789ecee4
This data set also has screenshots of the modifications that we wanted to use. The same setup works for analyzing MSFragger data in Skyline and gives, as said, really nice data.
It would be great, if you could give some input.
Best
Stephan

 modifications.local.xml  Error_Message.png 
view request
No spectra were found for the new library
(7 responses) joshuasmith 2021-12-03
Hi Skyline team,

I know that this issue has been the subject of several previous support tickets, but after reading through all of them and trying to figure out what is happening with my data, I am still having an issue with building a spectral library in Skyline using my DIA search results.

I have overlapping-window DIA data from a Fusion Lumos. I know the raw data isn't an issue, as I can import the .raw files into Skyline with a target list from a previous experiment just fine (see "DIAimported" screenshot). And I get valid .tsv output data from MSFragger. I used FragPipe for my search, first doing DIA-Umpire signal extraction on mzXML converted files, then searching the pseudo-DDA data with MSFragger in Open Search mode.

I have my interact.pep XML file in the same directory as the calibrated .mgf, .mzML, and .pepxml MSFragger output files.

I opened Skyline, saved my document, and navigated to File->Import->Peptide Search. I attempted to build a new library by adding my interact.pep search result file, with the DIA workflow selected. I hit Next, and then for a hot second, it looks like it is reading in the spectra from the .mzML files that are in the same directory as the interact.pep search result file (see "LibraryBuild" screenshot). But then it immediately returns the error below (also see "LibraryBuildError" screenshot).

Not sure what I'm doing wrong, any help would be appreciated. I have attached screenshots, my XML search result file, and the FragPipe/MSFragger log file.


Thanks,
Josh

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

Command-line: C:\Users\jossmith\AppData\Local\Apps\2.0\TZE299RM.CXM\MA2X4L6X.993\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i PAA3 -S "C:\Users\jossmith\AppData\Local\Temp\tmp5A85.tmp" "C:\Users\jossmith\Documents\Adductomics\ColeLabData\211109\PAA\DIANN\PAA3.redundant.blib"
Working directory: C:\Users\jossmith\Documents\Adductomics\ColeLabData\211109\PAA\MSFragger
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\jossmith\AppData\Local\Apps\2.0\TZE299RM.CXM\MA2X4L6X.993\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i PAA3 -S "C:\Users\jossmith\AppData\Local\Temp\tmp5A85.tmp" "C:\Users\jossmith\Documents\Adductomics\ColeLabData\211109\PAA\DIANN\PAA3.redundant.blib"
Working directory: C:\Users\jossmith\Documents\Adductomics\ColeLabData\211109\PAA\MSFragger
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 142
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
---------------------------
 DIAimported.png  LibraryBuild.jpg  LibraryBuildError.jpg  log_2021-11-17_16-36-35.txt  interact.pep.xml 
view request
Analyze Pan-Human Library using the human reference proteome fasta file
(5 responses) dtn074 2021-12-03

Hi,

I am Duong Nguyen. I am using Skyline for analyzing the Pan-Human dataset (link: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=1a91e137d235498aa865997e8d923348).
I imported the human reference proteome fasta file (human_proteome_UP000005640 from UniProt). After that I tried "import -> results" for some mzXML files but it takes forever to complete. The memory usage of my computer was >90% and CPU < 5% at that time (please see the file attached). Do you know what happened?

And a bigger question - have you ever been succeeded on using Skyline to analyze ALL Pan-Human files at the same time with a human reference proteome fasta file? Please give me some suggests if yes. That would be my final goal.

Thank you!
Duong

 Screen Shot 2021-12-03 at 9.47.19 AM.png 
view request
Error: No matching mod
(4 responses) valerie huhle 2021-03-25

Hey,
we were trying to open a MaxQuant search (version 1.6.17.0) with the new modifications Iodination (Y) with the "Import DDA Peptide search" from skyline (version 20.2). Before that we saved the new modifications.local.xml and modifications.xml into the same folder, where the msms.txt file is located. Nevertheless it still gives us the added error.
With chlorination (Y) we got a corresponding error.
If we try to open a MaxQuant search for only oxidations (M) skyline works fine.
I hope you can help us with that problem,
Thanks
Valerie

 msms.txt  Error-iodo.txt  modifications.local.xml  modifications.xml 
view request
modifications.xml
(5 responses) ziyang zhang 2020-12-22

Hi again skyline gods,

I recently had an issue when importing a DDA search (done by MaxQuant). The peptides carry a custom modification "ARS", which is defined in the modifications.xml file. When importing, Skyline prompts that there is no matching modification for "ARS".

I did the following:

  1. adding modifications.xml file to the data folder containing msms.txt
  2. adding modifications.xml file to the spectral library folder under the user home folder
  3. adding modifications.xml file to replace the skyline installation folder (with new App installation tool, this is now at C:\Users[user]\AppData\Local\Apps\2.0\xxxxxxxx.xxx\xxxxxxxx.xxx\skyl..tion_xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
  4. adding a modification in peptide settings named "ARS" with the same formula as in modifications.xml

But still cannot import the search result - the error persists. Could I please get some help...

Many thanks,
Ziyang

 Skyline_Mod_NoMatch.png  Mod_Def.png  modifications.xml  msms.txt 
view request
MSFragger error
(1 response) Shawn Rice 2021-12-01
Greetings,

I am trying to run MSFragger in Skyline and keep getting the following error. Any suggestions? I am running the newest version of Skyline-Daily (64) 21.1.9.334 (15641fb9e). The run log is attached.

---------------------------
Skyline-daily
---------------------------
Error
---------------------------
OK More Info
---------------------------
System.IO.InvalidDataException: while fixing scan numbers in PIN file, ran out of correct scan numbers from pepXML
   at pwiz.Skyline.Model.DdaSearch.MsFraggerSearchEngine.FixMSFraggerPin(String cruxInputFilepath, String cruxFixedInputFilepath, String msfraggerPepxmlFilepath) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DdaSearch\MsFraggerSearchEngine.cs:line 302
   at pwiz.Skyline.Model.DdaSearch.MsFraggerSearchEngine.Run(CancellationTokenSource cancelToken, IProgressStatus status) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DdaSearch\MsFraggerSearchEngine.cs:line 165
---------------------------

Thanks,
Shawn
 run_log.txt 
view request
Reporter ions Issue 352 update?
(1 response) Juan C. Rojas E. 2021-12-02

Hi Skyline team,

As I am finally going back to my bread and butter of derivatized peptides I wanted to inquire if Santa will bring a gift by allowing the annotation of modification-specific reporter ions in the Library Match window? I believe this has been discussed in issue 352:

https://skyline.ms/issues/home/issues/details.view?issueId=352&_docid=issue%3A352

Sincerely,
Juan C. Rojas E.

view request
Cannot import timsTOF PASEF bbCID data
(12 responses) michael witting 2021-10-01

Dear Skyline team,

I recently acquired in collaboration with Bruker some lipidomics data on the timsTOF pro, but I cannot read it into Skyline. I'm getting the following error:

At 13:42:
Failed importing results file 'H:\Data_Bruker\21010929_Celegans_ttPro\HESI - RP\neg bbCID\HESI_Celegans_MTBE-2_150ul_bbCID_4ul_neg_19_1_3206.d'.
[Reader_Bruker::createInstrumentConfigurations] no case for InstrumentSource 18
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'H:\Data_Bruker\21010929_Celegans_ttPro\HESI - RP\neg bbCID\HESI_Celegans_MTBE-2_150ul_bbCID_4ul_neg_19_1_3206.d'.
[Reader_Bruker::createInstrumentConfigurations] no case for InstrumentSource 18 ---> System.Exception: [Reader_Bruker::createInstrumentConfigurations] no case for InstrumentSource 18
bei pwiz.CLI.msdata.ReaderList.read(String filename, MSData result, Int32 runIndex, ReaderConfig config)
bei pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\pwiz_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 197.
bei pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:Zeile 291.
bei pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:Zeile 188.

Thanky you very much for your help.

Best regards,

Michael

view request
Removing automatically-loaded library
(4 responses) bsundberg 2021-12-01

Hello Skyline Team,
I received a Skyline document from Pierce along with their 7x5 synthetic peptide mix for evaluating system suitability data from those peptides. I would like everyone in our group to be putting data into this document, so it should be as uncomplicated as possible to use. However, the document automatically expects a spectral library that isn't provided, and Pierce's instructions tell you to just ignore the pop-up message about the missing library. The problem is that in the absence of the library Skyline does not import the data completely, resulting in the message "chromatographic information unavailable". Deselecting, removing, or resetting the library in Peptide Settings resolves the issue temporarily, but upon importing more data or making a copy of the document, the library shows up again! How do I permanently remove this "missing library" from the document? Problematic document is attached. Thanks for your help!

 7x5standard_Q3 MI.sky.zip 
view request
Skyline admin install - update does not work?
(1 response) pavel 2021-11-30

Hi,
on my computer (Win10, Skyline-daily 64-bit, v. 21.1.1.327) I have installed the Skyline software in the admin mode from the *.msi file („Skyline Administrator Install“). The installation of the Skyline program as Administrator from *.msi file worked nicely, the software runs, however checking for updates in the Help menu does not do anything. This holds both Skyline and Skyline-daily versions. By the way, I can install external tools there (e.g. Prego) as a common user when the appropriated accession rights for directories „C:\Program Files\Skyline“ or „C:\Program Files\Skyline-daily“ were set (i.e. Full access for Users using icacls command). Thank you for your help.
Best regards,
Pavel

view request
Download failed when trying to download jre-17.0.1 and crux-4.1
(3 responses) fcyu 2021-11-24

When I was trying to run MSFragger in Skyline, I got errors when it was downloading jre-17.0.1 and crux-4.1 (although I am not sure why needing crux. For Percolator and FDR estimation?):

Skyline version: 21.1.1.327-f21a06b12 (64-bit)
Installation ID: 605e00a1-46d6-417c-a8a9-dfc39063ff4e
Exception type: TargetInvocationException
Error message: Download failed. Check your network connection or contact Skyline developers.

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

System.Reflection.TargetInvocationException: Download failed. Check your network connection or contact Skyline developers. ---> System.Exception: Download failed. Check your network connection or contact Skyline developers.
   at pwiz.Skyline.Util.SimpleFileDownloader.DownloadRequiredFiles(IEnumerable`1 filesToDownload, ILongWaitBroker waitBroker) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Util\UtilInstall.cs:line 189
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\pwiz_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\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1941
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Alerts.SimpleFileDownloaderDlg.Show(Control parent, String title, IEnumerable`1 requiredFiles) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Alerts\SimpleFileDownloaderDlg.cs:line 124
   at pwiz.Skyline.FileUI.PeptideSearch.SearchSettingsControl.EnsureRequiredFilesDownloaded(IEnumerable`1 requiredFiles, Func`1 extraDownloadAction) in C:\proj\pwiz_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\SearchSettingsControl.cs:line 106
   at pwiz.Skyline.FileUI.PeptideSearch.SearchSettingsControl.InitSelectedSearchEngine() in C:\proj\pwiz_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\SearchSettingsControl.cs:line 125
   at pwiz.Skyline.FileUI.PeptideSearch.SearchSettingsControl.SearchEngineComboBox_SelectedIndexChanged(Object sender, EventArgs e) in C:\proj\pwiz_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\SearchSettingsControl.cs:line 55
   at System.Windows.Forms.ComboBox.OnSelectedIndexChanged(EventArgs e)
   at System.Windows.Forms.ComboBox.WmReflectCommand(Message& m)
   at System.Windows.Forms.ComboBox.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.SendMessage(HandleRef hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.UnsafeNativeMethods.SendMessage(HandleRef hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.Control.SendMessage(Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.Control.ReflectMessageInternal(IntPtr hWnd, Message& m)
   at System.Windows.Forms.Control.WmCommand(Message& m)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.UserControl.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.NativeWindow.DefWndProc(Message& m)
   at System.Windows.Forms.Control.WmCommand(Message& m)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ComboBox.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 System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.SkylineWindow.ShowImportPeptideSearchDlg(Nullable`1 workflowType) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 3139
   at pwiz.Skyline.Controls.Startup.ActionImport.OpenSkylineStartupSettingsUI(SkylineWindow skylineWindow) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\Startup\StartupActions.cs:line 111
   at System.Windows.Forms.Form.OnShown(EventArgs e)
   at pwiz.Skyline.SkylineWindow.OnShown(EventArgs e) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Skyline.cs:line 261
   at System.Windows.Forms.Control.InvokeMarshaledCallbackHelper(Object obj)
   at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
   at System.Windows.Forms.Control.InvokeMarshaledCallback(ThreadMethodEntry tme)
   at System.Windows.Forms.Control.InvokeMarshaledCallbacks()
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.Form.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\pwiz_x64\pwiz_tools\Skyline\Program.cs:line 307

Any clue?

Thanks,

Fengchao

view request
Retention Time shifts using deuterated internal standards.
(9 responses) alejandro.cohen 2021-03-23
Hi Skyline people,

I'm running a small molecule targeted (steroid hormone panel) analysis (LC-MS on a QExactive) using MS1 filtering (surprisingly better results than PRM... a discussion that I'm hearing quite a few users agree). I have spiked deuterated internal standards to my samples. Unsurprisingly, those with higher number of D atoms produce a noticeable shift in the retention times. However, peak boundaries can not be separately set for target and internal standard (setting the peak on one automatically sets the other). Is there any way to overide this feature? I'd like to manually determine the peak boundaries, as Skyline is getting a little confused establishing the peak start-end times. This creates a problem when integrating low abundance samples, as the 'wide' peaks of the internal standards overestimate the areas of the unlabeled precursors.

Thanks!!!

Alejandro
 Screen shot2.docx 
view request
How to replace comma for values on the Y-axis with the period
(2 responses) akulyyasov 2021-11-25

Dear Skyline team,
Please help me to set parameters in the graphical data presentation of Skyline.
How to replace the comma on the ordinate with a period. E.g.:”0,2”should be “0.2”

With best regards,
Arman

 Figure_4_periods.png 
view request
Skyline update - issue uploading method on LabSolutions (Shimadzu)
(8 responses) benoit fatou 2021-11-11

I updated the Skyline software on Monday and when I uploaded a new method to LabSolutions, I couldn't see the transitions table anymore by clicking on the associated tab (it basically doesn't allow me to open the tab). I found an alternative which is to first export a transition list from Skyline and import it to an old method in LabSolutions but it is not the best solution for me.

Let me know if you need more information as it may be a bit difficult to understand my problem.
Thanks,
Benoit

view request
Couple minor issues with the most recent skyline update
(8 responses) hs334 2021-11-04

First, synchronized integration is awesome! However, is it possible to update the software to allow us to click/drag to or use shift/click to select multiple samples under the synchronize integration tab? Currently when you go to synchronize integration if you want to select/deselect multiple sample items you have to individually check or uncheck each item. Often times I want to select/deselect upwards of 75 samples out of 100+ to synchronize together and its very tedious to select all of them individually by clicking.

Next, for some reason this update of skyline has made it so that I can’t double click on a skyline file and open with Skyline daily (i.e. if I go to a skyline file in my file explorer and double click it does not open it with Skyline daily.) The only way I can open these files currently is through using the open file function in skyline – which has prevented me from opening more than one skyline file at a time. It is really useful to have more than one skyline file open simultaneously for comparison purposes.

view request
Adding a transition without re-importing every file
(2 responses) nate 2021-11-22

Currently when I add a new transition to a document I go to Edit > Manage Results > Re Import. This removes all manual peak integrations that were previously curated.

Is there a command to only pull in the new transition's chromatograms?

Thanks
Nathaniel

view request
humble plea for peak boundary feature
(8 responses) Will Thompson 2021-06-21

Dear Brendan and Brian,

I have a plea for a feature that would save us a ton of time, and I hope would benefit many and would be pretty simple to implement. I would like to ask for a "Apply Peak Boundaries To All" feature in the right-click menu on the chromatogram window. I don't know what the back-end functionality of the "apply peak to all" or "...to subsequent" is, but it seems to be hopelessly broken and we spend a ton of time manually curating some peaks that have odd shapes or have not great S/N. My vision for this new feature is that if i integrate a peak and click the "Apply Peak Boundary To All" button, Skyline would just blindly apply those peak boundaries to that analyte across the document. No questions asked, no peak detection, just update the start and end time for the integration for every replicate in the document. Currently, in order to accomplish this task we need to integrate one replicate, then copy the table for the given retention time out from the doc grid, paste down for the replicates we need (make sure to not go too many replicates down!), save as a peak boundary file renaming key columns, then import the peak boundaries. The whole process seems such a waste of time for something that would seemingly be very easy to keep within Skyline. As with many things, I probably overestimate the simplicity but I hope it can be done. This would save us potentially hours in curation per plate of multiplexed quantitative metabolomics data.

Your humble beggar of features,

Will

view request
Frustrated with Library Build
(6 responses) jcarey 2021-11-19

Dear Skyline Support Team,

I have watched many of the webinars and completed most all of the tutorials successfully and still cannot build a library from MS data from a commercially available Shingrix vaccine. Data were aquired on a Waters QTOF. I've checked many of the library build Support threads and tried to follow the steps suggested by Skyline Support Staff, but still only get back error messages related to invalid file type.

As a last resort today, the data was redownloaded from our vendor and kept in the download folder for simplicity. The library Build function was used and the destination for the "to be created" .blib file was also chosen to be the exact same location in the download folder (have tried many permutations of file locations to no avail). The source file was specified using the Build dialog box and when the "Next" button was clicked the error message below was received. I have tried many, many ways to load MS results as a library and consulted lots of references. Seems like I need some hand-holding at this point. Thanks!


System.IO.IOException: ERROR: Error with '_FUNC001.DAT'. Invalid results (.dat) file.
ERROR:

Command-line: C:\Users\Jim\AppData\Local\Apps\2.0\JA9V9166.9KW\61QRGA69.CNO\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i Shingrix_19NOV21_blib -S "C:\Users\Jim\AppData\Local\Temp\tmpFE0A.tmp" "C:\Users\Jim\Downloads\Shingrix_19NOV21_blib.redundant.blib"
Working directory: C:\Users\Jim\Downloads\MC_20210909_ADES1407_Shingrix.raw
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 142
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157

view request
Synchronize integration
(2 responses) nate 2021-11-18

First off, thank you for the amazing piece of software. Synchronize integration is very useful in my workflows.

A bug to report, I believe. Wanted to post here first.

When the document contains files with only one of positive/negative polarity scans but files of both polarities there is an error. See attached. Things seem to work fine when only one polarity of files is selected in the Synchronize Integration dialogue.

Nathaniel

 Capture.PNG 
view request
File import failure
(1 response) andrew rowland 2021-11-16

Hi Support team,

I am trying to import MRM data files that were generated on an Agilent 6495 QQQ through MassHunter software into Skyline, but am having issues with the import generating an error:

At 1:45 PM:
Failed importing results file 'E:\210715 AR Pfizer GLP1\WorklistData-0000.d'.
[ReaderFail] don't know how to read E:\210715 AR Pfizer GLP1\WorklistData-0000.d

I have uploaded an example data file (WorklistData-0004.d) that is giving this error to the file sharing section and was wondering if you can shed some light.

Andrew

view request
Thank you for the Synchronized Integration!
harley robinson 2021-11-16

Hey, no questions, no concerns here. Just compliments!

Thank you so much for the new Synchronized integration option (on Skyline Daily).
I was dreading doing the next batch of MRM with so many samples, given that it usually takes 5-7hrs of dedicated attention.
But having just updated and used the new tool, it look a mere 1.5hr.
It was super easy, and super fast.

Thank you again, and keep up the fantastic work!
Harley.

view request
normalization using MSstats
(1 response) samyukta sah 2021-11-16

Hello,

I wanted to use the data normalization function in MSstats, however I am getting an error :
"Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :
** Please check annotation. Each MS run can't have multiple conditions or BioReplicates.
Can't finish analysis."

I have unique names and bioreplicate numbers for each file so I'm sure where I am going wrong.

Thank you

view request
“Compare Peak Picking” option
(2 responses) peter r mosen 2021-11-15

Hello Brendan and Team,

my post is linked to a previous issue of mine (Comparing Peak Scoring, 2021-06-17). I am still struggling with the “Compare Peak Picking” option. I acquired my MRM-data (400 peptides light and corresponding 400 heavy standards), built customized peak scoring model(s) using the 2nd best peak option and applied the models to my data. Peak scoring models (p- and q-value distribution) look good and q-value based refusal of peptides corresponds well with results from manual inspection. For comparison of the built models I want to use the ROC plots. And here my doubts start:

a) If I load my peak scoring model specific to the dataset (here CIL, which are isolated organelles), no ROC curve is visualized at first (slide1)
b) as soon as I load other models (which are actually not specific to the sample loaded in Skyline, here cell line) curves which look like ROC-curve are visualized and also for the CIL peak scoring model a trace is visualized (faint blue line directly on the y-axis)- at slide2. But these curves don’t really look like the ROC curves I know. I see that the shape of the curves look distorted (I see a retrograde trend for the curves (allCellLines and MEF)) as well as the vertical traces for the CIL.
c) Furthermore the ROC curves never reach 0.01 FP, which is the 1% FDR cutoff, right ? From my data I know that there are peptides with q-values higher than 0.01, so I would expect that the curve goes beyond this 0.01 FP point (slide 3)

To doublecheck I loaded DIA sample data from webinar 15 (slide4) and re-used the model you built. Also here the ROC curves for your model have this unusual shape. Maybe you guys can comment on this. What am I doing wrong? I didn’t find any documentation/tutorial on this.
Thank you for suggestions and your help,
Peter

FYI: I am using Skyline alpha (64-bit) 21.1.0.146

 comparePeakPicking_screenshots.pptx 
view request
Library Match - Prosit
(5 responses) blackmanbn 2021-04-16

Hi

I am unable to deselect (or remove) the Prosit library for library matching. It is not selected in the Molecule setting.

File attached.

 Fruit_Fly_lipds_ESI_NEG_IMS.skyP.zip 
view request
Export Scheduled methods for Agilent 6400 Series creates invalid method
(1 response) mmarx 2021-10-29

This is a new problem seen in the 21.1 release. Rolling back to the 20.1 release fixes the issue.

To reproduce the problem:
Export a scheduled method for Agilent 6400 Series.
Open the XML and observe that the scansegment includes this <isTriggeredMRM>true</isTriggeredMRM>.
For a scheduled method, this should be false.
There are no primary or triggered transitions in the method, so the method is an invalid method.

The root cause
The transition columns sent to the method creator includes the "IsTriggerMRM" column.
This causes the method creator interpret the data as a request to create a triggered method.

Please do not include the IsTriggerMRM column when exporting columns for scheduled methods.

view request
Error Pivot Editor
(2 responses) benoit fatou 2021-11-08

Dear Skyline Team,

I am working on a big project with about 4,000 samples.
I imported the samples in Skyline and now I would like to export the protein quantitation using the Document Grid settings.
However, when trying to use the pivot function to get all proteins in the first column and the sample name in the first row, I have an error message saying " The Pivot Editor cannot be shown because there are more than 2000 columns".
Could you please help me solving this issue?
Thanks a lot,
Best,
Benoit

view request
Exporting polarity-switching QQQ method from Skyline to Agilent-6495C
(2 responses) apickard 2021-11-09

Hi,

We have recently begun using skyline to process our metabolomics data. We've had a lot of success trying this on Agilent GCMS, Agilent LC-QTOF and both Agilent and Sciex QQQ data files. We believe it will really speed up our data processing workflow once we get the hang of it. The "Synchronize Integration" option for integrations in Skyline Daily is a tool we have been looking for for a while and it has worked great for us so far. Today we tried to export transitions we have from a polarity-switching QQQ Skyline method into the Agilent 6495C using Skyline 21.1.0.278. We've noticed a few issues.

  1. The Agilent 6495C can go down to a dwell time of 0.5ms (which we are currently running), however, skyline only allows integers in the Dwell time (ms) field when exporting a method. We changed it to 1 in order to export the method without an error, but if it is possible to allow lower dwell times in a future release, that would be helpful.

  2. If we export and choose Polarity = "All", it is exporting our entire list of transitions (that are designated as either [M-H] or [M+H] adducts in Skyline), but it is changing all of them to positive polarity while keeping the m/z values for positive and negative MRMs. Since this method has polarity-switching, we would have to individually search through the transitions and manually change those that are [M-H] back to negative polarity. If we choose Polarity = "Separated by polarity", it appropriately splits the transitions into a positive and negative method with the correct transitions and polarity in each, so we know that Skyline is able to decide the correct polarity based on the adduct. We only have this issue when trying to have both polarities in one method.

  3. When we tried to export the method as Method type = "Scheduled", the only options are to use the average retention time, or use the retention times from a certain replicate. We were wondering if there was a way to instead use the defined explicit retention time for the compound instead of basing the RT on files that are open, or if this could be considered as a feature in a future release. This is especially helpful when we are using Skyline to process hundreds of MRMs and determining the compound RTs out of many different data files with different standard mixes (where some will include the compound and some will not).

Thanks for your help,
Amanda

view request
Building libraries
(4 responses) luzjpaulo25041 2021-11-05

Dear MacCoss lab Team,

I am writing because I am trying to build a library and the result seems unexpected. I have some results from Peaks DB of a Bruker impact II TOF search. I feel like the quality of the library is not what is supposed to be. There are few peptides and the spectra matches are odd. I would like to know if there is a a problem with the run or something else. Could you help me?

Thanks for your attention.

Best Regards,

Joao Silva

view request
Full-MS/ddMS2 data import
(7 responses) ingus perkons 2021-11-08

Hello,
I have encountered an issue that I'm unable to solve.
So far I have used SkyLine (21.1.0.278) to process Full-MS small molecule data and I have found it to be the most user friendly and well designed tool so far. _Thank you very much, developers because SkyLine has truly helped to ease our lab's daily tasks _ :-)

However, I wanted to process some Full-MS/ddMS2 data acquired on Orbitrap using an inclusion list. The problem I encounter: I cannot extract (see) the corresponding ddMS2 product ion scans. The data files are not corrupt since I can extract ddMS2 scans in both XCalibur and MZmine and there are no import errors whatsoever in the Skyline itself.

I suspect the problem is either with (a) Transition list or (b) Transition settings.

I have attached an example data file that I want to process (" Example_Full-MS-ddMS2.mzML" ) and corresponding screenshots from the imported Transition list (Capture_a.png), Transition settings (Capture_b.png) + the screenshot of what I see once everything is imported and extracted (Capture_C.png).

_I have enabled View --> Transitions --> All. _

Interestingly, when I set the mass accuracy settings in " Transition settings --> Full-scan" to 100 ppm in both “MS1 filtering” and “MS/MS filtering” panels, I can see ddMS2 scans very well (see "Capture_D.PNG"). Yet, if I set 10 ppm in “MS1 filtering” and 100 ppm in “MS/MS filtering” or vice versa - ddMS2 data again disappears after re-import.

I'm puzzled because the raw data are of good quality and the mass errors are well below 10 ppm (see Capture_E.PNG). Tried to find the answer in tutorials and the forum, but, apparently, could not notice it.

 Example_Full-MS-ddMS2.mzML  Capture_A.PNG  Capture_B.PNG  Capture_C.PNG  Capture_D.PNG  Capture_E.PNG 
view request
Sure Quant quantification issue with thermo tune version 3.5
(2 responses) clemence balty 2021-11-09

Hi,

We are currently facing some problems with an existing custom SureQuant method that we had developed with the previous version of the thermo Tune software(3.3). With the current version (3.5) we are unable to reproduce previous results (see attached ppt). Peaks are present in the raw data (analyze with freestyle), but absent or troncated in skyline. I used the same method of analysis.

Do you think there might be a compatibility issue with the new version of the thermo Tune software ?

Thank you,
Clémence

 skyline support.pptx 
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Agilent QQQ data import failed
(1 response) stolltho 2021-11-07

Hi there,

With the update to the latest sky-daily v. 21.1.1.298, I am not able to import QQQ data any more.

Cheers,
Thomas

At 5:16 PM:
Failed importing results file 'E:\011_JasonLee\6470QQQ\01_Data\210630\210630_Lipids_1_dMRM_01.d'.
[MassHunterData::hasIonMobilityData()] Could not load file or assembly 'BaseDataAccess, Version=10.0.1.10035, Culture=neutral, PublicKeyToken=null' or one of its dependencies. The system cannot find the file specified.

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Error importing files
(2 responses) marbon 2021-11-07

Hi,
love your work, thanks so much!

I have successfully imported >20 .raw files from a Q-Exactive HF-X Orbitrap run into skyline (doing proteomics), but a few files from this batch of samples cannot be imported, see error below. Screenshot attached, I can upload / send the problematic file(s) if that's useful.

Failed importing results file 'C:\Users\mache_000\Dropbox (Sommerlab)\Mareike Bongers\Experiments\BILP\21\20210922_MK_MWN_70min_15cm_Mareike_SD4.raw'.
error reading spectrum controllerType=0 controllerNumber=1 scan=33698

I'm using Skyline 21.1.0.278 (64-bit). I'm not a super experienced user so a "as-simple-as-possible" explanation of what I could try or do would be awesome! :)

Thank you! Mareike

 Skyline error.png 
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Unable to finish importing chromatograms because the retention time predictor linear regression failed.
(3 responses) paga 2021-11-04
Good evening,

Ive been struggling with a diaPASEF (Bruker) analysis on Skyline. The experiment's goal is to find a set of 30 heavy labeled peptides on one DIA-file. I've produced a Skyline library using PEAKS but after loading the single DIA file it keeps fails. Ive been guiding the setting parameters based on the "Analysis of diaPASEF data in Skyline" document but including precursors as it is immunopeptidomics.

When specifying the use of Biognosys C-11 iRT peptides, I got the following error.

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

Skyline version: 21.1.0.278-fe50e66f2 (64-bit)
Installation ID: 5bc1d5e0-0694-49c2-87b5-7e376a14cafe
Exception type: InvalidDataException
Error message: Found invalid data while decoding.

System.IO.InvalidDataException: Found invalid data while decoding.
   at pwiz.Skyline.Model.ImportPeptideSearchManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\ImportPeptideSearch.cs:line 602
   at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
   at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
   at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
   at System.Threading.ThreadHelper.ThreadStart()
--------------------

After trying with the "Automatic" determination of iRT peptides, I got the following error


--------------------
Failed importing results file 'D:\DIA-PASEF_optimisation_2021\202109_Sr_DIAPSEF_EVAXheavy_RawData\10-7-2021_DIA_100ms_Im1.7_Evax_hela400_2sec_22-01-15_Slot1-10_3097.d'.
The calculator From_scratch_Automatic-iRT requires all of its standard peptides in order to determine a regression.
--------------------
 I will attached some of these file after the failed attempt.
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IM-TOF data will not import into newest release
(3 responses) Allison Stewart 2021-11-02

I am unable to import IM-TOF data in the updated version of Skyline-daily. The message I receive is as follows:

At 8:39 AM:
Failed importing results file 'C:\Users\akstewar\Desktop\BA FMT 2021\01Nov2021 BA Cal Curve\01Nov2021 BA Cal Curve\Cal_1.d'.
[MassHunterData::hasIonMobilityData()] Could not load file or assembly 'BaseDataAccess, Version=10.0.1.10035, Culture=neutral, PublicKeyToken=null' or one of its dependencies. The system cannot find the file specified.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\akstewar\Desktop\BA FMT 2021\01Nov2021 BA Cal Curve\01Nov2021 BA Cal Curve\Cal_1.d'.
[MassHunterData::hasIonMobilityData()] Could not load file or assembly 'BaseDataAccess, Version=10.0.1.10035, Culture=neutral, PublicKeyToken=null' or one of its dependencies. The system cannot find the file specified. ---> System.Exception: [MassHunterData::hasIonMobilityData()] Could not load file or assembly 'BaseDataAccess, Version=10.0.1.10035, Culture=neutral, PublicKeyToken=null' or one of its dependencies. The system cannot find the file specified.
at pwiz.CLI.msdata.ReaderList.read(String filename, MSData result, Int32 runIndex, ReaderConfig config)
at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\pwiz_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 197
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188
--- End of inner exception stack trace ---

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TMT balance
(4 responses) tehmina bharucha 2021-11-04
Thank you for all your work! I have been using Skyline to perform PRM of TMTpro (16plex) labelled peptides. Is it possible to set up Skyline to analyse transitions with TMT balance regions attached? i.e. look at the MS/MS spectra to identify transitions (product ions) that are modified by TMTpro balance regions?
view request
Unable to finish importing chromatograms because the retention time predictor linear regression failed.
(1 response) paga 2021-11-04

Good evening,

I've been struggling with a diaPASEF (Bruker) analysis on Skyline. The experiment's goal is to find a set of 30 heavy labeled peptides on one DIA-file. I've produced a Skyline library using PEAKS but after loading the single DIA file it keeps failing. I've been guiding the setting parameters based on the "Analysis of diaPASEF data in Skyline" document but including precursors as it is immunopeptidomics.

When specifying the use of Biognosys C-11 iRT peptides, I got the following error.

Skyline version: 21.1.0.278-fe50e66f2 (64-bit)
Installation ID: 5bc1d5e0-0694-49c2-87b5-7e376a14cafe
Exception type: InvalidDataException
Error message: Found invalid data while decoding.

System.IO.InvalidDataException: Found invalid data while decoding.
at pwiz.Skyline.Model.ImportPeptideSearchManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\ImportPeptideSearch.cs:line 602
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
at System.Threading.ThreadHelper.ThreadStart()

After trying with the "Automatic" determination of iRT peptides, I got the following error

Failed importing results file 'D:\DIA-PASEF_optimisation_2021\202109_Sr_DIAPSEF_EVAXheavy_RawData\10-7-2021_DIA_100ms_Im1.7_Evax_hela400_2sec_22-01-15_Slot1-10_3097.d'.
The calculator From_scratch_Automatic-iRT requires all of its standard peptides in order to determine a regression.

I will attached some of these file after the failed attempt.

view request
Unable to finish importing chromatograms because the retention time predictor linear regression failed.
paga 2021-11-04

Good evening,

I've been struggling with a diaPASEF (Bruker) analysis on Skyline. The experiment's goal is to find a set of 30 heavy labeled peptides on one DIA-file. I've produced a Skyline library using PEAKS but after loading the single DIA file it keeps failing. I've been guiding the setting parameters based on the "Analysis of diaPASEF data in Skyline" document but including precursors as it is immunopeptidomics.

When specifying the use of Biognosys C-11 iRT peptides, I got the following error.

Skyline version: 21.1.0.278-fe50e66f2 (64-bit)
Installation ID: 5bc1d5e0-0694-49c2-87b5-7e376a14cafe
Exception type: InvalidDataException
Error message: Found invalid data while decoding.

System.IO.InvalidDataException: Found invalid data while decoding.
at pwiz.Skyline.Model.ImportPeptideSearchManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\ImportPeptideSearch.cs:line 602
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
at System.Threading.ThreadHelper.ThreadStart()

After trying with the "Automatic" determination of iRT peptides, I got the following error

Failed importing results file 'D:\DIA-PASEF_optimisation_2021\202109_Sr_DIAPSEF_EVAXheavy_RawData\10-7-2021_DIA_100ms_Im1.7_Evax_hela400_2sec_22-01-15_Slot1-10_3097.d'.
The calculator From_scratch_Automatic-iRT requires all of its standard peptides in order to determine a regression.

I will attached some of these file after the failed attempt.

view request
Unable to finish importing chromatograms because the retention time predictor linear regression failed.
paga 2021-11-04

Good evening,

Ive been struggling with a diaPASEF (Bruker) analysis on Skyline. The experiment's goal is to find a set of 30 heavy labeled peptides on one DIA-file. I've produced a Skyline library using PEAKS but after loading the single DIA file it keeps fails. Ive been guiding the setting parameters based on the "Analysis of diaPASEF data in Skyline" document but including precursors as it is immunopeptidomics.

When specifying the use of Biognosys C-11 iRT peptides, I got the following error.


Skyline version: 21.1.0.278-fe50e66f2 (64-bit)
Installation ID: 5bc1d5e0-0694-49c2-87b5-7e376a14cafe
Exception type: InvalidDataException
Error message: Found invalid data while decoding.

System.IO.InvalidDataException: Found invalid data while decoding.
at pwiz.Skyline.Model.ImportPeptideSearchManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\ImportPeptideSearch.cs:line 602
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
at System.Threading.ThreadHelper.ThreadStart()

After trying with the "Automatic" determination of iRT peptides, I got the following error


Failed importing results file 'D:\DIA-PASEF_optimisation_2021\202109_Sr_DIAPSEF_EVAXheavy_RawData\10-7-2021_DIA_100ms_Im1.7_Evax_hela400_2sec_22-01-15_Slot1-10_3097.d'.
The calculator From_scratch_Automatic-iRT requires all of its standard peptides in order to determine a regression.

I will attached some of these file after the failed attempt.

view request
Specifying Integration Boundaries
(37 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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Problems while trying to optimize the CE while using Skyline and Agilent MassHunter Workstation
(1 response) rawel 2021-11-03

Hi Brendan,

i am posting this short observation since the exact same problem could not be found in the forum. We are using Skyline for a few years for targeted peptide analysis (thank you personally from us fro making that possible). We are using Triple QQQ (Agilent 6470) using the MassHunter Version Workstation Version 10.1. The Skyline version is the most recent one (Version 64-bit Skyline 21.1.0.278). Windows version is10 Pro.
While following the standard protocol for the method development, the Step A seems always to function perfectly (update of retention time). While executing the following Step B for the optimization of the CE, the instrument always goes offline with the following message "there are errors in the method" (Slide 1, attachment).
We have loaded an example of the method for step B in the offline option of the Masshunter methods (Slide 2, attachment). It seems the problem is with a new column "primary" and the triggered option.
In the old version of skyline, we did not see this column while optimizing the CE and it functioned very well. We tried to remove the option "triggered chromatogram acquisition" in the transition settings - still we get same response and the instrument goes offline. We talked with Agilent support and they could not help us out. The "bug" remained even after the update of the Agilent MassHunter Version Workstation software yesterday.. We set in step B , the max. concurrent transitions at 50 delivering two methods for altogether 95 transitions and it did not function.

Can anyone help us?

Kind regards,

H. Rawel

 CE-1.pdf 
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poor chromatogram
(2 responses) bysun 2021-11-01

We got strange peaks as shown below when the PRM of a precursor is scanned twice in one duty cycle of Q-Exactive. Is it possible to get a normal and smooth peak?

Thank you!

 skyline_question.PNG 
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DIA Profile vs Centroided data
(4 responses) boycea 2021-10-28

Hello Skyline Support Team,

I am currently acquiring my proteomics DIA data on the Thermo Orbitrap Exploris 480 using profile spectra because we were told from a colleague that this gives us more accurate data. Since we set it up this method, we have seen mixed opinions on whether centroided or profile data is preferred for DIA, and since the new Skyline daily update allows switching between both, we were wondering which of those the Skyline Team recommends. Another instrument we use for DIA acquisition is the Thermo Fusion Lumos. Thanks!

Aaron

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no files detected during import of library from fragpipe
(7 responses) kguehrs 2021-06-15

Hello Skyline team,

I tried to import MSFragger results into skyline. I followed the procedure given in tutorial on the following website by the develeopers of FragPipe.

https://msfragger.nesvilab.org/tutorial_skyline.html

I used the default method "DIA_Umpire_speclib" and the analysis by Fragpipe was successful. I can see the expected number of mzml, pepxml, calibrated.mgf, and interact....pep.xml files in the MSFragger subdirectory of the directory that also contains the raw files. This subdirectory also contains the protein.fas and tsv files generated by MSFragger.

The import of the interact...pep.xml files however failed with the error message shown in the attached file. For me, all the files described in the nesvilab tutorial are available and assume that here is either some file structure problem or some more syntax problem that I do not understand.

Best Karl-Heinz

 skyline_error.png 
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Skyline files not recognized after update
(1 response) mattkarasu 2021-10-29

Morning,
I updated Skyline-daily yesterday after which all the .sky files I had were no longer recognized by the program.
to be clear, I could still open the program from the start bar, but if I tried to start the program from a *.sky file, it didnt know how to open
attached is what I see when I open my folder

I uninstalled Skyline-daily, then reinstalled.
nothing changed.
I tried pointing the file format to skyline, but skylines location could not be found. note. I do not have admin access.
By searching for the application in explorer I found it in appdata but I cannot access that folder.
I tried changing the default, but where there should be a *.sky, *.skyd file format there was none.

Its like the program exists but the PC does not recognize the sky file format.

Thoughts?

Matt Karasu

 image.png  image (1).png 
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Issue importing waters RAW files, Unhandled Exception: Invalid Function Type
(3 responses) Allison Haase 2019-08-19

Hi All,

I have been getting error messages in skylines when importing raw files from a specific set of experiments on a Waters XEVO. Other files that have been imported from this instrument have worked fine in the past, and the raw files can be opened in MassLynx. The error is pasted below.

At 9:45 AM:
Failed importing results file 'C:\Users\allis\Desktop\redownload\20190809r001.raw'.
[pwiz::CLI::msdata::ChromatogramList::size] Unhandled exception: Invalid Function Type

Inner exceptions:
Exception type: System.Exception
Error message: [pwiz::CLI::msdata::ChromatogramList::size] Unhandled exception: Invalid Function Type
[pwiz::CLI::msdata::ChromatogramList::size] Unhandled exception: Invalid Function Type
at pwiz.CLI.msdata.ChromatogramList.size()
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasChromatogramData() in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 844
at pwiz.Skyline.Model.Results.ChromatogramDataProvider.HasChromatogramData(MsDataFileImpl dataFile) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 365
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 194

I have also attached one of the raw files in a zip folder.

Thank you very much for your time,
Allison

 20190809r001.raw.zip 
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RF Lens optimization in Skyline
(2 responses) roman sakson 2021-10-26

Hi Skyline team,

I was looking for a possibility to tune RF lens values for Thermo QQQs in Skyline similarly to CE values and I stumbled across an older issue (505) regarding that. Are there any plans about that at the moment? We were discussing with our Thermo application specialist for the TSQ Altis that tuning the RF lens value can indeed make a difference for the quality of your analyte signal. Skyline would, of course, be a great software to make this happen.

Thank you for considering it,
Roman

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Synchronize integration bugs
(2 responses) jrenders 2021-10-28

Hi there,
I have been using the new "synchronize integration" setting heavily. It reduces my processing time astronomically so THANK YOU!

However, I have observed some bugs I would like to report. The only working example I have of these bugs is in a batch that I am preparing for a manuscript, so if it would be of use I would prefer to share it privately.

-When synchronizing integration for a molecule that uses a surrogate IS, there seems to be some effect on the surrogate IS integration (which should not happen since the surrogate molecule is a completely different molecule).

-In the "synchronize integration" window, if you choose to synchronize according to analyte concentration Skyline encounters an unexpected error.

-(May or may not be a bug) - when you synchronize integration for a molecule that has a matched IS, the synchronization of the two is independent - i.e., you must synchronize the unlabeled version and then synchronize the labeled version separately. This is unlike how Skyline normally works where adjusting integration on the unlabeled set of a pair of matched analytes/internal standards is automatically applied to the matched IS. I actually kind of like that this is separate since at times the IS is deuterated (as opposed to having heavy carbon) and deuterons in place of hydrogens causes a shift in RT such that a matched analyte/internal standard pair may actually have slightly different RT's.

Please let me know if I need to elaborate more or share my skyline doc with you (I can share a more detailed explanation of the where the error is at that time). Thanks!

view request
About the AutoQC
(3 responses) hjl 2021-10-25

On the progress of installation of AutoQC, I've got some of questions about it.

Is the AutoQC only working on the instrument computer?
I tried to run the auto QC within my personal computer, but the Auto QC had the error like below:
"AutoQC Loader encountered an unexpected error. Error details may be found in the AutoQCProgram.log file in this directory:
C:\Users\user\AppData\Local\Apps\2.0\KNCPJLPD.WQA\YWDCAMM6JYB\auto..tion_e4141a2aa22107248_0015.0001_ed0ec6b30c3998a7"
*I would attach the AutoQCprogram.txt file

Is it possible to monitor other compounds except protein or peptides using AutoQC?
My compound is just a small chemical observed by MRM method using ALTIS of Thermo

I would attach the error message I got when I tried to open the AutoQC app and I wonder why the error message keeps showing(it had poped up few mins later since I closed)

As always, thank you for your support with a lot of appreciation.

Hope you have a great day.

 AutoQCProgram.log  AutoQC.PNG  AutoQCprogram location and the contents.PNG 
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Failed import from remote server
(4 responses) Jeff Whiteaker 2021-10-26

Hi Skyline team!
I am receiving the below error when trying to import results from raw datafiles stored on a remote server. The import is successful if I make a copy of the skyline document and raw datafiles on my local machine. I am otherwise able to work with other files directly from the server (e.g. MS Office, copy/edit, etc.). This is the case with multiple machines in the lab, but all accessing the same remote server.
We are also asking local IT about possible server issues, but like I said, we are able to read/write to the server in other applications. Do you think this is a Skyline issue or something else?
Please let me know what other information is needed.
Thanks,
Jeff

At 11:47:
Failed attempting to create a temporary file in the folder Z:... with the following error:
Win32 Error: 2

Skyline-daily (64-bit) 21.1.1.223 (7bb723fa8)

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MSstats error
(3 responses) laure bastide 2021-10-22

Hello Skyline team,

I have a new issu when I want to use MSstats as an external tools in Skyline, when I want to start the Data Processing, i have this warning message :
"** Loading the required statistical software packages in R .....

Registered S3 methods overwritten by 'tibble':
method from
format.tbl pillar
print.tbl pillar
Error : package or namespace load failed for 'MSstats' in loadNamespace(j <- i[[1L]], c(lib.loc, .libPaths()), versionCheck = vI[[j]]):
namespace 'tibble' 2.1.3 is already loaded, but >= 3.0.0 is required

Can't finish analysis."

I desintall and reinstall MSstats and R files and I try to install directly the "tibble" package in R but I can't because the package 3.0.0 is not available for R version 3.6.1...
The MSstats googlegroup suggested that you may have to make an update of R/packages/MSstats.
Alternatively, I can use it outside of Skyline but it was just for you know !

Thanks a lot for your great help !

Have a good day.

Laure.

What can I do ? Thanks a lot for your answer !

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Peak quantitation/ integration issue for high resolution small molecule data
(1 response) chevans 2021-10-26

Apologies in advance for what may be an elementary question. I’m trying to use Skyline for analysis of small molecule data collected on an Agilent TOF. I have a data set in which, for one or a few data files, the mass accuracy wasn’t as good as it should have been for a few scans (probable intermittent loss of reference ion due to co-elution with salts or other interfering species). Those scans fall in the middle of a few peaks I’m trying to quantitate; for example, acetylcarnitine. This particular peak has an ugly "split" appearance due to poor retention on the column, but Skyline handles it fine for most of the data, the issue only crops up when the mass accuracy for a few scans is poor. In those cases, skyline is integrating only one part of the peak. I’d like to widen the “mass extraction window” (not the "skyline" for it, I realize, but concept should be the same) so I can get integrate the whole peak. This works fine in Agilent Quant software. But no matter what parameter I try changing in the “transitions settings” menu, the split peak doesn’t change and the integration is wrong. The attached image illustrates the problem.

I could just use Agilent Quant for this project but I’m hoping to use it to analyze data on multiple different instruments and I like the “unifying” nature of Skyline software. Obviously an even better fix would be to solve the mass accuracy issue, but this crops up often enough that I'd like to be aware of a workaround, if one exists.

Thanks very much for your help!

Charles

 Skyline hi res quant question.png 
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FDR for Skyline Integrated MSAmanda Searches
(3 responses) jorge peinado 2021-10-25

Dear Skyline team,

I would like to know more about how the Skyline cut-off score and the MSAmanda integrated search tool interact.

I have been using Maxquant as my main search tool when working with Thermo Orbitrap raw data.
When reading some previous responses in this forum about how Maxquant FDR (PEP based) and Skyline cut-off interact i thought that a 0.99 Skyline cut-off would be the equivalent to Maxquant 1% FDR. However, i found out that when using a 0.99 Skyline cut-off i was missing some peptides identified in my 1% FDR Maxquant search.
After testing different Skyline cut-offs i found out that 0.9 cut-off is the cut-off that allows me to import all my results from Maxquant without additional cut-off from Skyline.
I don´t understand why 0.9 is the Skyline equivalent to Maxquant 1% FDR but i was happy with it as i trust Maxquant FDR.

However, i have recently received some data from a Waters Synapt which i haven´t been able to analyse via Maxquant.
I have found that performing my DDA peptide search using Skyline integrated MSAmanda is an alternative pipeline that could work for me, but i´m doubtful about what cut-off value would be the most appropiate to use as my experience suggest that 0.9 cut-off would be the equivalent to 1%FDR while logic makes me think that 0.99 cut-off should be the equivalent to 1%FDR.
I´m aware that MSAmanda uses a different scoring system than Maxquant, so i´m not sure about the cut-off value to use.

Thanks in advance

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Heavy Modifications Label:13C(6)15N(2) (C-term K) and Label:13C(6)15N(4) (C-term R) from PLGS Final Fragment File Recognized as 13C
(6 responses) dwwilk02 2021-10-25

Hi,
I've been trying to build a library in Skyline 21.1.0.278 from a Waters PLGS 3.0.2 final fragment file (Waters MSe data from Synapt G2-Si), but have not been able to get Skyline to identify the heavy labels Label:13C(6)15N(2) (C-term K) and Label:13C(6)15N(4) (C-term R) correctly. These PTMs are identified as 13C in my attempts to build the library. The original string for the peptide.modification from PLGS in the attached file was Label%3A13C%286%2915N%282%29+%28K%29(15), but Skyline does not recognize this. I tried to modify it, but have not been able to get the correct PTM. Thanks for your help,
Daniel Wilkey

 210812_Merchant_05_IA_final_fragment.csv 
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Normalization by TIC
(6 responses) caminha marcelle 2021-10-22

Hi,
How the normalization of PRM data by TIC is calculated?
Thank you in advance.

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Dealing with huge sample set in Skyline
(4 responses) carmen.gonzaleztejedo 2021-10-17

Dear Skyline Support Team,
We have just finished a PRM experiment comparing different treatments and time points, in total, 396 samples.
I have already uploaded all the files in Skyline and now, as I usually do, I would like to confirm the peak picking by manually checking all the peaks (correct peaks at expected RT and peak boundaries).
However, as I have 90 target peptides and 396 samples that can be endless. Is there any tip or suggestions from your side to deal with such a huge set of targets and samples?
Many thanks in advance.
Kind regards,
Carmen

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Chromatogram Information Unavailable - The document must be fully loaded before it can be saved to a new name.
(1 response) leaptrkl 2021-10-22

Hello and thanks in advance for your help!

I was working in Skyline daily with one document (no issues) and tried to open another, but none of the chromatograms would load in the second document. I closed both documents (saving the first before closing), and now neither will show chromatograms. I tried saving the document as a new name (received message "The document must be fully loaded before it can be saved to a new name.") , waiting 10 minutes for the file to load (nothing appeared to be happening), tried re-importing the files (received message "All results must be completely imported before any can be re-imported."), and tried re-scoring in place (received message "All results must be completely imported before they can be re-scored."). I closed and opened the file 5 times, since another someone with this issue in another support thread said the issue resolved after 3 attempts.

The software still shows my retention times, mass errors, calibration curve, etc., but no chromatograms. I tried exiting out of all the chromatograms and then bringing them back up with View>Arrange Graphs but that didn't do it either.

I was able to see chromatograms for an unrelated Skyline document I created a few months ago during a Skyline Online Course, but I tried to open my document of issue afterwards and still no chromatograms.

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DIA Umpire error
(8 responses) Francis Beaudry 2021-07-08
Hi,

I am setting up a DIA analysis workflow using DIA raw to build a library. I follow the tutorial using your dataset or my dataset but I obtain the same error message. I tried on 2 different computers. Please advise ?

---------------------------
Skyline
---------------------------
Error
---------------------------
OK More Info
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System.IO.FileNotFoundException: Le fichier 'C:\Users\franc\AppData\Local\Temp\gyo1ftie.mkn.mz5' est introuvable.
Nom de fichier : 'C:\Users\franc\AppData\Local\Temp\gyo1ftie.mkn.mz5'
   à System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
   à System.IO.File.InternalMove(String sourceFileName, String destFileName, Boolean checkHost)
   à pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) dans C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:ligne 148
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installation of Sprocop
(1 response) hjl 2021-10-21

Hi

First of all, Thank you for providing this great tool for us.

I've tried to install the additional tool called Sprocop within a Skyline on 3 computers but they all had the same error message.
(I did tried both R software, and skyline)

warning: unable to access index for the storage of http://cran.us.r-project.org/bin/windows/contrib/3.0
"package 'qcc' is available as a source package but not as a binary.
package 'qcc' is not available (for R version 3.0.2)
same as the car.

Is there any chance to get a solution for me to use the Sprocop tool to monitor the quality of my batches..

Waiting for your response :)

Thank you
hope you have a great day.

P.S I attached the warning screen shot

 Sprocop error(within Skyline).PNG  Sprocop error(within R 3.0.2).PNG 
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