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 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
small molecules: Error with calibration curve: all of the external standards are missing one or more peaks
(7 responses) ayson 2020-03-27

Hello! What are some of the criteria of having calibration curve? Currently, I only have standards and also blanks but no quality control samples. I have already labeled my standards as standards and blanks as blanks and I have also put in the concentration. But whenever I am opening calibration curve this is what I get: Error: All of the external standards are missing one or more peaks

Is it because some of my standard samples especially the lower region do not have any peaks detected? Thank you!

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Error reading iRT standards transition list
(2 responses) HENG 2020-03-27
Hello. I am learning your Skyline Importing Assay Libraries documents. And when I proceed to the following steps (see my attachments) and click OK button. The skyline showed this Error reading

Error reading iRT standards transition list: Product m/z value 171.11 in peptide TPVISGGPYEYR has no matching product ion.
OK More Info
System.IO.InvalidDataException: Product m/z value 171.11 in peptide TPVISGGPYEYR has no matching product ion.
   at pwiz.Skyline.FileUI.CreateIrtCalculatorDlg.OkDialog() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\FileUI\CreateIrtCalculatorDlg.cs:line 194

I am sure I was following every steps of the document. So how can I solve this problem?


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Small molecule - custom report - cannot get light and heavy on same row
(4 responses) MartijnvF 2020-03-27

Dear all,

I am working on getting my custom report to show both the areas of the light and heavy molecule on the same row in the report. However, I only get the areas on different rows, see attachment.


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Existing Quantitative Experiment Tutorial_Peptide Setting problems
(2 responses) doloph 2020-03-27

Dear Ladies and Gentlemen,

I am currently trying to get familiar with your excellent software and am therefore working through the provided Tutorials. However, while I was working on the Existing Quantitative Experiment I am facing some problems. The user interface looks quite different when setting up the data and the program does not allow to pick the expected "Labels:13C(6)15N(4) from the drop down menu. Additionally, when typing in the parameters manually the, software gives back several error codes. Does someone know what I have to change?

Kind regards,

 Screenshot (350).png  Screenshot (351).png  Screenshot (352).png  Screenshot (354).png  Screenshot (355).png 
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Small molecule: Skyline cannot properly analyze Agilent QQQ with waste diverter in the beginning of the run
(4 responses) ayson 2020-03-26

We have acquired HILIC data in Agilent QQQ with diverter valve to waste from 0-0.2 mins. From 0.2 to 5 mins, mass spec was turned on. When I analyze the data in Skyline, I am only seeing the chromatogram from 0-0.2 mins but never 0.2 to 5 mins. The data looks fine in Masshunter Quant but not in skyline. I have sent you more detailed report in the attached word document.

It is vital that skyline can process these types of data because more small molecules will be diverter their initial flow to waste to protect mass spec instrument. Thank you so much!!!

Best regards,

 032620_skyline_not properly analyzing Agilent QQQ data with waste diverter on.docx 
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Small molecule - calibration curve error: Matrix must be positive definite
(2 responses) MartijnvF 2020-03-26

Dear all,

I am trying to use Skyline to see if it will be suitable in our clinical laboratory for quantification and monitoring of MS performance. I am getting an error I cannot find anything previous help on. The calibration curves are not showing:

Error: Matrix must be positive definite.

Cheers for any help, much appreciated.

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backwards compatibility skyline v20 vs skyline v19
(1 response) kguehrs 2020-03-25

Hallo Skyline team,
I generated some dta with the newest version of skyline. I wanted to reload them on an other computer running Skyline from an external disk. However, skyline refused to open the data because they are generated with a newer version. I do not have administrator rights for this computer and it is therefore somewhat complicated the upgrade skyline in short time in these days. Moreover, there is the question of the general backward compatibility of skyline files.

Wish you all the best and hope that we all can keep going.

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Increase in m/z of fragment ions when exporting methods for collision energy optimisation
(2 responses) sarah lennon 2020-03-24

Dear Skyline team,

I have just noticed that when exporting a Waters method for collision energy optimisation from Skyline Daily, the m/z of the fragment ion is increased slightly (+0.01 Da, +0.1 Da from first to last) with increased collision energy. Is there any reason for that ?

Thank you very much for your help,
Kind regards,


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Prosit in skyline
(11 responses) Tobi 2019-12-05

Dear all,

thank you very much for integrating Prosit into Skyline. I noticed that the dotp in the new library match mirror view does not treat y ions from deferentially labeled peptides as equivalent. Is it possible to extract the Comparison dotp via results grid for all peptides at once?


 Prosit in Skyline.png 
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Peak quality score for small molecules
(4 responses) bwidner 2020-03-17

Hello Skyline Team!

I am working with small molecule data, and I would like to put a number on chromatographic quality for each peak. I would like this score to only be for MS1 peaks (or MS2, if I decide that's what I want to look at) and to include things like peak shape (i.e. gaussian), signal to noise, agreement with predicted retention time, etc. I have not so far found this in Skyline.

I looked over the Advanced Peak Picking Models tutorial since it includes scoring, but I do not think this is exactly what I am looking for. It seems more complicated than what I need; perhaps I am overlooking something?

I am new to MS and get a little lost in the terminology, especially when it is peptide terminology, as I have never worked on peptides. My apologies if I am overlooking something obvious, and thanks for your assistance!


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LOD and LOQ calculation for Small Molecules
(1 response) h ravuri 2020-03-19

Hello Skyline Team

I am currently working on small molecules. I am trying to work out the LOD and LOQ on the molecule. I followed all the steps in the small molecule tutorial document. But could not figure out where I can get the steps or information on required samples sets to calculate the same.

Can you please provide any information on this?



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Question on Limits
(6 responses) Tobi 2019-08-06

Dear Skyline team,

we have an outstanding project and want to work with DIA and Spectral libraries on a atrget list generated from huge fasta files in the GB range. Are there still any (fixed) limits for the number of peptides and transitions in the target list or the number of peptides in a spectral library that skyline can hendle? Older posts here yield different answers.

Best regards,

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Skyline for MAC OS
(1 response) ana normando 2020-03-17

Is there an available version of Skyline for MAC OS?

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Skyline-daily and Skyline 20.1 crashed in loading pepxml from MSFragger
(17 responses) fcyu 2020-01-30

In loading pepXML or ineract.pep.xml from MSFragger/FragPipe using the "Import DDA Peptide Search", skyline crashed with the following error. Looks like it can find the mzML file which is in the same folder, but crashed somehow. You may find the mzML, pepXML, interact.pep.xml, and fasta file in from file sharing. Could you please help to take a look?

ERROR: No spectra were found for the new library.

Command-line: C:\Users\yufe\AppData\Local\Apps\2.0\KG99EWD8.1KH\L9HYCN5B.KLC\skyl..tion_e4141a2a22107248_0014.0000_e9276d4fd631b09e\BlibBuild -s -A -H -o -c 0.95 -i test -S "C:\Users\yufe\AppData\Local\Temp\tmp6570.tmp" "E:\dev\msfragger\dev2\test.redundant.blib"
Working directory: E:\dev\msfragger\dev2
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\KG99EWD8.1KH\L9HYCN5B.KLC\skyl..tion_e4141a2a22107248_0014.0000_e9276d4fd631b09e\BlibBuild -s -A -H -o -c 0.95 -i test -S "C:\Users\yufe\AppData\Local\Temp\tmp6570.tmp" "E:\dev\msfragger\dev2\test.redundant.blib"
Working directory: E:\dev\msfragger\dev2
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 160



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Issue with the skyline cmd.exe with the latest release version of Skyline
(1 response) shobhabr20 2020-03-16

Hi Brendan,

An error related to command line runner is observed. I downloaded the Release version of Skyline 20.1.76. But the error description indicates that there is a dependency on Skyline daily.

Please find attached the screenshot for more details.


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Points Per Peak
(3 responses) sa825 2020-03-11

Good morning,

I am conducting PRM on the Thermo Q Exacitive and I wanted to ask if there was a setting in Skyline that allowed you to select the number of points per peak for the peptides of interest?



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export a method with each transition CE optimized
(3 responses) tongzhang 2020-03-12

In a project to optimize CEs for each transition, I created methods files with the option of optimizing collision energy selected. I ended up with 50 injections for 391 transitions on a TSQ. I followed a Skyline tutorial (link below) to upload the raw data, and would like to export a method with the best CEs for each transition. I enabled the "use optimization values when present" option, but can not get the optimized CE exported. One thing I noticed is that a schedule method was used in the tutorial, but I still want to export multiple methods because my peptides elute in a pretty narrow window.
I also attached the skyline files if that helps.
Thanks very much! 
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Import Results no longer works for bruker.d DIA data in version 20.1 (works fine in version 19.1)
(6 responses) dkueltz 2020-03-04

Hi Brendan,

We recently noticed that the import of bruker.d folders that contain DIA raw data from Bruker UHRQTOF instruments does no longer work in Skyline v20.1. It took us a while to track this problem to the version of Sklyline. We tried all other possibilities (spectral library. assay list, raw DIA files) and finally were able to determine that the results import works without any problem in v 19.1 but not at all in v 20.1. It would be great if you could look into this. Let me know if you don't have bruler.d raw data to test this with.


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adding modifications to peptide sequence
(6 responses) kbagramyan 2019-12-17

Hi Brendan,

I am glad that I found the question that I wanted to ask you too. We recently performed an MRM analysis using 6495 LC-MS Triple Quadrupole (Agilent). We optimized the MRM transitions for the quadruply charged precursor ions of the light and the heavy peptides. Here, are our transition settings used in the method: 545.3 (precursor, +4) and the transitions are 509.0 (b, 4+); 640.0 (b, 3+); 678.3 (b, 3+). The Skyline recognized the two of transitions such as 508.5155 and 639.67. The third transition of 678.3 in our method was 677.6849 in Skyline transitions giving 0.6151 Da difference. Unfortunately, this was the most intense peak but wasn't accepted by Skyline because I couldn't extend the method match tolerance above 0.6.
The data look very good and I was wondering if there is a way to extend the method match tolerance, so I can include that transition for accurate quantification?
I hope I was clear and was able to describe the problem.
Thank you in advance,


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Unable to export method to Masslynx 4.1 SCN950
(10 responses) Tran Tran 2020-01-16

Skyline ( 64-bit)
Masslynx V4.2 SCN895/ Waters Xevo TQD
Windows 7 professional 64 bit

Skyline is installed at the same computer as the instrument . I was trying to export CE optimization using the File/ Export /Method/Multiple methods but received an error ( screen shot attached). However, I was able to export into the .csv format using File/Export/Transition List

Waters Tech Support was contacted but they don't have a solution at the time. Masslynx security update isn't installed either.
Wonder if anyone else observed this issue and was able to resolve it.
Thank you,

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Generation of spectral library using Prosit
(8 responses) benoit fatou 2020-03-03

Hello Skyline Team,

I am trying to use Prosit to generate spectral libraries and I realized that Prosit does not recognize "propionamide" as a fixed modification on the cysteine residues even when I set this modification on Skyline.
Is there a way to modify the modifications into Prosit?

Thanks for your help,

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Error generating spectral library from FragPipe in Skyline v20.1.0.31
(12 responses) Chinmaya k 2020-02-13


I have not been able to generate a spectral library in Skyline from FragPipe/MSFragger output. Please find the attached error file. The same error has also been reported recently as well. (

I tried to generate a spectral library separately using BlibBuid.exe and it was also ended up with the same error. Please find the attached screenshot as well.

Is there any other way to rectify this and generate a Skyline compatible spectral library?


 FragPipe_SpecLib_BlibBuild.exe_Error.PNG  FragPipe_SpecLib_Skyline_Error.PNG 
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Peaks image export
(5 responses) davidz 2020-03-09

Hi there,

Is there a way to export all the peak images to a PDF file?


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Setting to limit the tolerance to be 5 ppm in peak finding
(2 responses) davidz 2020-03-06

Hi there,

Which setting would be the one to set so the ppm tolerance is 5 ppm or a given ppm value during peak finding?


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Showing Trendlines in Skyline
romeally 2020-03-06

I have a project where I am looking at change over 5 time points of a reaction. I ran 3 replicates of each and wish to show the changes over the 5 time points with variance. What is the best way to represent this in Skyline? I am calling each time point a replicate, but the document grid only allows a comparison of two conditions. Any suggestions?
Thanks! Bob

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Skyline not recognizing FIAMS CV rough tune data
(4 responses) cmwats2 2020-03-03


I used Skyline to export SRM transition list for compensation voltage rough tune. I acquired the data on an Altis instrument. I imported the data selecting optimizing compensation voltage -> rough tune. I attached an image of the imported data. However, when I try to make an optimization library containing the peptides and best CVs, skyline says there are no new optimizations. I can successfully create the optimization library for collision energies. What am I missing for Skyline to select the optimal FAIMS CV for my peptides? I cannot make a medium or fine tune method because I do not have the rough tune data.

Thanks in advance,

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fail to import peptide search into skyline due to the retention time problem.
(3 responses) chengkang 2020-03-04

I was trying to import my maxquant peptide search into the DDA in skyline, however, after I selected the .raw file of the search, an error message popped out:"The document specific spectral library does not have valid retention times. Please check your peptide search pipeline or contact Skyline support to ensure retention times appear in your spectral libraries." Can anyone help me out?


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Calibration curve of Sodiated Adduct not appearing
(5 responses) alejandro.cohen 2020-03-04

Hi Skyline people!

I'm running a MS1 filtering (Molecule Interface) of three catecholamines on a Vanquish QExactive system. Two of our targets (Epinephrine and Phenylephrine) work fine, however, I cant get Norepinephrine to show the Calibration Curve with all other parameter being identical. Interestingly, I only see the sodiated adduct on this target... wondering if this could be the issue?

Attached the corresponding file.


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Separating HCD and EThcD spectra
(9 responses) jonasbecker 2020-02-26

Dear Skyline Team,

I ran a PRM experiment which used different fragmentation modes (HCD and EThcD) on the same peptides in the same run, i.e. HCD and EThcD spectra were recorded alternately. When loading these data into Skyline, it seems for some spectra that the MS2 chromatogram is "oscillating" (see attached picture1 & picture2). In contrast, this does not occure for precoursers fragmented only with HCD (see attached picture3).

Is there any filtering/data processing option available to separate the HCD and EThcD spectra so that Skyline can interpolate the peak areas correctly?

Thanks in advance and best regards,

P.S.: If you could provide a private link, I could share you the skyline project.

 picture1.png  picture2.png  picture3.png 
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Error in using Skyline command line
(10 responses) minliecn 2020-02-18

Hi Skyline team,

I am trying to practice Skyline command line with data downloaded from Webinar 15. I modified the MultiAnalysis.bat and Skyline template files to reflect programs/file path. The rest files are used as downloaded. When I try to run the cmd line, it worked a few times. Then when I tried to change mass accuracy and RT window in the Skyline template file, it stopped working. After I restarted cmd program, it worked again. Then I changed the mass accuracy and RT window and restart the cmd program to retest the program, it stopped working. I am using Windows 7 and running Skyline v20.1.0.28. I am wondering what could I do differently so that the program will work better for me?

Thanks very much for your help in advance!

 MultiAnalysis_webnar15.bat  SkylineAnalysis.bat 
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QuaSAR download error
(1 response) jung165 2020-03-02

Hello, Skyline team

I have one question about downloading QuaSAR.
I am currently using Skyline 20.1.

When I try to download QuaSAR, it automatically downloads R 3.0 and continue to download QuaSAR related package.
However, the error message appealed with mentioning I need R 3.5 or higher version to download the packages.
So, I downloaded R 3.5 but, error message still appeals and it seems that those packages were still trying to be downloaded in R 3.0 folder.
May I ask your help for this issue??

I appreciate for your help


The following packages failed to install:


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Tried uploading mzML and mzXML files does not work
(3 responses) rodriguezj5 2020-02-27

I have proteowizard and tried uploading mzML and mzXML files. I also tried converting my raw files using the peak picking option on proteowizard. Peak picking option says "Vendor (does not work for...."
Please let me know how to upload mzML files to skyline.

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(2 responses) roman sakson 2020-02-27

Dear Skyline team,

I have noticed some time ago that there seem to be a newer type of Skyline document, which I have not seen before, with the ending .skyl. I am just wondering what type of information is stored in there and whether it can be deleted without much damage done, as for example, the sky.view file?

Thank you,


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Different types of skyline files
(4 responses) heyang 2020-02-27

Hi Skyline team,

Just want to know details about different types of skyline files, e.g document, view, skyl and chromatogram data. if I want to upload a result to panarama, I need to upload all 4 files, or just document file. thanks,


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Peptide Quantitation Using Unlabled Peptide as Internal Standard
(4 responses) Richard Lam 2020-02-26

I would like to create calibration curves for 3 peptides based on the peak area ratio of the peptide and a common unlabeled peptide (see attachment for the Skyline document).
For each BSA peptide (a total of 3), I would like to use the PSA peptide as internal standard and create the standard curve for each BSA peptide.


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unable to load a spectrum from Sciex Triple-TOF 5600 data
(1 response) jech 2020-02-25
I have analysed a tryptic digest of a sample on a Triple-TOF 5600 in TOF mode.
I have loaded the wiff file into skyline to look for the tryptic digests.
When i click on a chromatographic peak, to see the spectrum i get an error, saying that the scan ID was not found in the wiff file.
Am i doing something wrong, or is there a problem with the wiff files from sciex
I have attached a screenshot of the error message that i get
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Error importing Thermo Quantiva raw files
(4 responses) Simon11 2020-02-21

After changing our LC setup for our Quantiva, we can no longer directly import raw files into Skyline. We have to convert to mzXML first.

The error given is below:
Failed importing results file 'C:\Users\footes\Documents\Skyline\VBPB140220R1602
[RawFileImpl::setCurrentController()] Instrument index not available for request
ed device
Parameter name: instrumentIndex

I checked the raw metadata for a pre-LC change file and a post LC change file and have attached the 2 dumps. Looks like the error is caused by the recording of the LC method 1st in the postLC instance, which is assigning an Instrument: nano3000. The preLC file doesn't record any LC info.


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Multiple injections in a single LC-MRM file, aka RapidFire
(2 responses) echan 2020-02-21

Hello Skyline team,

Skyline has worked out great for our small molecule analysis by target MRM/PRM.

However, we're having trouble figuring out how to import data from our screening datasets in a RapidFire-like workflow as described in Figure 5 of this SCIEX technote.

We're monitoring the same 4 transitions (for molecules) in 30 second injections across one 96-well plate (4 * 96 = 384 transitions), with all the results saved to one .wiff file. We can process the data using SCIEX MultiQuant as shown in the attached screenshot.

However, the closest I have come to with Skyline is to import the .wiff file as 96 .sky files, each with different explicit retention time values (incremented by 30 seconds). I can manually duplicate the same molecules 96 times and edit the RT (and RT windows) in the XML document, but all of my edits get overwritten with all the RT (and RT windows) reverting to the same values after re-importing my results (i.e. re-importing my .wiff file).

It would be fantastic if we can use Skyline to manage our MRM data, regardless of whether we're monitoring a single molecule/single injection or single molecules/multiple injections in a single file. Could you give us some pointers? Thanks. -Eric

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'Chromatogram information unavailable' small clinical molecule DIA
(7 responses) Mus 2020-02-06

Hi all
Over the past week, I have been successfully extracting and exporting AUC info of precursor masses from batch replicates (x10) of small molecules to determine precision across 10-13 Calibration points.
I am using DIA and all ion fragmentation (bbCID) mode on a Bruker QTOF - Impact II.
Using precursors and quantifiers and MS/MS fragments as Qualifiers only.

However, yesterday morning, after an autoupdate of skyline to, any analysis I attempt is displays the message, ‘Chromatogram Information Unavailable’.
The only other change I have made is to the transition table, updating RT’s for 4 precursors and their heavy isotopic internal standards
No other settings have changed. I cant analyse old data with new update either.
I understand, from the skyline discussion forums that this could be a ‘matching’ issue.
The data is definitely acquired and definitely in the correct datafiles.
I have appended a screenshot for help with diagnosis.

Any advice?
Thank you kindly, in advance!


 screenshot 1.png 
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timsTOF Scheduled PRM Data Import
(8 responses) robwsprung 2020-02-19

Dear Skyline Team,

Thank you for your continued development of an excellent software package.

I am having trouble importing scheduled PRM data from a timsTOF Pro instrument. When I look at the results in Compass DataAnalysis, it appears that the method worked as expected - MS1 signals for the expected isotope labeled peptides are observed and MS/MS spectra are acquired at the retention times of interest. However, when importing into Skyline Daily ( no precursor and no product ions are observed.

I've successfully imported other PRM runs from the instrument. Only this scheduled method is causing problems. Any insight you could provide would be appreciated. Thanks.


 timsTOF MHC  timsTOF MHC settings.pptx 
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Export Method from Skyline Daily to Waters Xevo TQ-XS equipment
(3 responses) danielacgranato 2020-02-20

We have been using Skyline in computers that are not connected to the mass spectrometer to export our methods to Waters Xevo TQ-XS. To do so we installed MassLynx 4.1 version in our computers and it has been working fine like that. Actually it was a suggestion given by you in 2017. Recently, we have tried to export the method from one of our computers that did not have MassLynx installed (we just installed it in a non operative mode) and it did not work.

Do you have any other suggestion in how I can export the method without having the need to install Skyline in the computer connected to the mass spectrometer? In case that is the only way, do you have information if the new version of MassLynx 4.2 currently allows us to export the method from Skyline? Thank you very much. Best, Daniela

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Using more than one calibration curve in a single skyline document
(4 responses) hogan 2019-10-30


I have been using skyline smoothly to run a quantitative small molecule assay with a calibration curve and check standards and the whole nine yards for a few months now and it is great!!! Thank you all so much for your hard work in bringing small molecule functionality into this great platform!

Now the issue.
Skyline works fine for 1 analyte with 1 standard curve. I have recently tried to add a second analyte with a separate calibration curve to the same skyline document and I am only able to input one value in the "Analyte Concentration" column of the document grid for each replicate that is set as a "Standard". (I have multiple analytes in my calibration curve and their concentrations are different in the CAL levels). I can only load up one analyte curve worth of values and then they repeat over and over again in the grid even if the "Molecule Name" or "Precursor" are set to different values.

Is it possible for skyline to have multiple calibration curves set in the same document? Can you add a custom sample type like "Standard2", "Standard3", etc. and assign separate calibration values there?

My current workaround is to pull the data files into two separate skyline documents, with different analytes. I then import the annotations separately in each one and combine the calculated concentration data afterward using the replicate name as the index to join them on. Please let me know if there is another way to do this or if there is something in the works!

Again. Thanks so much for your work!


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Pressure output?
(4 responses) alejandro.cohen 2020-02-20

Hi Skyline people,

I work with an Agilent 1290 coupled to a Qtrap5500 running MRM metabolomics which I analyze using Skyline (.dam methods, .wiff datafiles). Analyst is recording the column pressure output signals, however viewing these using Analyst is cumbersome and time intensive when working with big sample batches. We have encountered erratic behaviour of our chromatography, accompanied by unusual pressure profiles for samples, which tend to correct themselves after a few runs.

As part of our QC, it would be great if skyline would be able to add (overlap) the pressure traces on each chromatogram. Is this possible? I know each vendor 'hides' the pressure traces in different output and channels... but the information is there :)

I know this request has been done in the past... here another kind 'reminder'



Alejandro Cohen, Ph.D.

Scientific Director
Biological Mass Spectrometry Core Facility. Room N-105
Life Sciences Research Institute

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How to automatically export skyline results to email?
(1 response) kaylee xu 2020-02-20

Hello skyline team,
Could skyline export csv. format result to my email automatically? If yes, then how?

Thank you!


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Skyline runner dependencies
(1 response) shridevi patil 2020-02-20

Hello All,
We are working on automation, this software generates mrm methods. Internally we use SkylineRunner.exe to generate experiment files in various steps. We are packing skylinerunner.exe( version) in to intallers. Once installed software picks its path but during execution we get generic fail: check for skyline full installation. We have skyline full installation on our place. Can you please help us to know what is dependency.
Thank you

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Copying Skyline Charts or Chromatograms
(2 responses) Richard Lam 2020-02-13


How to copy charts or chromatograms generated in Skyline for presentation purpose?


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Build spectral library with propionamide modification
(6 responses) benoit fatou 2020-02-19

Dear Skyline Team,

I have an issue with building a spectral library from Maxquant search using propionamide modification on the cysteine residues.
It seems Skyline does not recognize it. I attached the screen shots from Maxquant and Skyline and I don't really see the problem.

Thanks for your help,

 MaxQuant Window.jpg  Skyline Window.jpg 
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Long import duration of DIA MSE data with iRT
(2 responses) david gomez zepeda 2020-02-19

Dear Skyline Team, I'm trying to analyze data acquired in a Synapt HDMS (G1) in MSE (DIA) mode, but file importing is taking around 12 hours for one single file from a single protein (BSA) analyzed in a 1 hour gradient (1.5 GB file), despite using RT prediction from iRT peptides. I'm intending to analyze complex proteomes from 2 hour gradients (8 GB files), so this time for importing would be a problem.
Is there something that I could do to reduce the import time?

Here is the description of the experiment and what I have done:

  1. Injected a BSA in-home digest in nanoLC-MSE using a Synapt G1 with a 1 hour gradient.
  2. Obtained a Prosit Library of the CRAPome (including BSA) and used it for peptide identification
  3. Created a Skyline file including the peptides for BSA only
  4. Imported the RAW data to Skyline without using RTs
  5. Calibrated an iRT model with the iRT prediction in the Prosit Library, using 13 BSA peptides as standards. I'm doing this because I want to use the BSA peptides for iRT calibration in the analysis of complex proteomes.
  6. In a new file I imported the RAW data to Skyline using predicted RTs from the iRT calibration described above, using 5 minute windows. This took about 12 hours.
  7. I have attached the RAW data and the Skyline complete share folder after results import, without further peptide refinement.
  8. PC specifications
    Processor: Intel Xeon CPU E5.2620 v2 @2.10 GHz (2 processors)
    RAM: 64 GB
    OS: Windows 7 64-bit 
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ERROR on imported spectral library generated by Spectronaut software
(3 responses) Winnie 2020-02-17

Skyline team,

Hi! I have problems in importing the spectral library (.csv) generated by Spectronaut software (file >import> assay library) int skyline. The detailed error information attached. According to the error information, the Modification tab in the Peptide Settings, the m/z types on the Prediction tab and the m/z match tolerance on the Instrument tab of the Transition Settings were re-checked. See the attachment for a screenshot of the parameter settings. I did not find any errors.

Is there any wrong in importing the spectral library? The attachment for a screenshot of the spectral library was also upload.

Thanks in advance,

 ERROR_information.txt  Skyline_settings.docx  Screenshot of the spectral library.docx 
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Maintaining Integration Windows When Re-importing Data
(2 responses) meyermr 2014-02-28
Hi Skyline Team,

I'd like to know if Skyline can maintain integration windows manually set by the user when re-importing a data file? For example, when I manually set the integration window for a given peak, add additional fragment ions to the peptide tree, and then re-import the data...Skyline will shift the integration windows slightly. Is there a way to override this so I don't have to manually go through and reset the integration windows?

Thanks for the help,
view request
Not displaying all files in the skyline document View Tiled
(1 response) rle041 2020-02-18

Dear skyline team,

First of all, thank you for a great program.
I am currently working on a PRM project where I have approximately 200 PRM runs in the same skyline document.
To check the peak integration, I can only look at 100 at a time if I click view->arrange graphs->tiled,
I am very sure that the remaining .raw files are loaded into the document, and the "missing" files are displayed if I click on them in the retention time- replicate comparison view. Also, when the graphs are tabbed, not every file is displayed in the tabs.
Noticably, not all peaks require manual integration, but some do, and it is also nice to check wether some have been cut etc. I was therefore wondering if there is a way to organise my skyline workspace so that I can access all easily? For instance, have two pages of 100 files each that I can easily switch between?

Hopefully you can help me!

Ragnhild Reehorst Lereim

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System.IO.IOException: ERROR: No spectra were found for the new library
(6 responses) Pavan Kumar Dappili 2020-02-12

Hi all

I am trying to DDA search by importing to the library from msmsscans.txt file generated after Maxquant analysis. But the skyline is showing the following error.

System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\admin\AppData\Local\Apps\2.0\1QE2T2VZ.QYZ\8XBYAZMR.OMP\skyl..tion_e4141a2a22107248_0014.0001_bc11b87fcb56f175\BlibBuild -s -A -H -o -c 0.99 -i Four_cell_lines_trial -S "C:\Users\admin\AppData\Local\Temp\tmp82BA.tmp" "D:\Important Information\Pavan\Skyline Protein Analaysis\Four cell lines_trial.redundant.blib"
Working directory: D:\Important Information\Pavan\Protein Analysis\Agilent Raw data\Cell analysis_Trials\200201\combined\txt
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 160

Please suggest how to resolve this issue.

I have used the latest version of the skyline and maxquant for this analysis.

Thanks and Regards
Pavan Kumar Dappili

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No calculated concentration values randomly
(6 responses) Serge 2020-02-13

I use the software version 20.1 (64 bit).
I use two transition (Analyte and internal standard). I generate calibration curve using peak area ratio but for some samples, they didn't calculate the concentration. Those samples with uncalculated concentration had appropriate peak ratio value.
In attachment, you have different print screen of the method.
I'm waiting your feedback

 Skyline bug.pdf 
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About Msstats error
(2 responses) Quanhuiwang 2020-02-10

Dear Skyline team,
I have successfully installed MSstatas from the tool store, but it doesn't work! When I tried to use it, an error would always be reported in the immediate window as I shown in the attached file. When I changed another data, the error is the same. I hope someone help find the reason. Thank you!

Quanhui Wang

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Error when importing .wiff file
(5 responses) aleksandr stotland 2020-02-13


I am trying to import the results file and Skyline throws up the error that I have attached. Previously the data loaded correctly. Thank you,


view request
How to caculate the Precision,Extraction recovery or Matrix effect with skyline?
(2 responses) kaylee xu 2020-02-11

Dear skyline team,

Q1: Recently, I am using Skyline for small molecular quantitative analysis. At methodological validation, I don't know how to calculated the Precision, Extraction recovery or Matrix effect with Skyline? Can you give me some steps?

Q2: If I build 10 compounds' calibrations at a time with different concentrations, eg. Compound-1( 1, 2, 5, 10, 20, 50, 100ng/mL),compound-2(2,4,10,20,40,100,200ng/mL) How to enter the concentration of these different compounds in the skyline?

Q3.What does these black error bar mean? Please see attach file.

Q4:Can I quantity one analyte in unknow samples with other analyte's standard curve line ? For example, I want quantity SM18:1 in unknown samples with SM18:1(d9)'s standard curve line.

Thank You!


 Q3_blank error bar.PNG 
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Importing peak boundaries for all raw data file for a molecule
(1 response) viragsagikiss 2020-02-11

Hi all,

I am using Skyline-daily, on Windows 7. Small molecule multi-targeted metabolomics.

First, just want to say how great is Skyline. We are using it for small molecule method development in the last year and I really enjoy both the simplicity the visualisation capability and the flexibility of the software; it is a life saver in many ways.

Now, after put together a multi-targeted LC-MS method to monitor a few hundred compounds in the same time i have run into new questions. I understand maybe not a good idea to bundle up my questions, but if you can help with one of them that's already great!

  1. Peak boundaries! Importing peak boundaries are essential for A; isobaric species (on a QTRAP and TQ in my case) and B; irregular peak shapes (ion-pairing LC)
    After reading through support, i have found this tutorial:

Is there a way to apply Peak boundaries to all Experimental files (*.wiff) in a certain or i have to create a "10.000" line csv every time I analyse a new data set?
Also, can i set t1 and t2 to look for the peak only between those parameters? I assumed that's what "Explicit Retention Time Window" does, but regardless what i set there, in case of blanks Skyline picks up peaks minutes away from the RT?

  1. Overlay peaks/anlaytes from the same samples
    to deal with shifty retention time AND/OR isobars, i understand the 'retention time plot' with 'replicate comparison' is almost the same, but for presentation and visualization i still would like to show overlayed chromatograms, do you plan to include this?

  2. Dropbox/Data storage in the cloud [to share data between Skyline Windows/data collection Windows/user's Mac]. Because of the size of the dataset i work on the same *.sky file over the time course of days, leave Windows sleeping, and after continue with the same file I notice some chromatogram disappeared (i.e. Main Window displays 'Chromatogram information unavailable', where used to be data the day before. Same happens time to time when I insert a new transition list (same values, but using molecule list names), or i rename/resave the .sky.
    Do you recommend data is stored in the same folder (or just computer) where Skyline files, or just avoid storing the files in the cloud? Anyone reported similar? I thought i am making a mistake but after this occurred enough times I wonder if it is because all Original Files and Skyline files are in the cloud.

I appreciate the help.

view request
Points per peak removed from Skyline?
(4 responses) melechco 2020-02-10

We´ve noticed that the indication of number of points per peak is not displayed anymore in the newest versions of Skyline. Was this funcionality removed from Skyline? Thanks

view request
Metabolite settting up
(3 responses) sgler 2020-02-06

I’m self-learning on how to use skyline for metabolite. I was going through the tutorial but question is how did you achieve the dot product?

Did you create library for metabolite study just like PRM study in protemics?

view request
Is the light version of the peptide present?
(10 responses) sa825 2019-11-11


I imported some heavy labelled PRM results into skyline and for the light version of a peptide I am interested in, only 2 fragment ions were detected.

In the heavy version of the same peptide, all 3 fragment ions were detected.

Can I assume from this that there is close to 100% labelling, in the sense that the light peptide is no longer present in the sample? And its only the heavy version left in the sample?



view request
Skyline for fully labelled metabolomics
(1 response) adelabriere 2020-02-10

Dear Skyline team,

We are trying to use skyline for fully lablled cell extract for lipidomics analysis, with the isotopic pattern going up to +20 in some case.

Is there a good setup to do so ? It seems at the moment that skyline does not allow us to go above 10 peaks and has a threshold which prevent the detection of heavier isotopes.

Best regards,

Alexis Delabriere

view request
How to import a custom spectral library
(2 responses) s26guan 2020-02-07

Dear Skyline team,

Could you show me the steps to import a custom spectral library, say in the msp format? Thank you.

view request
Log iRT error but almost working fully
(3 responses) Chris Ashwood 2020-02-05

Dear Skyline team,

Thanks to Kaipo for putting the logarithmic iRT feature in Skyline, I think I will use it a lot.

I've attempted to use it but I had two issues in the implementation that might be related (one is a limitation with how it's currently set up to be used and the second is a crash I get). The roadblock (and subsequent workaround) is that the calculator only supports linear calibrations (picture attached) but after tweaking how I edit the calculator, it crashes on the final step.

The procedure that hits a snag is:

  1. Calibrate the iRT calculator in linear space (only option) with my standards (Calibrate iRT Calculator window)
  2. Save the calculator then edit the calculator by changing the iRT values to what they are meant to be (Edit iRT Calculator window)
  3. Set regression type to logarithmic and then add results in the bottom right.
  4. Once i click OK, the results field is populated with correct iRT values but then I get an index out of bounds crash.

The crash can be found below.


Skyline version: (64-bit)
Installation ID: 586d68dd-c361-4392-a8c5-ad0df1faa1e9
Exception type: IndexOutOfRangeException
Error message: Index was outside the bounds of the array.

Stack trace:

at pwiz.Common.Collections.ImmutableList`1.SingletonImpl.get_Item(Int32 index) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\Collections\ImmutableList.cs:line 310
at pwiz.Skyline.Controls.Graphs.RTLinearRegressionGraphPane.GraphData.get_XAxisName() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\Graphs\RTLinearRegressionGraphPane.cs:line 1397
at pwiz.Skyline.Controls.Graphs.RTLinearRegressionGraphPane.GraphData.Graph(GraphPane graphPane, PeptideDocNode nodeSelected) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\Graphs\RTLinearRegressionGraphPane.cs:line 1132
at pwiz.Skyline.Controls.Graphs.RTLinearRegressionGraphPane.UpdateGraph(Boolean selectionChanged) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\Graphs\RTLinearRegressionGraphPane.cs:line 429
at pwiz.Skyline.Controls.Graphs.RTLinearRegressionGraphPane.<>c__DisplayClass65_0.<UpdateAndRefine>b__0() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\Graphs\RTLinearRegressionGraphPane.cs:line 593

Exception caught at:
at pwiz.Skyline.Controls.Graphs.RTLinearRegressionGraphPane.<>c__DisplayClass65_0.<UpdateAndRefine>b__0() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\Graphs\RTLinearRegressionGraphPane.cs:line 593
at System.RuntimeMethodHandle.InvokeMethod(Object target, Object[] arguments, Signature sig, Boolean constructor)
at System.Reflection.RuntimeMethodInfo.UnsafeInvokeInternal(Object obj, Object[] parameters, Object[] arguments)
at System.Delegate.DynamicInvokeImpl(Object[] args)
at System.Windows.Forms.Control.InvokeMarshaledCallbackDo(ThreadMethodEntry tme)
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 System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
at pwiz.Skyline.SkylineWindow.ShowPeptideSettingsUI(IWin32Window parent, Nullable`1 tab) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Skyline.cs:line 3394
at pwiz.Skyline.SkylineWindow.peptideSettingsMenuItem_Click(Object sender, EventArgs e) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Skyline.cs:line 3371
at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.ToolStrip.WndProc(Message& m)
at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at pwiz.Skyline.Program.Main(String[] args) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Program.cs:line 306

 iRTcalibration.png  irtAlmostOkay.png 
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Issue importing waters RAW files, Unhandled Exception: Invalid Function Type
(2 responses) Allison Haase 2019-08-19

Hi All,

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

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

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

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

Thank you very much for your time,
view request
Chromatogram information unavailable
(6 responses) joaopcnunes 2020-02-05


I analysed some data files in Mascot and then import the .dat files into Skyline (v. but i can´t see the chromatogram (Chromatogram information unavailable). I already changed the name of the .dat file for the same of the raw file, but it didn´t solve the problem.

Could you please help me on this problem?

Thank you very much


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MRM triggered MSMS
(3 responses) heyang 2020-02-04

Dear Skyline team,

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



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SKYLINE BUG for display and peak area reporting when Non-Quantitative transitions are included
(13 responses) Will Thompson 2020-01-14

Hi Brian, Brendan et al

Please see attached to this message a reasonably detailed account of a pretty bad bug in peak area reporting which seems to occur when non-quantitative transitions are included in Skyline documents. It affects reported total peak areas, such that external measurement of results (such as stats or against calibration curves) will not match up what is returned by Skyline.

As always, let us know if you have any questions.


Will  SKYLINE BUG reporting when Non Quant Transitions Included.docx 
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SRM-triggered MS2 for QTRAP
(2 responses) tobias.kockmann 2013-07-16
Hi there,

does skyline support method development for SRM-triggered MS2 on ABsciex QTRAP instruments? There is a method type "triggered" in the Export window, but I always get the message:

"To export a scheduled method , you must first choose a retention time predictor in Peptide Settings/Settings".

To my understanding this does not quite make sense for a SRM-triggered MS2 experiment...could this be a software bug, since the same message also appears when selecting the type "Scheduled"???

I am running skyline-daily

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Exporting mirror plot dotp values
(1 response) sven.van.bael 2020-02-04

Hi Skyline team

When comparing two spectral libraries using the mirror plot option, a dotp comparing the individual spectra from each library is calculated (see example attached). Is there a way to export these comparison dotp values for all peptides at once? (i.e. is there an option to do this via Document Grid?)

Many thanks!

view request
Negative iRT values
(1 response) smanda 2020-02-02

Hi Brendan,

I am using irt calculator to in Skyline to calculate the normalized iRT values for my spectral library based on a set of 30 standard peptides. I see that in the results some of the normalizedRT values are negative. What could the reason? Any pointers?


view request
Importing MRM method *from* MassLynx
(4 responses) Koen 2020-01-30
First post here!

I'm unable to find any info how to import an existing MassLynx method with MRMs *into* SkyLine. Most tutorials, etc are the other way around. We're using MassLynx 4.2.

Is this possible?


- Koen.
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not being able to see product ions when importing ddMS2 data
(2 responses) ana 2020-01-30

Hi Skyline team,

I ran a few standards on QE Exactive using DIA and ddMS2 for comparison. I have no problems seeing both precursors and products for DIA but when I import ddMS2 data (having the settings to DDA) I can't see the products. Also, the peak are bars for the products are "grayed out"
If I import ddMS2 data having DIA settings, I can see the product ions but peaks are getting cut (can't see the whole RT) although I don't have retention time windows enable.

I have attached a picture showing what I get, I have a library for BSA but not for the other proteins. I am also only selecting y ions (not b). Could the library (or lack of) be an issue when processing DDA?


 ddMS2 data_20200130AI.png 
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Can I use SkylineDailyRunner to remove check mark on libraries in settings>peptide settings>library?
(1 response) Gao 2020-01-28

Thank you!

view request
Command line SRL generation
(3 responses) smanda 2020-01-20

Hi Brendon,

I have search engines results as mzid file which I import into Skyline and create a spectral library. I then map the peptides to proteins and export the library as a OpenSWATH report. Is it possible to do all these steps using the command line? I have several of these and interested to automate the process of library generation using Skyline.

  1. Import mzid and clean (probably bibliospec does this)
  2. Calcualte iRTs using standards
  3. Map peptides to proteins
  4. Import into project and export as a report.

Any help appreciated.


view request
Problem with Importing a Particular Document
(4 responses) roman sakson 2020-01-23
Dear Skyline-Team,

I am trying to import one Skyline document ( into another ( When I do that (I choose to add new replicates and to merge matching peptides), I get the following error message:

Data cache files with different score types cannot be joined.
OK More Info
System.IO.InvalidDataException: Data cache files with different score types cannot be joined.
   at pwiz.Skyline.Model.Results.ChromCacheJoiner.JoinNextPart() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheJoiner.cs:line 135

In the resulting file, it seems that the import worked for one of the two peptides but not for other. Also, things like "order according to acquired time" do not work anymore but when I manually import the corresponding wiff-files, everything seems fine. I cannot share the resulting file, when I try I get another error message:

The document must be fully loaded before it can be shared.
OK More Info
System.IO.IOException: Settings.MeasuredResults Not all chromatogram sets are loaded - No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R1_Ctrl.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R1_0h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R1_1h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R1_6h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R1_12h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R1_24h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R3_Ctrl.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R3_0h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R3_1h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R3_6h_II.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R3_12h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\190516_43_degree\19.07.19_RS_Arsenite_R3_24h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R2_Ctrl.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R2_0h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R2_1h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R2_6h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R2_12h.wiff;No ChromFileInfo.FileWriteTime for C:\Users\Roman Sakson\Desktop\Sophie_London\SK_Uebergabe\19.07.19_Arsenite\Wiff\19.07.19_RS_Arsenite_R2_24h.wiff
   at pwiz.Skyline.SkylineWindow.ShareDocument() in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1183

I used to move those Skyline-files quite a bit after I have imported the results for the first time and if I get it right, at some point the link between the skyd file and the wiff files got lost somehow? I would just like to know whether I am allowed to relocate my Skyline documents to be able to import them somewhere else later on.

Thank you!

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Question about Data upload to panorama
hogan 2020-01-28

When targeted MS/MS proteomics data is uploaded to panorama with the Normalization to global standard setting on, is the RatioLightToGlobal standard value uploaded to the panorama database? I have been unable to find that value anywhere.

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instalation and framework
(1 response) vbesada2020 2020-01-28
Hello, I am trying to install version 19.1 (64 bits) in a new laptop with windows 10 but the setup ask for a .NET framework 3.5 SP1 which is not able to be installed. It neither function if I try to skip this step as the laptop comes with framework above 4.0. How can I get rid of this? Thanks in advance
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New Feature Request
(1 response) Peter Jackson 2020-01-24


Just wanted to again request that a future version of Skyline be able to go directly from document to method on an Agilent QTOF. It's very helpful for our Agilent QqQ and we would get a lot of use out of the ability to do it on our QTOF. Thank you.

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Using Skyline image/logo in graphical abstract for publication
(5 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.

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Input of GC-MS SIM data
(3 responses) Joerg 2020-01-21

i want to import SIM-data from our GC-MS from Agilent. However, i repeatedly get the following error message upon import of the runs:

Failed importing results file 'C:\Daten\Skyline\GC\Allopregnanolone\10µmolar A -sim.D'.
Invalid chromatogram ID SIM SIC Q1=215.15 start=5.087066667 end=14.497416667 found. Failure parsing m/z values.

Could it be that my transition list is flawed or i this a problem of the import file structure?

Many thanks in advance!


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Adding measured peptides to iRT standard curve
(12 responses) romeally 2020-01-16

I have 224 peptides into which I spiked a pierce iRT standard mix and ran it as 3 methods to cover my retention time calibration. When I go to add the non-iRT peptides to the iRT calculator I get an error saying I have insufficent correlation and no peptides will be added to the iRT database. This is PRM data and I am not using a library at this point, so I do not have any fragment correlation but the dotp for the precursors all looks pretty good. The tutorials are all based on triple quad with transitions calling the peptides. Is this something I have to do? I ran these all back to back on the same column with the iRT peptides spiked in so I don't know why there wouldn't be correlation. Is my data bad or am I doing something wrong??? I am attaching a screen this a different procedure with PRM than MRM data? I do see my intercept for my peptide mix is lower than the intercept for my iRT standards...could this be my issue?

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Scheduling Lumos Targeted Method Development
romeally 2020-01-15

So I am trying to build a scheduled method for 224 peptides which are all +2 charge. When I go into build the multiple methods to get the scheduling I selected 30 precursors maximum per run thinking this should be about 8 injections, but under Thermo Fusion it says I need 172 methods to get there. If I switch it up to QE or Agilent QTOF it says 8. I did a test export and I found 172 .csv files some with only 1 peptide in them. Is this a bug? Should I use the QE settings? What is the most isolation precursors I should use in method building for a Lumos instrument? In any event 172 methods can not be I have some weird setting I am missing? This is with fixed carbamidomethyl and all +2 charge so it should be exactly 224 precursors.

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Skyline does not recognize all peptides identified with MaxQuant
(14 responses) Sven F 2014-08-21
Hi, I like to do some quantification of phosphorylated peptides with the Skyline software. For identification I use the MaxQuant software v1.4.1.2 To quantify my data, I import the msms.txt from the MaxQuant search into Skyline. Because I need just the data from one single protein, I erased data of all other proteins from the .txt file. Skyline builds up a peptide library based on the MaxQuant search, but also there are some peptides, which can be found in the library but are not identified by Skyline in the MS spectrum. So there are some phosphorylated peptides identified by MaxQuant but are not found by Skyline. So in the end, MaxQuant gives me a sequence coverage for this protein about 86%, wheras Skyline is approximately at 20%.
I already checked the parameters like modifications ect, but I could not find a solution. Also I tried to import peptide boundaries using a modified verdion of the evidence.txt as described formerly here on this forum, which occurs in an error, saying the peptide could not be found.

I attached the Skyline an msms file so hopefully you can take a look on them.

Thanks a lot.

Best regards,
 msms.txt  Pex5_CK2_200u_100u_20140729.skyd  Pex5_CK2_200u_100u_20140729.blib 
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mProphet issues
(3 responses) Tobi 2020-01-13

Dear Skyline Team,

in the attached skyline daily I want to highlight some things I would be happy to get your feedback.

When exporting mProphet features unfiltered it seems to list all potential peaks for a single peptide at various retention times and with different z-scores and p-values, however, the q values for all peak candidates are the same despite different p-values, is that a bug?

Also, the checkbox to filter for highest scoring peak can be highly useful but also lead to errors. Problem here is that when correcting peak picking (by hand or with Avant-Garde) this export still reports the highest scoring candidate but not the actually picked peak. Having this filter ( only integrated peak instead of highest scoring or both) would be very nice as it would deliver desired results for both automatic and manual peak picking.


 mProphet unfiltered.csv 
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Exploris PRM data import into skyline
(5 responses) Fynn 2020-01-08

Dear Skyline-Team,

I am using a PRM method with multiplexing (MSX) for targeted measurements on a "Q Exactive HF-X" mass spectrometer. Here everything works well and I can observe precursors as well as transitions. After transferring the method onto the "Orbitrap Exploris 480" platform and loading of the raw files into skyline (and Skyline daily) I still see the precursors, but no transitions. When I open the raw files with "SeeMS" I can see both, the MS1 and MS2 scans. Since the skyline method worked for the HF-X runs I am a bit lost why it is not working for the Exploris runs. Do you have any suggestions how to solve the data import problem?

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Wrong position of amino acids?
(1 response) fcsigloch 2020-01-13

Dear Skyline team,

I find the way of numbering amino acids of a protein in Skyline somewhat confusing: The first amino acid is given as position 0. This behaviour is especially misleading, when trying to locate point mutations or PTMs, where the Skyline position is reported to be one less than the normally reported one.

An example: if, say, a deamidation is detected on the first N of F2QME5 (see attached picture), it would be reported to be at position 3. But the N is actually amino acid #4.

Is this a bug or a feature of Skyline?


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Add q value annotation check box missing in the Reintegrate form
(2 responses) minliecn 2020-01-10

Hi Skyline team,

I am trying to add q value annotation so that I can export the q value in Skyline report. But I do not find the Add a value annotation check box in my Reintegrate form. I am running Skyline(64-bit) on Windows 7. Do you know how can I fix the reintegrate form issue? I attached the expected reintegrate form and the actual integrate form in case these are useful.


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Customs Adducts // GC MS Agilent import
(2 responses) Christina lucas 2020-01-06

I try to import GC MS Data (SingleQuad) to skyline, as I really like the software. The approach presented by Pawel Sadowski for Shimadzu and Thermo does not work for Agilent Data.

So far my I was to create different adducts for the basepeak molecule, but this is not possible if you just want to create a mass additon that is not in the adduct list.
I tried [M(-5.0)+H] or likewise, it does not work with the actual version.

If I try to add more than one precurser, skyline starts to label them

I attach a file of the agilent data, maybe that makes it aesier to solve the problem... 
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The document format version 19.12 is newer than the version 19.1 supported by Skyline (64-bit)
(2 responses) Sadr 2020-01-08

Please can you help me with file format version error below;



Failure opening I:\core\proteomics\data\group_folders\Mass Spectrometry DATA\Q_Exactive_HF\2019\12_December\QE_HF_PR1271_REB_12122019\SKYLINE\
The document format version 19.12 is newer than the version 19.1 supported by Skyline (64-bit)
OK More Info
System.Reflection.TargetInvocationException: There is an error in XML document (2, 2). ---> System.InvalidOperationException: There is an error in XML document (2, 2). ---> pwiz.Skyline.Util.VersionNewerException: The document format version 19.12 is newer than the version 19.1 supported by Skyline (64-bit)
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 532
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2099
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass948_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 311
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Util\UtilUI.cs:line 123
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1868
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 132
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 305
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Importing peak boundaries with Skyline Runner
(2 responses) estancliffe 2020-01-06

Hi Skyline Team,

I have begun using the command line version of Skyline to automate the processing of a large metabolomics dataset. With the user interface, I am able to import the peak boundaries to make sure the correct peaks are being integrated; however, with Skyline runner I am unable to do this. Right now, I am able to import my transition list and all of my datafiles and generate a skyline document with Skyline runner, but then I have to manually go in and open the skyline file and import the peak boundaries and then export the final report of the peak areas. If I was able to import peak boundaries through the command line, I would not need to open the interface at all. I don't know if this is a simple fix on your end, but it would help me out a lot!

Thank you!


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Peak picking and transitions
(1 response) aqassab 2020-01-06

I am currently working on a project designed to discover peptide bio-markers using SRM. I observe some peptides peptides with five well represented transitions, other peptides with less transitions. Based on the changes observed between samples, a few peptides show one transition better than other transitions selected in the method. I have a few questions related to this:

  1. What is the minimum number of transitions recommended to use in the data analysis?
  2. For peptides with maximum of three transitions selected, one or two transitions are not located under the main peak area (the area under the transition with the highest intensity), is it recommended to delete those "poor" transitions and use only two or maybe just one transition for some peptides?
  3. When transitions in actual samples (please see the figure attached) do not line up with the transitions found in the heavy "spiked" peptide, should I extend the boundaries to cover the area of all transition selected in the endogenous peptide in the sample, or should I place the boundaries based on the transitions observed for the heavy "spiked" transitions?



 peak picking issue.emf 
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Adding to my transition list (small molecules) does not show the new molecules
(1 response) mazzouny 2020-01-06

I hope these questions are not redundant. Whenever I add to my transition list, everything I add, is not getting analyzed. I need to re-insert a new list and import the RAW files from scratch in order to quantify a new compound in my small molecules list. Are there any re-analyze option ?
Also, when I export the transition list, it shows that there is no CE chosen (because I'm doing MS1 quant) and it export it as a generic format. When I import the same file again, I get an error. Any idea?
I'm using version 19.10.193, small molecules installation.

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Targeted Calibration Curve Value Import
(1 response) gsbyram 2020-01-06

Hey Everyone,

I have a targeted steroid assay which quantifies roughly 35 compounds. The compounds contained in the calibration curve have different concentrations based around their reference range. Is there a way to individually assign concentration values for each compound in a calibration standard instead of a blanket value for all compounds?

West Coast Metabolomics Center
UC Davis
Oliver Fiehn Laboratory

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CE optimization library different precursor and product z
(2 responses) Erik 2020-01-03

Hi All,

I more often measure peptides that have a predominant breakage point (mostly proline) and thus the most abundant ions are formed from the same ion but in two different charge-states. Can I store the CE value of multiple product ion charges from the same peptide ion fragment (y6+ and y6++ for instance) in the library?
Similarly, but less relevant can I do the same for a peptide sequence with different precursor charge?

I was not able to find this anywhere in the tutorial or support forum. Hopefully this is possible without changing the software too much. I suggest one or two additional columns with precursor z and product z. Or is it possible to provide the charge in the "product ion" column?


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Error while running MSstats and AvantGardeDIA external Tools
(4 responses) Chinmaya k 2020-01-02


I have installed both MSstats and AvantGardeDIA with all the R versions and libraries successfully. However, I am facing some issues while running both of these external tools right now. Such as;

MSstats - As you can see in the attached file. The MSstats function exports the results to a Temp directory with all the basic column information in it. But, the Fragment Ion column contains both fragment ions and precursors identified in the DIA search with respective "Area". According to MSstats, precursor information is not required for DIA analysis and how to exclude precursors from being exported by the MSstats as MSstats input file?

AvantGardeDIA - Whereas here in AvantGardeDIA, this tool is not able to change the working directory. I do not know what exactly is the reason here. I have also attached the screenshot of the current set working directory for AvantGardeDIA\Run_AvG_Module\GlobalRefinement below. Why this tool is not able to change the working directory and how can we resolve this issue?

Thank you,

 AvantGardeDIA_Error  AvantGardeDIA_Working_Directory.PNG  MSstats_Error.txt 
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Different peak height, area in Skyline and in Xcalibur
(3 responses) hilaire 2020-01-02

I am analyzing Q-Exactive HF PRM Data with Skyline.
In Skyline after importing of raw data, for compound G, the Skyline XIC is totally different from what I can see in Xcalibur.
For 78.9585, the height in Skyline is 880, while in Xcalibur it is 5.02e4.
For 492.9919, the height in Skyline is 75346, while in Xcalibur it is 1.98e4.
What is the cause of the error and how can I improve it??

I have attached the skyline file, transition setting and the XIC, MSMS snapshot from Xcalibur for your reference.

 Test.skyd  QExHF03_438 493 XIC.PNG  QExHF03_438 286 MSMS.PNG  QExHF03_438 79 XIC.PNG  QExHF03_438 Skyline XIC.PNG  Transition setting.PNG 
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xuezhangzhi 2020-01-02
Dear Skyline team
 I am trying to analyze hundreds of MRM dataset to use skyline, but I didn’t acquire the transitions of Decoy peptides in the MRM experiment in prior as suggested by the mprophet method. Can I still use the mProphet model for peak picking, or could you provide me some suggestions?Now, I just following the tutorial for manual evaluation.
I am wondering if can I automate pick manual peak and avoid of the bias (such as ( how good RT, ppm accuracy etc.)) by any statistical model? Do you have any suggestion to automate manual peak picking with statistical model for MRM dataset using Skyline?

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Poor System Utilization by Skyline
(15 responses) Chinmaya k 2019-11-27

Hello Skyline Team,

I am trying to perform DIA analysis with 2 DIA raw files against a quite large spectral library having 581197 precursors corresponding to 477997 peptides using Skyline 19.1 in our Windows workstation (Please find the attachment for details).

However, I find Skyline hardly utilizing CPU and RAM for any small changes with occupying all the available disk space (Currently the disk containing this skyline document has 1.8 TB space).

It would we great if there is an option in the skyline to set the number of processors/cores to be used, I have not seen any.

Thank You,

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mass tolerance
(2 responses) zhengemma 2017-03-29
Can the method match tolerance be set above 0.6 under the Transition settings? Is there a way to do?

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Analyzing timsTOF data with FragPipe/MSFragger search results
(9 responses) nesvi 2019-12-13

Dear Skyline team,

I am trying to extract MS1 quantification from timsDIA PASEF data. I have 4 raw files (.d folders) that were processed using FragPipe/MSFragger/Philosopher. Which, by the way, works great for these data.

I am not able to build a spectral library using the DDA-MS1 quant workflow, with Import - Peptide results etc for building the list form the search results. Neither using MSFragger pepXML files nor using interact-.pep.xml files after PeptideProphet work. When using interact-.pep.xml files, I get an error that it cannot find spectral files. When using pepXML, it finds the files to get the spectra (from file_calibrated.mgf files produced by MSFragger) but gives an error at a later stage.

However, I am able to trick Skyline into extracting MS1 quant from DDA files by going through the DIA workflow. When going that route, I am able to select pepXML files, Skyline finds the corresponding spectral files, and it can build the library. I can also select .d DDA files and perform quantification. However, with this trick, Skyline tries to quantify both precursor and y/b ions (because it is a DIA workflow), which takes a lot of time. I just need precursor quantification from MS1, but removing b,y ions from the list of ions to quantify gives me an error.
Also not that using interact-*.pep.xml files (instead of pepXML from MSFragger) still does not work when using the DIA workflow.

So, ideally, I would like to being able to

  1. Load PeptideProphet pep.xml files, and not just the MSFragger pepXML files.
  2. Being able to use DDA-MS1 quant route rather than DIA route when working with DDA data.

Admittedly, I am an inexperienced Skyline user. So I may be doing something wrong. However, Brett Phinney seemed to confirm the errors I am getting with DDA files.

I could get someone in my team to work directly with the Skyline team to resolve these issues, or to learn how to do this analysis properly.

Thank you,
Alexey Nesvizhskii
University of Michigan

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