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
Show total ratio next to precursor mass
(1 response) sarah joestl 2022-01-28

Dear Skyline Team,

since today I've got problems with my skyline program. I'm using isotopic labeled standards for quantification. Normally, right next to the mass of the precursor ion, rdotp and total ratio are shown in brackets, e.g. 514,5931++ (rdotp. 1, total ratio 1,52), but my program does not show this anymore, there is only the mass. If I export the data, the ratio is calculated. Do you have any suggestions on how to reactivate this in the settings? I cannot find anything on that in the tutorials or settings.

Thank you in advance for your help!
Kind regards, Sarah

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Registration problem
ab598059 2022-01-28

My name is Abir Lefsay

Abir Lefsay, MS, MS, PhD

Assistance director

Health and Environment Research Centre (HERC) Lab

Faculty of Medicine, Dalhousie University

I am having trouble registering for Session 2 - Advanced Small Molecule Topics - February 10-11
I would appreciate your assistance

My Visa is not accepted."???????

 Capture.PNG 
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Problem inserting small molecule transition list
(3 responses) Sangram 2022-01-27

Hello Skyline Team / Experts,

I am trying to execute a small molecule detection on our Sciex DDA files. The objective is to find if there is any contamination in our samples coming from plasticware or due to any mishap in best lab practices.

For this I am using the MaConDa database [Attachmentment 1] to come up with the Transition list. I follow the steps elucidated in this document.

Now skyline keeps giving this error : The precursor m/z 61.0284054 is not measureable with your current instrument settings.

I tried giving lowering the minimum m/z at instrument transition settings, still it did not work. What am I doing wrong ? Or is there any problem with the transition list ? Please help

Regards
Sangram

 Popular_MassSpec_contaminants_TL.txt 
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Transition signals integrate to 0
(4 responses) jfoe 2022-01-26

Dear Skyline team,

I am working with low intensity peptides in PRM and try to get as much evidence as possible.
Recently I came across a peptide that had some transitions always marked in red (area 0) even though there is some signal.
I tried to force reintegration or reimporting the data and had no success.
I am attaching an image showing this should not be related to the background subtraction.
Maybe you can tell right away what is up.

I could also provide the skyline file if needed.

Skyline (64bit) 21.2.0.369

Best,
Jonas

 peak_area_issue.png 
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Diferentiate dda and dia
(1 response) luzjpaulo25041 2022-01-26

Dear Skyline team,

I would like to ask if one could use Skyline to find out if a raw file was generated by dda or dia. I have a file that is supposed to be DDA but I fear it is not.

Best regards
Joao

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How to set the same (matching) peak boundaries while integrating in Skyline
(4 responses) MNezvedova 2022-01-20

Dear Sir or Madam,
sometimes it happens that when moving peak boundaries for light peptide, there is no change in peak boundaries for heavy peptide. So then they are not matching, and it makes the integration quite tricky. I couldn't find anything in the Skyline settings where this can be changed?

I appreciate your support,

Marketa

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Unable to generate calibration curves
(4 responses) r yagoubi19 2022-01-22

I am trying to generate a calibration curve but after following all the steps I get an error message: "Error: All of the external standards are missing one or more peaks. The selected replicate has missing or truncated transitions." I don't understand this error message as all of my standards have clear well-defined peaks.

I have designated all of my external standards as "Standard" and filled in their concentrations in the document grid.

I also paid attention to light and heavy parameters.

Can anyone help me? Did I forget something?

Thanks

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Using Skyline image/logo in graphical abstract for publication
(8 responses) Alok 2020-01-08
Hello Skyline team,

Is it possible to use Skyline image in graphical abstract of a paper? We think it is good to include the logo as we will be making all Skyline files publicly available after the acceptance.

Thanks,
Alok
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Is it possible to get mProphet scores at the precursor level rather than the peptide level?
(1 response) brian24641 2022-01-22

In the mProphet paper it looks like they generate scores at the precursor level i.e. one score for each potential peptide charge (typically +2 and +3). However when I look at the exported mProphet features they appear to be at the peptide level. When I then generate a report with Detection Q Values for each precursor I see that all precursors associated with a peptide for a replicate have the same value, which implies that mProphet results were rolled up to the peptide level from the precursor level implicitly. Thus, is it possible to get these scores at the precursor level, and also what the algorithm is that is being used to roll up mProphet scores (or Q values) to the peptide level?

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Keyboard Shortcuts
(13 responses) thomlau 2011-09-16
Hi all,

Is there a file or list somewhere with all the keyboard shortcuts for skyline? I'm going through a large batch of peptides and replicates and my hands feel like falling off!

Thanks!

Tom
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CE Optimization - Product Ion m/z is different in exported transition list
(7 responses) Peter Jackson 2022-01-21

Hello,

I'm using the molecule interface and performing some CE optimization experiments. When I export my transition list, my product ion values are being changed along with the CE value. I've attached an example to this post.

Thanks,

Peter

 20220121_Namide_NMN_NR_CEopt.csv 
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error message for the impro
(3 responses) diletta piana 2022-01-20
I downloaded from Peaks the peptides in a format readable by skyline, but once attached I get the error message that I report below and attached.
---------------------------
Skyline
---------------------------
ERROR: While searching for spectrum file for the search results file 'peptides.pep.xml', could not find matches for the following basename with any of the supported file extensions (.mz5, .mz5, .mzML, .mzXML, .raw, .wiff, .wiff2, .d, .lcd, .ms2, .cms2, .bms2, .pms2):
ERROR: Sample1BIS_RUN1(180919)
ERROR:
ERROR: In any of the following directories:
ERROR: D:\Diletta\SKYLINE
ERROR: D:\Diletta
ERROR: D:\
ERROR:
ERROR: While searching for spectrum file for the search results file 'peptides_1_1_0.mzid', could not find matches for the following basename with any of the supported file extensions (.MGF, .MGF, .mzXML, .mzML, .mz5, .raw, .wiff, .wiff2, .d, .lcd):
ERROR: Sample11_RUN1(180919)_VUS
ERROR:
ERROR: In any of the following directories:
ERROR: D:\Diletta\SKYLINE
ERROR:

Command-line: C:\Users\Urbani\AppData\Local\Apps\2.0\TTENHC55.79M\37MD986X.ZC4\skyl..tion_e4141a2a22107248_0015.0002_b6130d78fa8ea9a3\BlibBuild -s -A -H -o -c 0.99 -i DDAtoMS1_prova1_190122 -S "C:\Users\Urbani\AppData\Local\Temp\tmp3650.tmp" "C:\Users\Urbani\Documents\DdaSearchMs1Filtering (1)\DdaSearchMS1Filtering\DDAtoMS1_prova1_190122.redundant.blib"
Working directory: D:\Diletta\SKYLINE
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: While searching for spectrum file for the search results file 'peptides.pep.xml', could not find matches for the following basename with any of the supported file extensions (.mz5, .mz5, .mzML, .mzXML, .raw, .wiff, .wiff2, .d, .lcd, .ms2, .cms2, .bms2, .pms2):
ERROR: Sample1BIS_RUN1(180919)
ERROR:
ERROR: In any of the following directories:
ERROR: D:\Diletta\SKYLINE
ERROR: D:\Diletta
ERROR: D:\
ERROR:
ERROR: While searching for spectrum file for the search results file 'peptides_1_1_0.mzid', could not find matches for the following basename with any of the supported file extensions (.MGF, .MGF, .mzXML, .mzML, .mz5, .raw, .wiff, .wiff2, .d, .lcd):
ERROR: Sample11_RUN1(180919)_VUS
ERROR:
ERROR: In any of the following directories:
ERROR: D:\Diletta\SKYLINE
ERROR:

Command-line: C:\Users\Urbani\AppData\Local\Apps\2.0\TTENHC55.79M\37MD986X.ZC4\skyl..tion_e4141a2a22107248_0015.0002_b6130d78fa8ea9a3\BlibBuild -s -A -H -o -c 0.99 -i DDAtoMS1_prova1_190122 -S "C:\Users\Urbani\AppData\Local\Temp\tmp3650.tmp" "C:\Users\Urbani\Documents\DdaSearchMs1Filtering (1)\DdaSearchMS1Filtering\DDAtoMS1_prova1_190122.redundant.blib"
Working directory: D:\Diletta\SKYLINE
   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
---------------------------

I was wondering how I could read my library of peptides to make a DDA search to MS1.
I tried to save the files in mz5 format but at the end it gives me the error that it doesn't read or doesn't find the corresponding match.
 Capture_error.JPG 
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Export report
(2 responses) georgios mermelekas 2022-01-20

Dear Skyline team,

I would like to ask you if there is way to export dotp for both heavy and light peptides in separate columns in the report file.

Thank you very much for your time and consideration,

Best regards,
Georgios

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Help with bromide modifications
(7 responses) bianca dempsey 2022-01-18

Hello!
We have been looking for bromide modifications in Tyr residues and we are facing problems with the spectral library. We intent to use DIA to find with proteins are modified, but apparently, we don't have support for bromide modifications. Did anybody have the same problem?
Thanks!

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Wishlist: Indicator for transitions that are diagnostic of PTMs
(2 responses) 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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Selection of transitions that diminish precursor dotp
(2 responses) 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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Theoretical peptide z-ion mass appears to be incorrect
(11 responses) JHamey 2018-11-14

Hi there Skyline team,

I've come across a problem to do with the theoretical mass of z-ions in Skyline. They appear to be consistently off by 1 Da - they are predicted to be 1 Da lighter than they actually are. E.g. a z13 ion is predicted to be 1316.7 Da when it is actually 1317.7 Da. When I searched for threads related to this issue I came across this one (Issue 498):

https://skyline.ms/issues/home/issues/details.view?issueId=498&_docid=issue%3A498

It appears as if the "fix" that was made to make z-ions 1 Da lighter was done in error. The 1 Da heavier masses for z-ions I am getting from MS-Product match what I see experimentally for a normal peptide (it was noted in that thread that the peptide where the "error" was observed contained an abnormal gamma linkage at the position where the mass shift was thought to be wrong). C-ion masses are perfectly ok. I see this consistently for at least 5 different z-ions in the peptide.

Thank you in advance for your help.

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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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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 
view request
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

view request
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

view request
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

view request
How can I connect to Spectrum Mill Server and what is my IP address
xzhmuxrl 2021-12-23
view request
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.

view request
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 
view request
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

view request
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

view request
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 
view request
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 
view request
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 
view request
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
---------------------------
view request
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

view request
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

view request
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.

view request
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

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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 
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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

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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.

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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 
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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 
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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.

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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

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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

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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

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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 
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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?
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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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