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.  

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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.

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Showing: limited to 100 requests
extract chromatograms from tims TOF
(1 response) tleonova 2021-06-18
Dear Skyline team support,

I am quite new to Skyline and I try to use it for MS1 filtering and spectra library building.
I have data acquired on Bruker tims TOF. I used MGF files created by Bruker software for the search in Proteome Discoverer 2.2.

I want to import peptide search from Proteome Discoverer (.msf). But I got stuck at the Extract Chromatograms window. The MGF files were missing.

I found that you already answered to the similar request. I realised that I have to add RAW files for chromatogram extraction. In my case RAW files are .d folders. But when I try to locate these files by clicking on the Find button, navigating to the RAW file location, and clicking these .d folders, nothing happens.

Am I doing something wrong?

Thanks in advance for your help!

Kind regards,
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Skyline failure_metabolomics_methods containing high number of transitions
anna illiano 2021-06-18

Good afternoon.
I have a couple of problems in using Skyline for metabolomics.

  1. The software does not allow me to simultaneously monitor all the transitions present in the method (more than 400). I need to create multiple methods to analyze a single sample.
    When I try to upload a sample by inserting all the target molecules in the skyline, I get this error message:
    "Failed importing results file
    An item with the same key has already been added. "
  2. Even if I prepare methods containing fewer transitions, for some molecules Skyline does not allow me to evaluate the TICs even if by using the Analyst Software (Sciex) I am able to see and integrate that transitions and TICs.

Can you help me?


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Comparing Peak Scoring / Sample Specific Peak Scoring Models
(1 response) peter r mosen 2021-06-17

Hello Skyline team, hello Skyline community,

Is there any tutorial etc. which deals with Skylines “Comparing Peak Scoring“ function? I would like to understand the plots (e.g. the different y-axis options) as well as its application better. The tutorials 14, 15 and 18 cover peak model building in general but I didn’t find anything on the “Comparing Peak Scoring” option.

Briefly about my situation: In my targeted MRM assay I am monitoring peptides across multiple organs (9x) and cell lines (5x). For data analysis I am using mProphet peak scoring models. I was now wondering if it is best to use for each organ type its own peak scoring model, or if a mixed-organ respectively mixed cell line peak scoring model can be used for the individual organs. Anyone with any experience in that? Any suggestions, any hints ?

Best, Peter

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More proteins shown than library in SWATH analysis
(8 responses) heyang 2021-06-16

Hi Skyline team,

I just started to learn using Skyline to process SWATH data acquired by Sciex 6600. Library was created by multiple DDA runs searched by ProteinPilot software. Following your instruction, input FASTA files and spectra library. More pep/Proteins than number in library are shown in Skyline. How to explain this?


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Crosslinking Quantification
(14 responses) swatibanerjee060 2021-06-04
I am using Skyline for quantifying the cross-link and in many cases the precursor mass is not matching with raw file's precursor mass, though I am putting right modification. What could be the reason?
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Skyline command runner fails to write the exp file.
(1 response) shridevi patil 2021-06-17

Hello ,

We are working on tool which automates the manual process of creating MRM method using skyline and MassLynx.
we are using Water's Xevo TQ S instrument.
test was to find the min dwell supported to create write MRM methods.
Attaching the file where command passed to Skyline is shown and output of command runner also been provided.
Does Command runner splits the method into multiple experiment files?
Kindly suggest if anything is wrong with using command runner.

Thank you

 Refer_Step3 error.txt 
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Skyline runs quite slow in extracting chromatograms from DIA raw files
(11 responses) ref p 2021-06-14

Hello Skyline team,

Recently, I have been performing a real whole-proteome DIA analysis of which the raw data was acquired on a QE-HF-X MS instrument both in DDA and DIA mode a week ago. According to your provided DIA tutorial, I started doing this by using Import Peptide Search wizard under "Import Results" "One at a time". At the very beginning, because of the very large dataset (35*60min run, a total of ~25 GB), I think, Skyline v21.1 presents a very low performance in extracting chromatograms for 9795 proteins including 6 transitions per peptide with automatical mProphet model training at the same time.

At present, after nearly 20 hours raw data importing, this step was still not completed yet, which clearly makes me astonished very!

My questions therefore come across in this way, they are:

  • How to make Skyline work faster?
  • Is Skyline-daily more helpful? In Skyline support forum, I learned that someone got good results after being changed form Skyline 20.2 to Skyline-daily. []
  • Is there an optimal workflow available? As an alternative approach, I think, I will try to import the raw data by Import Result one by one using default peak picking algorithm. Subsequently, I will train an mProphet peak picking model using the imported DIA dataset and apply it for the same dataset. Finally, if I am a lucky man, I would get an expected quantitative results which then will be used in R analysis. How about this idea?

Guihua Jia

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Can we do LFQ analysis in Skyline?
(3 responses) shelkarmicrobio23591 2021-06-14

Dear Skyline,
Can we do LFQ analysis in Skyline?

Please suggest workflow.

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no files detected during import of library from fragpipe
(1 response) 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.

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

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Creation of spectral library using DIA without DDA
(1 response) mjgou 2021-06-15
Dear Skyline Team,

In our lab, we are currently using a 6560 Ion-Mobility qTOF mass spectrometer from Agilent for untargeted proteomics studies and Spectrum Mill is used to generate peptides/proteins identification lists. We are particularly interested in using Skyline to create and manage specific spectral libraries for our clinical applications. However, our workflow does not include the prior acquisitions in DDA to build the library due to the limited amount of samples. We would like to directly use the DIA acquisition files to create this library. Do you think this workflow could be compatible with Skyline?

Thanks in advance,

Best regards,

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about Edit Report
(3 responses) minyg98 2021-06-14

Hi, I want to know about a few options in Edit Report.

What is 'Peptide Peak Found Ratio' and how 'RatioLightToHeavy' is calculated?


Minyeong Ji.

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Retention time decimal place
(2 responses) muluneh 2021-06-14

Is there a way to set up retention time decimal places? I use a windows computer and it displays only one decimal place. 14.3. Can I set up Skyline to display 14.32 or something like that?
I use RT +/- 0.02 min between the analyte and heavy labeled standard, and it's making the comparison a bit difficult. I know the skyline displays two decimal places when I export the report in Excel.


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Peak Integration
(2 responses) muluneh 2021-06-10
Hi, I have been using Skyline for the last 8 months and I really liked the way it works. I am analyzing MRM data generated with the AB SCIEX instrument. Some of the proteins I work with are less abundant and have a high background. So I need to manually integrate the peaks. We use two fragment ions for each peptide we collect MS data for. Skyline displays the two peaks simultaneously and when I try to edit a peak (Peak B in the Figure) for one of the fragments by moving the vertical line, all the peaks (Peak A) are being affected. Are there any means that I can process the two fragments separately/independently? For now, I am manually deselecting the peak I don't edit, then export the data, and then process the second peak. I am working with several proteins, and this app[roach is taking too much time. Just looking for an efficient approach... Thanks! Muluneh
 Peak Intergation.pptx 
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Error while installing LipidCreator via Skyline Tools
(1 response) komal kedia15918 2021-06-14

Skyline-daily (64-bit)

Hi Skyline Team,

While attempting to install LipidCreator from Skyline Tool Store, we keep receiving this error. Any familiarity with this error? Any help to resolve this error would be greatly appreciated. Thanks!

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Unable to see isotopes in blue while checking Spectral library BLIB
(2 responses) orynbek azhar 2021-06-10

Hi, I followed the guideline of working on the spectral library. However, it is not showing me isotope labels.
Attached screenshots

 v1.png  v2.png 
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Request: Visualize reporter ions in "Library Match" window
(2 responses) Juan C. Rojas E. 2021-06-11


I was wondering if it is possible to visualize modification specific reporter ions in the library match or if it is a feature that could be implemented.

I have managed to show some modification specific neutral losses (Slide 1; e.g. y7-331), but there are several other ions that I can annotate to derivatization tag specific fragments that I regularly check and would be really nice if this were also highlighted for my validation steps and consideration for fragment transitions to track.

As always, thanks for the awesome work and support.

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Specifying height and width of chromatograms
(1 response) jaltman 2021-06-11

My lab is new to Skyline (and peptide MS), which we're using to analyze VERY simple peptide mixtures eluted from engineered MHC molecules as part of our reagent quality control.

I've asked members of my lab to capture chromatograms in a database as in the attached file. For database display, it would be helpful if all of the chromatograms had the exact same height and width (say 4" wide by 0.75" tall). I've tried to find this in Skyline and on the Support pages, but I haven't been able to find how to do it (assuming it's possible).

Help with this would be much appreciated.

John Altman
Emory University

 Skykine to Filemaker.png 
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small molecule sMRM - chromatogram information unavailable
(4 responses) paul leonhardt 2021-06-09

Hi Skyline team,

i currently use skyline as open-source tool to analyse data that has been acquired on a Sciex 5500 qqq instrument. My research focuses on nucleotides and their modifications, that's why i often need to detect the same [M+H]+ mass during different retention times. Currently I only want to get a quick overview of the data, so i need the chromatogram, peak area and retention times. The problem i have is that skyline most of the time only recognizes one of my isobaric sMRM transitions.

I have acquired my data as full Range MRM scan and also as scheduled MRM scan. When creating the transition list for the full range MRM data i did not have so many problems - apart from adjusting the integration manually. But now when i want to use scheduled MRM data most of the time skyline does not find the corresponding chromatogram. Is there a way to assign this manually?

Attached you find a file for demonstration which includes 4 results, two of them are full range and two scheduled - you will see that some transitions are only detected within the full range files even though the peaks are present.

I'd really appreciate if you take a look into it

Kind regards,

 Nucleosides MRM skyline chromatogram not 
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An error occurred attempting to export. the mobility range 0.775548 - 0.829034 is not within the limits 0.780000 - 1.600000
(2 responses) akshada gajbhiye 2021-06-10
Hi Skyline team,

I'm trying to export a prmsqlite method for tims tof, however when I try to export it the error below appears. Kindly help to resolve this issue.


An error occurred attempting to export.
the mobility range 0.775548 - 0.829034 is not within the limits 0.780000 - 1.600000
OK More Info
System.Reflection.TargetInvocationException: the mobility range 0.775548 - 0.829034 is not within the limits 0.780000 - 1.600000 ---> System.Exception: the mobility range 0.775548 - 0.829034 is not within the limits 0.780000 - 1.600000
   at pwiz.CLI.Bruker.PrmScheduling.Scheduler.AddInputTarget(InputTarget inputTarget, String external_id, String description)
   at pwiz.Skyline.Model.BrukerTimsTofMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor, TimeSegmentList& timeSegments, SchedulingEntryList& schedulingEntries, Boolean getMetrics) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3281
   at pwiz.Skyline.Model.ExportProperties.<>c__DisplayClass131_0.<ExportBrukerTimsTofMethod>b__0(IProgressMonitor m) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 613
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2116
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peptide sequences - Goserelin, Ipamorelin, Desmopressin
(2 responses) heather eastwood 2021-06-10

This is my first time working with peptides, so please forgive my confusion here!
So, I am trying to get the Precursor Ions for the following 3 peptides listed below. I am unsure if I have my sequences wrong, but the values that Skyline is returning don't seem to be correct. I'm using a Agilent Ultivo QQQ. And when I analyze, I don't see those masses. Also, a quick literature search leads me to believe they are incorrect.

Goserelin: XHWSYXLRP. Precursor Ion: 590.808319

Ipamorelin: XHXFK. Precursor Ion: 327.183703

Desmopressin. CYFQNCPRG. Precursor Ion: 601.2475

Also attached, is the full Transition List that Skyline returned.

Any feedback will be greatly appreciated!


 Transition List 2021.06.02.xlsx 
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Skyline for peptide search using Waters MSe data
(3 responses) ywu1 2021-06-07

Hi there,

Curious to the ability of Skyline to perform a peptide search using MSe data generated on a Waters Synapt G2Si. Reading some of the older posts, it sounds like PLGS was the preferred route prior to Skyline, just curious if this was addressed since you have added so many features as of late. Additionally, If this is indeed possible, would skyline be able to read the ion mobility data as well?


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Linear fit in log space
(5 responses) schen19 2021-06-08

Dear Skyline team,

I have a question about the Linear in Log Space regression fit in Skyline. My initial understanding is that the equation of the calibration curve using this fit is Log(Y) = slope*Log(X) + Y-intercept (Y is RatioLightToHeavy, X is Analyte Concentration). However, when I tried to use this equation to calculate X from Y using this equation, the result does not agree with the Calculated Concentration, which is the quantification value given directly by Skyline.

I tried to do a similar regression fit (Log-transformed both X and Y, then do a linear regression fit) in another software and it gives the same slope but different intercept. When using the slope and intercept given by that software and the Log(Y) = slope*Log(X) + Y-intercept equation, I get the same quant value as the Calculated Concentration given by Skyline. Based on this observation, the problem seems to be related to the Y-intercept value given by Skyline.

I am trying to understand what equation is Skyline using for the Linear in Log Space regression fit. Could you provide some guidance on that?

Thank you,

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about ms/ms filtering
(7 responses) minyg98 2021-06-06

Dear Skyline team

Hi! This is Minyeong Ji from the Center for ProteoGenome Research in Korea University.

I'm using SureQuant Method but I want to customize it.

Let me explain about how I want to customize it first.

First, the SIL peptides are scanned with 7,500 resolution

Once the Quant Mode is triggered, the SIL peptides are scanned one more time but this time with 60,000 resolution.

I already gave it a try this way, and when I opened the raw file on Skyline, the profile looked like the image I attached.

So I want to know if I can give a filter with resolution so only scans with 60,000 resolution can be used for drawing profiles.

I tried to use the MS/MS filtering in Transition Settings but I don't think it filtered scans with 7,500 resolution.

Could you help me please?

Best regards, Minyeong Ji

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Regex filter tab
(5 responses) af1234 2021-06-07


I have been trying to remove peptides containing specific AA, but I am having some issues.
I imported a msms.txt file from MQ then imported all the dda raw files.
I then went to the refine>advance> select all precursors/transition/peptides and I went to the filter tab selected> filter C/M added [Q|B|W] and then autoselect all matching peptides, but there are still peptides with C/M and also containing either Q, B or W.
Am I missing something obvious?

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(5 responses) Linwei Li 2021-06-07

Hello Nick,
I tried search results using FragPipe and raw files. However, when I tried to import the search results in skyline, there is an error report showing that I need to have mzxml files instead for import.
I am not sure how to solve this. I think the reason is because I have run mzxml format files before and it saves the previous cached data. I am not sure if I need to remove previous cached data because they are from a separate skyline file. At the same time, I really need skyline to import raw files instead so that I can start a ion mobility library (if mzxml files are imported, ion mobility library will show no result.)
I have attached the error report below. Thank you so much!


 Screen Shot 2021-06-07 at 14.56.16.png 
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about ms/ms filtering
minyg98 2021-06-07

I asked about ms/ms filtering yesterday and I was asked to upload my skyline file here

so I uploaded my skyline document and a raw file.

Can you check on them please

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Error with method export for SCIEX QTOF after upgrade to Analyst TF 1.8.1
(2 responses) stephan kamrad 2021-06-03
I have been using Skyline very successfully for a while to generate .dam acquisition methods for our Sciex 6600.
We recently upgraded to Analyst TF 1.8.1 and now the method export crashes Analyst and produces the following error.
I am doing this on the instrument computer with the correct hardware profile activated and I am using .sky and template .dam files which have worked before.
Can you help?

Problem signature:
  Problem Event Name: BEX
  Application Name: Analyst.exe
  Application Version: 3.7323.0.2
  Application Timestamp: 5d69ae47
  Fault Module Name: MSVCR90.dll
  Fault Module Version: 9.0.30729.6161
  Fault Module Timestamp: 4dace5b9
  Exception Offset: 0006ccd5
  Exception Code: c0000417
  Exception Data: 00000000
  OS Version: 6.1.7601.
  Locale ID: 1033
  Additional Information 1: 0120
  Additional Information 2: 0120e0cf538c5533ae847dad19fdbdc5
  Additional Information 3: 9694
  Additional Information 4: 969458d6bc7742c30d364858d5a7bf8e
-System.IO.IOException: ERROR: Failure creating method file D:\Analyst Data\Projects\Stephan_June20\Acquisition Methods\20210603_K_gluC.dam. The remote procedure call failed. (Exception from HRESULT: 0x800706BE)

Command-line: Method\AbSciex\TOF\BuildAnalystFullScanMethod -s -m "D:\Analyst Data\Projects\Stephan_June20\Acquisition Methods\20min_Skiline_template_40acc.dam"
Working directory: D:\Analyst Data\Projects\Stephan_June20\Acquisition Methods
Standard input:
D:\Analyst Data\Projects\Stephan_June20\Acquisition Methods\~SKB61F.tmp
D:\Analyst Data\Projects\Stephan_June20\Acquisition Methods\20210603_K_gluC.dam
   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 65
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 4291
   at pwiz.Skyline.Model.AbiMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 2447
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1938
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2116
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optimization for small molecule
(11 responses) carlos penno 2021-06-03

Dear all,

I am exploring the software for small molecule work.

During the CE optmization I noticed the following issues

When I export transition list in csv value the precursor name is changed to something like Oxylipin.15-KETE.[M-]Ion [112_999451_112_999451][M-].CE_-17.0.light.

Another problem is that when I try to export a method the DP values are changing and therefore the data collect cannot be re imported in the software?

Why I am experience these and how can I solve it?

Thank you very much, Carlos.

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in silico trypsin digestion from a FASTA file
(2 responses) nicolas drouin 2021-05-27

Dear Skyline team,

I encounter a problem with the in silico digestion of my FASTA file with trypsin (KR/P).
For unknown reasons, the first generated peptide starts at residue 77 where the first K residue is. But there should be many more peptides with the R cleavage site before this peptide.

Another problem I see is the count of the residue. Indeed, this first peptide K.RFDNPVLPFNDGVYFASTEK. Indeed, according to the fast file, the residue R is located in position 78 but skyline indicated in position 77.

I'm running the last version of skyline-daily.

Please find enclosed the skyline file I'm using.

Many thanks in advance.
Best regards,

 Skyline digestion 
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Spectral Library Building – Option: Filter for document peptides Fails
(3 responses) peter r mosen 2021-06-02

Dear Skyline team,

for a MRM-assay I tried to generate a “reduced/tailored” spectral library covering only the peptides of interest using as input multiple, already existing *.blib-files (generated from DDA-data). To do so, I used the option: Filter for document peptides. The merged, resulting spectral library gets generated however it does not only contain the MS2 spectra corresponding to my peptides of interest. I.e. the “merged, reduced” spectral library (which got filtered for my 70 document peptides) comprised as many peptides as the original, full spectral library. No filtering takes place.

According to Kaipo’s post ( I understand the “filter for document peptides” option, that it will consider only the activated (ticked off peptides per protein) in the target window and it will ignore deactivated peptides, right ? I am using Windows 10, Skyline (alpha), however I had the same problem also with your latest Skyline daily version. Thank you for your help.
Best Peter

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Skyline-daily (64-bit) (6dbba07d9) cannot load the library.tsv file from EasyPQP
(1 response) fcyu 2021-06-02

The library.tsv file from EasyPQP is in OpenSWATH format. But when loading it in Skyline, it says "the file F:\dev\msfragger\test_skyline\library.tsv is not a supported spectral library file format.". According to, it should support OpenSWATH tsv file.

I put an example of library.tsv in the attachment. Could you please take a look when you have time?


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Having Issues with Chosen Peaks Being Removed
(4 responses) kaitlyn b donohoo 2021-06-01


I'm having an issue where those transitions that are shown to be orange will switch which trace is red and which is green if new peaks are chosen on the red trace. It removes the peaks from the now red trace. This is an issue I've never encountered before but is now happening across all my skyline files. I have included the first file to have this issue.

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Chromatogram information unavailable
(2 responses) m anna monika 2021-06-01


I am currently setting up a PRM method, but I am having some issues with one of my files. I have 42 peptides and set up a PRM run on a QExactive without full-scan or scheduling.
The first time the sample was loaded in to Skyline I was only able to detect peaks for 3 peptides. I was not sure if it is an instrument method setup issue, so I repeated the run this time only including 13 peptides in the inclusion list (with two detected in the previous run included), but this time I have no chromatograms for any of the peptides. Looking at the raw file there is no visible problem and the two runs look very similar. I also did a DDA run to see if I made a mistake with the setup, but it does not seem to be the case.

I must be missing something in the setup but I am not able to find where the problem is.

Please find attached the raw files as well as the Skyline document (Skyline version: 64-bit,

Thank you very much for the help in advance!


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MSfragger splib to blib?
(2 responses) dkueltz 2021-05-31


I wonder if there is a way to convert MSfragger generated splib to blib for use in skyline? I tried generating blib in skyline from MSfragger mzml, pepxml and calibrated.mgf output files but only get blibs that have proteins but 0 peptides/ precursors. and transitions - this seems similar to a previous thread report where the upgrade of Skyline 19 to 20 was reported to fail in including precursor spectra in the speclib. ANy suggestins would be appreciated.


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File Not Reopening After Saving and Exiting the Program
(13 responses) kaitlyn b donohoo 2021-05-28


I'm having an issue where my file does not open once I have saved and closed out of the program. It keeps popping up with a window stating "The file contains an error at line 114 column 9". I have tried remaking the file with the same transition list only to see the problem occur a second time. I will be uploading both the file and the transition list (in excel format) under the file name KBD_RiboB. I believe the issue must be in the transition list but I used the same format for other samples (not the exact same list but the molecule name, adduct names, and adducts are the same as in other lists) and had no issue. 
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how to perform unspecific enzyme digestion
(1 response) hui-song pak 2021-05-28

Dear Skyline Team,
I would like to use Skyline to analyze immunopeptidomics DDA, DIA and directDIA (DIA-Umpire) data. I'm using the wizard to do it but I'm stuck at the creation and importation of fastA file. We usually use "unspecific enzyme digestion" for normal Discovery approach and I don't know how to do it especially for directDIA (DIA-Umpire).
Your help to overcome this would be great.

Thanks in advance for your help


Hui Song

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Skyline export to Chromeleon
(4 responses) Andrew 2021-05-12


I was wondering if you could help me. I would like to export out my PRM transition list from Skyline and import directly into the processing method of Chromeleon. I was wondering if this is possible. I have tried all formats and nothing seems to be direct? If it is not possible is this something you are looking into?



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Export method to MassLynx 4.2 version
(8 responses) danielacgranato 2018-11-27


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

Thank you very much. Best, Daniela

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impossible to read wiff2 files
(1 response) nicolas drouin 2021-05-27

Dear Skyline team,

i remembered a newsletter indicating the wiff2 from Sciex can now be read with skyline.
However, I'am encountering difficulties to do so. Only solution i found is to convert the wiff2 in mzml using MS convert, but for some reasons, i have to convert the data one by one for the conversion fails (not all the chromatogram is converted in batch mode :( )
I have proteomic data from a X500r QTOF running SciexOS (version

Please find enclosed one of my file containing full scan data of a BSA digest.

Many thanks in advance for your help.

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comparison between groups with heavy peptides spiked
(2 responses) io 2021-05-06

Dear all,
I have samples of 5 different conditions
I have spiked heavy peptides into different amounts in each group of samples (to have a ratio light/heavy of 1 approx) but not a calibration curve.
What is the best results table to do the quantification and to see the significant differences?
Thank you in advance

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Can a differential protein be validated by PRM measurement of a single peptide?
(1 response) alima 2021-05-05

Dear all,
I hope you are doing well.
We performed a discovery proteomics experiments comparing a WT and a KO bacterium proteome. We found very interesting differential proteins and we wanted to validate some of them by PRM. We chose some proteotypic peptides using Skyling but when we run targeted experiment we found that for a protein, just one peptide was detected in the run. We wonder if we can validate the difference in abundance of a proteins just the measurement of one peptide. I wanted to mention that the biological sample is very difficutl to obtain and we have not more material.

I would really appreciate your comments

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eliminating contaminated fragment ions in DIA processing
(2 responses) Tomas Vaisar 2021-05-25

Skyline team,
I wonder if there is a way to filter out contaminated transitions/fragment ions (on a large scale). I have run into many instances in processing DIA data that out of 6 top fragment ions some are clearly contaminated and often they are one of the higher ranked ones. Looking at mass accuracy they are clearly off (mass error >10ppm) while the rest of the fragment ions are spot on (<5ppm). Ranking clearly does not remove these. Sometimes they are just slightly shifted in retention or show a shoulder on the peak. I attach an example.


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Unable to build spectral library and import results
(1 response) S.K. 2021-05-25

I am unable to build a spectral library or import results during my proteomics analysis. I had followed all the instructions from the tutorials as well. An error message shows up whenever I try to perform either of these tasks. Could you please guide me so that I can resolve these issues? I have attached a screenshot the two error messages that I encountered.

Thank you very much!

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Importing daughter scan data from a Xevo TQ-S
(6 responses) matthew daly-3 2021-05-11

Hello. I was hoping there was a way of importing daughter scan data acquired on a Xevo TQ-S? My precursors are selected for but then all product ions are monitored. I notice there was a similar question posted in 2017 but I wasn't sure if it had been resolved. Many thanks.

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Missing Graph Legend
(1 response) alaine garrett 2021-05-24

Hi All,

How do I get the legend for the peak area graph to reappear?

Thank you!

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MSP Spectral Library import for small molecules failed
(12 responses) stolltho 2020-05-17
Hi there.

Wanted to import .msp spectral libraries from:

.msp import works for HMDB, MoNA and ReSpect but NOT for GNPS, Fiehn HILIC and LipidBlast (throws an error message, see example attached) from

Spectral library import from
does not work at all, i.e. shows zero molecules after import


 lipidblast error.png 
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Peptide integrated peak area ranking filtering
(1 response) boycea 2021-05-21


We were wondering if there was a way to rank and filter out peptides within proteins based on integrated peak area among all the replicates, as opposed to based on library? I currently have a document with 160 proteins and 2,600+ peptides, with some proteins with 20+ peptides, which is more than what we need (10 being more ideal). We want to streamline the number of peptides to manually go through, and we want to keep all the peptides with the best integrated peak areas among all the replicates before we go through them. I'm assuming when refining the max peptide peak rank is based on the library ranking, and not total replicate integrated peak area. Thanks!


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Spectral library explorer - question/suggestion about search functionality for small molecules
(2 responses) Emmanuel 2021-05-20
Dear Skyline support team,

I've a question/suggestion for the spectral library explorer when searching compounds through the "Molecule" text entry.

Is it possible to use regular expressions in the search to find compound names with similarities?

In a next release, would it be possible to search also the other fields like, formula, Inchi key, precursor m/z, etc and use a kind of advanced search?

Maybe these features exist already and I haven't seen them so feel free to blame me for not watching/reading all your nice tutorials and documentation :-)

Thanks again for your support.


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about pq500 analysis template
(2 responses) minyg98 2021-05-20

Hello I'm minyeong Ji from center for proteogenomic research in Korea University.

I should first thank you for all your service since Skyline has been very helpful for my research.

The reason why I'm writing this is because I want to get a little help from you.

I'm currently interested in doing experiments with SureQuant - PQ500 method

They say there's the PQ500 Skyline analysis template provided but I don't know where to find it.

Also, should I use Skyline-daily for the analysis of surequant survey run?

I look forward hearing from you soon


Minyeong JI.

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How to define your quantifier fragment ion
(1 response) Orla Coleman 2021-05-20

Dear Skyline team,

I am currently measuring 3 transitions per peptide of interest in my MRM on a LC-TQ. Firstly, is the community preference to use all transitions or the most intense (peak rank =1) transition for quantitative proteomics? Secondly, if the answer is to use the peak rank=1 transitions I am finding that different samples from the same experiment and MS run have different peak rank=1 fragment ions for a given peptide, as I don't want to compare different transitions as this is surely unfair can I define which transition is the quantifier ion to be used for all calculations? And/or define 2 or more other transitions as just qualifier ions for helping peak picking etc.


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Library matching
(2 responses) atable 2021-05-20


I was wondering if it is possible in Skyline, to match a peak spectra to that of a library reference spectra?

Kind Regards,

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DIA-NN .speclib support
(28 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.

With best regards,

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Invalid cvParam accession 200000854
(4 responses) anita liu 2021-05-18


I was trying to create a new Skyline template/file but encountered issues when I was trying to import a "Peptide search" and build a new spectral library. Upon importing the .mzid file and selecting "next", the following message pops up: "ERROR: Invalid cvParam accession "200000854", "ERROR: reading file xxxx.mzid". I had used Protein Metrics Byonic (v4.0.12) software to generate the mzid file, with modifications "Label: 13C(8)15N(2) and 13C(6)15N(2)". I have seen similar cvParam accession number issues in the past and I think it is linked to modifications listed in Protein Metrics when generating the mzid file, but I am not sure why this is the case.

Could you please advise as to what this invalid cvParam accession code refers to, and how to resolve the mzid importing issue?

Thank you!

 20210518 Skyline Error with cvparam accession 200000854.png 
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export to pdf
(1 response) dwebb 2021-05-19


I was wondering if there was a way to export all the chromatograms (for each analyte and sample) to PDF similar to how Sciex Analyst software can export chromatograms. We need PDF copies for reports and internal audits. Unfortunately the Panorama platform won't be allowed for our purposes.


view request
difference between normalization methods in group comparison
(1 response) Linwei Li 2021-05-18

Hi Nick,
I was looking into the different calculation methods using group comparison and not sure which one should I choose to process my data. Among them, I am not sure about the total ion current and ratio to heavy method. The total ion current yielded no result for my data.
I looked into the tutorial and I only found the differences of normalization methods in MSstats, which is slightly different (quantile and relative to global methods). Do you know if there is more tutorial if possible? Thanks so much!


 Screen Shot 2021-05-18 at 13.03.55.png  pg7.png 
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differences in the volcano plot data generated from skyline
(8 responses) Linwei Li 2021-05-02

Hello! I used two ways to generate a volcano plot from skyline, and both of them produced data that are somehow different.
The first way I used is to us the group analysis in MSstats, which produced a viable result. However, despite my target of interest that is down-regulated, the rest of the data points look very flat.
The second way I used is try to extract the data of volcano plot out, however, they produced the same adjusted p value, and all my data points look flat including my target of interest.
I have attached the pdf using the first method (group analysis). The standard I used is "equalize to all medians."
Also, When I set my standard to "relative to global standard," group analysis from MSstats failed to generate one. I wonder what I can do to improve.
Thanks so much!


 data extract plot.png 
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L/H Area Ratio Mismatch
(2 responses) germolus 2021-05-17

Hi there.

I am working on the same data which I mentioned in this earlier post but now a bit further down the pipeline. I'm quantifying small derivatized metabolites, and using internal standard additions (13C6) to make ratios.

Skyline does this, as well as a cal curve and quantification, but I'm working with an external (MATLAB) pipeline to process the exports. Upon checking the peak area ratios that Skyline exported, and those which I calculate manually by using the ratio of light-to-heavy area, the two answers are slightly different. For example:

ReplicatemoleculeIsotope Label TypeAreaRatioLightToHeavy
1 ng/mlglutamic acid 1'light'80.4385
1 ng/mlglutamic acid 1'heavy'170.4385

8/17 = 0.4705

This probably wouldn't be a big deal if the peak areas were really large, but what I'd like to know is if there's something I missed. I used glutamic acid because, unlike some of the other molecules, I only have the precursors for light and heavy in the doc. As far as I can tell, there isn't another molecule that would be incorporated as in cases where quantifying peptides via multiple transitions.

When Skyline exports peak areas, are they not the same as the ones it used to calculate the light/heavy ratios? 
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Issue with Library creation using DIA-NN based 'report-lib.tsv' file (SPECLIB file type)
(10 responses) abhinav bioc1213 2021-05-13

Hi, I am not able to create a library using the DIA-NN generated library (report-lib.tsv' ). I get the attached error message, I have been trying this for many times now and still get the same message. The DIA-NN data shows about 4500 proteins IDs at 1% FDR.

Can you please suggest if I am doing it in wrong way?


 Lib creationn issue_13May2021.pptx 
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Waters SONAR data conversion for use in EncyclopeDIA
(6 responses) david schmidt 2021-05-11


I'm acquiring SONAR data using a Waters Xevo G2-XS. These don't have ion mobility information. They consist exclusively of alternating low- and high- collision energy scans (saved as independent files). Since I can't seem to get good information from waters: What is the recommended way to use these for building chromatogram libraries, for example in EncyclopeDIA?

Thank you very much,


view request
Does Skyline support auditing and User password restriction to lock down analytical methods ? Is also GLP / GCP ?
(1 response) capocherokee24500 2021-05-13

Hi All,

May you please advice on the following:

  1. Does Skyline support auditing and User password restriction to lock down analytical methods ?

  2. Is also GLP / GCP approved ?

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Does Skyline perform RT alignment of EI-GC-MS chromatograms spiked-in with deuterated internal standards
(3 responses) capocherokee24500 2021-05-11

Hi All,

I am interested in doing untargeted and targeted metabolomics using Ei-GC-MS Orbitraps and I would be keen to know if :

  1. Skyline does RT alignment of EI-GC-MS chromatograms spiked-in with deuterated internal standards and

  2. If skyline can be used to do the following:

    • Import in Skyline EI-GC-MS thermo files as .raw then
    • RT align the files then
    • Perform targeted feature extraction in Skyline then
    • Export the EI-GC-MS thermo files KEEPING THE ALIGNMENT ALREADY performed by skyline then
    • Import the RT aligned thermo files as .raw in compound discoverer (Thermo Scientific) and lastly
    • Perform untargeted feature extraction in Compound discoverer

I am using Compound Discoverer (Thermo) for untargeted analysis but the software does not provide a RT alignment tool that works well with EI-GC-MS data.




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Error library-free analysis
(2 responses) sstoychev 2021-05-13
Hi Skyline team, I get the following error when trying to do a library-free data extraction from SWATH runs. This happens at the 1st raw data conversion step, any clues to what might be causing this?:

OK More Info
System.IO.IOException: Error occurred running process.

Command-line: C:\Users\6600 PROCESS\AppData\Local\Apps\2.0\NNEX7B00.JX1\W572QYT8.HKO\skyl..tion_e4141a2a22107248_0015.0000_7c1413170fdadb9e\msconvert -v --32 -z --mz5 -o "C:\Users\6600 PROCESS\AppData\Local\Temp" --outfile tcof2ros.nkp.mz5 --filter "diaUmpire params=C:\Users\6600 PROCESS\AppData\Local\Temp\4rrusmmt.q4q.params" "D:\Analyst_data\Proteomics\2021_05_05 Non TDF Cohort\Data\Hela_QC\20210503 100ng Hela_1.wiff"
Working directory:
   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 65
   at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 139
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How does skyline handle the origin when performing calibration
(1 response) jrenders 2021-05-13

Hi there,
Pretty easy question I hope, but I couldn't find any documentation on this topic in previous requests:

I would like to understand how Skyline handles the origin when performing calibration of analytes. I noticed that in my calibration curves the regression line extends below my lowest accepted calibrator (and I am not accustomed to seeing this in MS quantitative plots). This got me thinking that perhaps skyline considers the origin when performing these calibrations. Typically, the choices for how to handle the origin are "Include", "Force", or "Ignore" (I usually use "ignore" in small molecule quantitation) but I cannot see any indication of what is used or how to change this setting in the "Molecule Settings" --> "Quantification" window. Thanks for any info!

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Mismatch in PPP for experiment file viewed in Mass Lynx
(1 response) kartik kundgol 2021-05-13

Hello Support Team,

We are using SkylineRunner.exe to create Experiment files and form which we create Sample list which is summited to the Mass Lynx as batch.
We use Xevo TQ S instrument with SCN 1001.
Now when Method editor in MassLynx opens the experiment files, PPP(point per peak) value is less than expected and this results in less number of methods to be created.
Does SkylineRunner.exe does any optimization for give PPP value ??

Please suggest.

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Collision Energy Optimisation on Agilent Ultivo QQQ
(4 responses) a grayson 2021-04-12


I have been using an Agilent Ultivo triple quadrupole instrument to perform MRM analysis of peptides. I used Skyline to create a method which optimises the collision energy of 5 transitions for each peptide precursor. In Settings - Transition Settings, I selected Agilent QQQ from the dropdown menu for collision energy, which made a transition list with collision energy step size 3V and step count 3.

Upon running this method on the Ultivo and importing the data into Skyline, some transitions only show 5 or 6 out of 7 collision energy results, and the Retention Time and Peak Area - Replicate Comparisson graphs will only show a select few of these results. For example, in the screenshot attached, the transition selected shows data for 6 different collision energies was recorded (not 7, why?) and the graphs for Retention Time and Peak Area on the bottom only plot four of these.

I have double checked the method I ran and confirmed all seven collision energies were meant to be analysed for all transitions. Could you please advise why I cannot see all 7 collision energies for some of the transitions, but can for others? There does not seem to be a pattern in which transitions it did not record all data for.

Kind Regards,

 Skyline - CE optimisation troubleshooting.tiff 
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Running peptide search with MsAmanda from commandline
(2 responses) fcsigloch 2021-05-12

I am using Skyline form commandline (using SkylineCMD.exe). So far, I performed the peptide search with MSGF+. Now I would like to switch to MsAmanda. Can I run it directly from the Skyline executive or do I have to run the search separately?
I could not find an appropriate command in the CLI documentation.
Greetings, Florian

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Skyline not compatible with Shimadzu LCMS-8060
(8 responses) William 2021-04-19

I am porting the method to our new purchased Shimadzu LCMS-8060. I exported transition list from Skyline (choose Shimadzu QQQ) to txt file. However, when importing compound list to Shimadzu, it is completely chaos. I then check the title of each rows and find they don't match. Here I attach the compound export file from Shimadzu QQQ. Pls update Skyline so that exported file can be recognised by Shimadzu.

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Error with MSstats_Bruderer.R script
(1 response) dayne0208 2021-05-12

I am attending 2021 May Institute proteomics workshop. I am following Ali skyline batch tutorial and received an error message during the MSstats_Bruderer.R script phase. What did I do wrong? I've also attached a full copy of the log and MSstats Input-plus.csv files.

[5/11/2021 10:21:57 PM] Saving MSstats Input-plus.csv
[5/11/2021 10:22:22 PM] "C:\Users\dayne\Documents\May2021\MacLean_L3\NEU21_Skyline\scripts\MSstats_Bruderer.R" "C:\Users\dayne\Documents\May2021\MacLean_L3\NEU21_Skyline\Bruderer\Bruderer_ppm15c_rt10\MSstats Input-plus.csv"
[5/11/2021 10:22:28 PM] Reading MSstats Input-plus.csv
[5/11/2021 10:22:28 PM]
[5/11/2021 10:22:28 PM] Attaching package: 'dplyr'
[5/11/2021 10:22:28 PM]
[5/11/2021 10:22:28 PM] The following objects are masked from 'package:stats':
[5/11/2021 10:22:28 PM]
[5/11/2021 10:22:28 PM] filter, lag
[5/11/2021 10:22:28 PM]
[5/11/2021 10:22:28 PM] The following objects are masked from 'package:base':
[5/11/2021 10:22:28 PM]
[5/11/2021 10:22:28 PM] intersect, setdiff, setequal, union
[5/11/2021 10:22:28 PM]
[5/11/2021 10:23:02 PM] Finished reading
[5/11/2021 10:23:03 PM] [1] ProteinName PeptideSequence PeptideModifiedSequence
[5/11/2021 10:23:03 PM] [4] PrecursorCharge PrecursorMz FragmentIon
[5/11/2021 10:23:03 PM] [7] ProductCharge ProductMz IsotopeLabelType
[5/11/2021 10:23:03 PM] [10] Condition BioReplicate FileName
[5/11/2021 10:23:03 PM] [13] Area StandardType Truncated
[5/11/2021 10:23:03 PM] [16] DetectionQValue
[5/11/2021 10:23:03 PM] <0 rows> (or 0-length row.names)
[5/11/2021 10:23:03 PM] Converting to MSstats format
[5/11/2021 10:23:14 PM] ERROR: Error in SkylinetoMSstatsFormat(raw, filter_with_Qvalue = TRUE, qvalue_cutoff = 0.01, :
[5/11/2021 10:23:14 PM] ** Please check annotation. Each MS run can't have multiple conditions or BioReplicates.
[5/11/2021 10:23:14 PM] Execution halted
[5/11/2021 10:23:15 PM] ------------------------------ Bruderer: Error -------------------------------
[5/11/2021 10:23:15 PM] ------------------------------ Runtime: 16:31:03 -------------------------------

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ERROR: Error in SkylinetoMSstatsFormat
(5 responses) dayne0208 2021-05-07

I am attending 2021 May Institute proteomics workshop. I am following Ali skyline batch tutorial and received an error message at the end of log. What did I do wrong?

[5/7/2021 8:26:59 PM]    97% - Writing row 4,075,679/4,208,580
[5/7/2021 8:27:01 PM]    98% - Writing row 4,119,980/4,208,580
[5/7/2021 8:27:04 PM]    99% - Writing row 4,164,281/4,208,580
[5/7/2021 8:27:06 PM]    100%
[5/7/2021 8:27:06 PM]    Report MSstats Input-plus exported successfully to C:\NEU21_Skyline\Bruderer\Bruderer_ppm15c_rt10\MSstats Input-plus.csv.
[5/7/2021 8:27:07 PM]    Converting MSstats Input-plus to invariant format...
[5/7/2021 8:27:07 PM]    Reading MSstats Input-plus.csv
[5/7/2021 8:27:10 PM]    Updating MSstats Input-plus.csv
[5/7/2021 8:27:12 PM]    Saving MSstats Input-plus.csv
[5/7/2021 8:27:14 PM]    "C:\NEU21_Skyline\scripts\MSstats_Bruderer.R" "C:\NEU21_Skyline\Bruderer\Bruderer_ppm15c_rt10\MSstats Input-plus.csv"
[5/7/2021 8:27:18 PM]    
[5/7/2021 8:27:18 PM]    Reading  MSstats Input-plus.csv 
[5/7/2021 8:27:18 PM]    Attaching package: 'dplyr'
[5/7/2021 8:27:18 PM]    
[5/7/2021 8:27:18 PM]    The following objects are masked from 'package:stats':
[5/7/2021 8:27:18 PM]    
[5/7/2021 8:27:18 PM]        filter, lag
[5/7/2021 8:27:18 PM]    
[5/7/2021 8:27:18 PM]    The following objects are masked from 'package:base':
[5/7/2021 8:27:18 PM]    
[5/7/2021 8:27:18 PM]        intersect, setdiff, setequal, union
[5/7/2021 8:27:18 PM]    
[5/7/2021 8:27:55 PM]    Finished reading  
[5/7/2021 8:27:55 PM]     [1] ProteinName             PeptideSequence         PeptideModifiedSequence
[5/7/2021 8:27:55 PM]     [4] PrecursorCharge         PrecursorMz             FragmentIon            
[5/7/2021 8:27:55 PM]     [7] ProductCharge           ProductMz               IsotopeLabelType       
[5/7/2021 8:27:55 PM]    [10] Condition               BioReplicate            FileName               
[5/7/2021 8:27:55 PM]    [13] Area                    StandardType            Truncated              
[5/7/2021 8:27:55 PM]    [16] DetectionQValue        
[5/7/2021 8:27:55 PM]    <0 rows> (or 0-length row.names)
[5/7/2021 8:27:55 PM]    Converting to MSstats format 
[5/7/2021 8:28:07 PM]    ERROR: Error in SkylinetoMSstatsFormat(raw, filter_with_Qvalue = TRUE, qvalue_cutoff = 0.01,  : 
[5/7/2021 8:28:07 PM]      ** Please check annotation. Each MS run can't have multiple conditions or BioReplicates.
[5/7/2021 8:28:07 PM]    Execution halted
[5/7/2021 8:28:09 PM]    ------------------------------ Bruderer: Error -------------------------------
[5/7/2021 8:28:09 PM]    ------------------------------ Runtime: 01:06:47 -------------------------------

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Kalibrierkurve für die Linearität erstellen
(1 response) alexandria balac 2021-05-11

Hallo Nick,

ich wollte im Skyline eine Kalibrierkurve für die Linearität erstellen.
Könnten Sie mir bitte sagen, wie das geht.
Ich habe drei Standards und wollte die Kalibrierkurve erstellen.
Anbei schicke ich Ihnen einen Printscreen von meiner Auswertung.

Vielen Dank für Ihre Unterstützung

Alexandria Balac
LC-MS Service

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DDA to DIA (MSE) Peak Peaking Errors
(6 responses) Juan C. Rojas E. 2021-05-06

Hi All,

I'm following this general workflow from data generated from a Synapt G2-Si using multiple DDA and MSE methods:

  1. Measure same sample in DDA and DIA sequentially
  2. Create Skyline project with IDs from DDA results
  3. Filter bad IDs and correct any integration errors
  4. Import DIA results into same document (after changing appropriate transition settings) using RT allignment (2 min)

What I have observed in multiple datasets is that in many instances Sykline is defining the peak boundaries that fit a good dotp with respect to the spectral library but ignores the fit of the peak boundaries to the precursor ions. This happens when there are closely eluting peptides with similar sequence missing or having extra one or two amino acids (using semi-specific digest results) where the peptide happens to have higher total area for the fragments AUC but there is no detectable signal for the peptide in the precursor scan (low energy scan; Slide 1). The result is in inflated coefficients of variation just because of bad, automatic peak picking (Slide 2, Fig. 1) that can be resolved (Slide 2, Fig. 2) if I check for all peptides in the document.

Unfortunately, I don't work with SWATH-like data, but I would assume this is not an issue that that approach would have (or at least less frequently) since the fragments would not be binned with the m/z range of the "interferring" peptide. Is this an issue specific to MSE or is there something I am overlooking in the data import? If indeed this is an all-ion-fragmentation issue, is there any solution that could be implemented?

I have prepared two sample set examples that can be found in our U of Leipzig Panorama space under seMSE called:

Raw import:

Curated results:
"seMSE_EColi_MPDS..." (work in progress)

I can think of some "tricks" to avoid these issues using the IMS information collected (for the files containing IMS data), but I would like to explore the only m/z dimension solutions that can be applied on the initial data import without having to train an mProphet model (this is pending to test on this data to see if it would help).

As always, thanks for the time and help.
Juan C.

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Normalization method ratio to heavy
(2 responses) io 2021-05-10

Hi all,
A very short question. In peptide settings Normalization method I only have the option equalize medians but I want to choose the ratio to heavy option.
Where can I find this?
Thank you!

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Error message when trying to copy and paste phospho peptides from one file into another file
(7 responses) rschoenh 2018-11-08


we're sometimes having trouble copying and pasting phospho peptides from one Skyline file into another Skyline file. Skyline basically doesn't let us do the pasting, but instead we get an error message saying "The modification Phospho (ST) already exists with a different definition." As far as we can tell, we have the same definitions in both Skyline files. Thanks very much in advance for your help!


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Skyline Batch Download
(5 responses) heyang 2021-05-04

I am trying to download Skyline Batch and keep getting the following error message that is attached. Has anyone encountered this problem?

 skyline error.PNG 
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DIA-NN Support
Shawn Rice 2021-05-05

What files do you need for DIA-NN to support DIA-NN results? I can supply some files.


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Skyline is slow on my computer
(2 responses) cmwats2 2021-05-03

Below are my computer specifications. I am only doing targeted peptide methods. I noticed a few months ago that my imports were taking a long time and telling me that the import is taking longer than it should. I figured I was pushing my computer too hard and the data files uploaded so I didn't address it. Now I am trying to save a skyline file with 950 data files and 750 transitions. Skyline saves the file and then says it is "optimizing data file..." This always take a long time so I let is optimize overnight and it had not finished by this morning. I end up canceling the process so I can use skyline. There is quite a response delay when I select a different replicate. The data files and skyline files are saved on the C: drive in My Documents. I am uploading Thermo Raw files that are about 20MB each (scheduled MRM 15min method). I am using Skyline (64-bit) The long imports and optimizing data file happens in multiple saved skyline files that are created from one another. I have removed all the data and re-imported but that did not resolve any issues. I am sure I forgot to include something important.

Can someone tell me how to improve skyline performance on my computer? I've searched the supports pages but all related issues involve users doing things that seem more complicated than my situation.

Device name LAPTOP-P29U4S18
Processor Intel(R) Core(TM) i7-8565U CPU @ 1.80GHz 1.99 GHz
Installed RAM 16.0 GB (15.8 GB usable)
System type 64-bit operating system, x64-based processor

Thanks in advance,

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How To Delete Replicates?
(1 response) dperez2697 2021-05-05


this is my first post, I didn't manage to find an answer elsewhere, I'm not very familiar with the request platform for now. So my question is very simple, I do not know how to delete a replicate. In fact I can close the window where my replicate is by clicking on the cross on top right but it don't delete the replicate. It is possible to do it? I tried by searching in all the tools on the bar on the top (EDIT, REFINE, VIEW, TOOLS...), but I found anything.

Thank you in advance for your answer, have a nice day.

David Pérez

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"Missing" Transitions
(4 responses) germolus 2021-05-04

Hey there. This is my first time posting a question, and I've gotten bits and pieces of an answer from other posts, but here's the gist:

The lowdown

I'm working with metabolites (so, molecule settings), and with .RAW files from a Thermo Orbitrap Fusion Lumos. We're running it in targeted mode, so MS2s are only collected when the precursor shows up. We're quantifying based on MS1 ratios to heavy (13C6), and verifying the location and identity of the EIC based on MS2 products. The fragments in question generally seem to be in the files--I have attached a screenshot of MZmine, which shows the rt locations of several MS2 scans and the expected product ions for pantothenic acid.

Skyline, however, does not show these ions. This is not always the case. When a really concentrated standard is run (>100x my highest standard curve point; not included in the attached doc), Skyline picks up on some of the transitions; however, I would assume that since the peaks in a "normal" sample still can appear at e4-level intensity, I am suspicious that there is an over-restrictive baseline calculation algorithm hidden somewhere I just haven't found.


  • Transition list items denoted with a "1" are the focus. Those denoted "0" are those that do not have the 13C label, and cannot be quantified as a ratio.
  • I do still have an unresolved issue that may or may not matter. In Settings>Transition Settings>Filter>Fragment adducts, I cannot change the product adducts to [M+]. This doesn't seem to matter as the transitions themselves (right click on transtion, Modify...>Ion chemical formula) all show [M+] as they should. If I try to adjust the global setting; however, it reverts back to [M-] as soon as I close the window.
  • I have been reading through this post and this post. I tried a couple of things, including setting my m/z match tolerance really high (0.5). No dice.
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iRT peak pickinpicking wrong peak of some replicates)
(2 responses) boycea 2021-05-03

After setting up our iRT predicted calculator, chrom libraries, replicates uploaded, and reintegrating by the mProphey peak scoring model and 0.01 significant q values, we noticed that some peptides don't have the same, nor correct peaks picked automatically among all their replicates. On top of that, those incorrect peaks had higher ppm (>10), lower dotp (<0.7), and further from the predicted time than other peaks that seems like they should be automatically choosing (on predicted RT, dotp=0.99, ppm<1). Is there a filter or refinement in skyline that can automatically pick the peaks that seem more likely based on certain dotp, ppm, and RT thresholds, and consistently across all replicates of all peptides? Also, more specifically, is there a filter for eliminating or reintegrating peptides that don't have peaks within a certain ppm threshold. I have a document with a couple of examples, if needed.

In the example provided, for gene CLU, peptide ASSII, most replicates chosen is the small peak in the middle of 3 at RT ~25.3, dotp >0.98, and ppm<10. However, other replicates (e.g. 1, 13, 27), with less ideal ppm, dotp, and RT peaks (the one on the right, ~RT 25.5) were chosen, and when we manually integrated the middle peak in the those replicates, it gave us favorable ppm and dotp like the rest. Is there further refinement we can so that more peptides will have more consistent peaks chosen among all the replicates without having to go through each of them?



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Is BlibSearch still supported?
(5 responses) Chris Ashwood 2021-05-03

Dear Skyline team,

I hope you are all well. I am trying out the BlibSearch tool from Bibliospec and am getting no matches to spectra. The mzml being searched is one of the files which was used to make the blib file so there should certainly be matches.

I believe this error below (from the program run in verbose) may reveal the reason why I am getting no matches to spectra. The blib itself works fine in Skyline, just not with BlibSearch:

At line:1 char:1
+ .\BlibSearch.exe -w 5 .\Redacted2.mzML .\Redacted. ...
+ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
   + CategoryInfo          : NotSpecified: (Using library(s) .\Redacted1.blib .:String) [], RemoteException
   + FullyQualifiedErrorId : NativeCommandError

DEBUG: Creating reader for library .\Redacted.blib.
DEBUG: Highest lib spec ID is 501.
DEBUG: Creating PwizReader. 

An exerpt of the console output in verbose can be found below, the final line is always scoring 0 matches:

DEBUG: PwizReader looking for index 17416.
DEBUG: Searching spectrum 17417
DEBUG: Adding spectrum 274, precursor 739.27.
DEBUG: Adding spectrum 275, precursor 739.27.
DEBUG: Adding spectrum 276, precursor 739.27.
DEBUG: Adding spectrum 277, precursor 739.27.
DEBUG: Found 4 spec between 738.37 and 748.37.
DEBUG: Scoring 4 matches.
DEBUG: Scoring 0 matches.

The actual output (.tmp file) looks like this:

# Date: Mon May  3 21:20:05 2021

# query file: Redacted2.mzML
# Library file list:
# libID1        Redacted.blib
# Options:
# clear-precursor = true
# topPeaksForSearch = 100
# mz-window = 5
# low-charge = 1
# high-charge = 5
# report-matches = 5

Query   LibId   LibSpec rank    dotp    query-mz        query-z lib-mz  lib-z   copies  candidates      sequence        TIC-raw bp-mz-raw       bp-raw  lbp-mz-raw      num-peaks       matched-ions


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another Skyline install problem
(2 responses) kvancott2 2021-05-01
Brand new Win10 Enterprise computer, 64 bit. This is a acquisition computer for a Sciex 6600+ TTOF-MS.

See attached screen-capture for error message.

I've looked through several pages of support requests regarding installation problems, and didn't see anything similar.

I get this error with both the downloaded setup and the off-line setup.

Install.log is also attached
 SkylineInstallError_May012021.JPG  install.log 
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Feature Score Generations Fails: “Signal to noise missing from chromatogram peak” for particular replicates
(4 responses) peter r mosen 2021-05-02

Dear Skyline team,

During the generation of a peak scoring model Skyline (64bit, v21.0.9.118) reports for one peptide in two of the nine loaded replicates that it is missing the signal to noise for the chromatogram peak, excluding this feature score form the scoring model. However, as you can see (screenshot, lower chromatogram panel the two replicates L01-05 and L01-10) Skyline integrated for the two reported replicates the peak in the light and heavy channel properly (“Integrate all” is active). Also after manual peak integration for the two replicates the noise to signal feature score is still greyed out/inactive. If I exclude the two problem replicates (or delete the affected peptide) and I can build my peak scoring model having the noise to signal feature score included.

Interestingly Skyline is missing the signal to noise only for the two of the nine loaded replicates, though all nine loaded replicates were acquired with the same instrument method in the same acquisition period.

Is there anything what I am missing? What is causing the problem with these two replicates?
Thanks for your help,


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Problem importing Brucker Tims-TOF Pro data in docker container
(3 responses) m j noga 2021-04-23

Dear Skyline Support team,

With a larger team of bioinformaticians and developers we are prototyping data processing and analysis workflow based on Skyline. In one of the use cases we performing targeted MS1 feature extraction using from the TimsTOF Pro data. We already successfully tested the workflow on Windows, both via GUI and command line and we are right now at the stage of transferring it to containerized environment on our Linux cluster.

As a first stage we are just using official docker image (, but we experiencing a high number of import failures with the following exception:

Unhandled Exception: System.AccessViolationException: Attempted to read or write protected memory. This is often an indication that other memory is corrupt.
   at System.Collections.Generic.GenericArraySortHelper`2.Swap(TKey[] keys, TValue[] values, Int32 i, Int32 j)
   at System.Collections.Generic.GenericArraySortHelper`2.PickPivotAndPartition(TKey[] keys, TValue[] values, Int32 lo, Int32 hi)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.Sort(TKey[] keys, TValue[] values, Int32 index, Int32 length, IComparer`1 comparer)
   at System.Array.Sort[TKey,TValue](TKey[] keys, TValue[] items, Int32 index, Int32 length, IComparer`1 comparer)
   at System.Array.Sort[TKey,TValue](TKey[] keys, TValue[] items)
   at pwiz.Skyline.Util.ArrayUtil.Sort[TItem](TItem[] array, TItem[][] secondaryArrays) in Z:\pwiz\pwiz_tools\Skyline\Util\Util.cs:line 945
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.SortSpectrum(SpectrumInfo spectrumInfo, Int32 i) in Z:\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 997
   at pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in Z:\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:line 185
   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(Object obj) 

When attempting to import a single file multiple times it fails 30-40% of the cases, at random stage during import (see example import log attached).
We invoke Skyline by:

docker run -it --rm -v /Users/m/surfdrive2/Glycopeptides/data:/data \
proteowizard/pwiz-skyline-i-agree-to-the-vendor-licenses wine SkylineCmd --dir=/data \
--in=output/ --out=output/ --timestamp --memstamp \
--import-file=Controle_16_Slot1-36_1_3174.d --full-scan-precursor-res=40 \
--log-file=output/import_log.txt > data/output/import_console_log.txt 

Setting up options like '--import-process-count' or '--import-threads' doesn't seem to have any influence.

We already tested this using docker on Linux and OS X hosts with similar error rates.

We do not see this issue when running the same workflow on Agilent Q-ToF data, which may point out this is somehow linked to reading Bruker data.

Despite high failure rates we are able to import our 40-sample test dataset in the end (with up to 9 re-tries) and the final results are identical as on Windows.

I am wondering if you could help us with fixing this problem. Is this a Proteowizard issue or Wine? Should we contact proteowizard support directly? Our software engineer is eager to get involved, but would need some support and feedback to get started.

We will appreciate any help you might provide.


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Unable to convert .wiff format file to mzml format
(1 response) sumukha hegde7 2021-04-30

Hi all,

I was doing lipidomics data analysis and wanted to convert .wiff file mzml format for further analysis. I am unable to convert via skyline. Did anybody face the same problem? Is there any solution?

 P_POS.wiff  P_NEG.wiff  N_NEG.wiff  N_POS.wiff 
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Quantifying with surrogate standards
(3 responses) Joerg 2020-11-24

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

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Skyline for Lipidomics
(7 responses) aravandi 2021-04-27

Hi all,
Have couple of questions to see if Skyline can help our work flow and should we invest the time.

  1. Can skyline import scheduled MRM files from AbSciex for lipidomics?
  2. Can Skyline help with peak picking and flag transition that are falling outside of integration parameters in data sets with more than 2000 runs?
  3. Can we intergrade our batch normalization algorithm within Skyline?


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Spectral library building with open source/free MS/MS identification software
(5 responses) Erik 2021-04-21

Hi MacCoss-members,

I've been trying different free/open-source MS2 identifcation software to identify my MS2 spectra from my thermo altis triple quadrupole (only MS2 no MS1) and use their output for a Skyline spectral library.

So far I've tried Peptideshaker/SearchGUI with X-Tandem, MSAmanda, comet, omssa and msgf+ without any success. Somehow the mzid/pepxml/mzIdentML are not able to be read by the Skyline spectral library builder.

Tried maxquant but it does not work because I think it needs MS1 spectra.
Tried PD 2.4 demo (with MSAmanda and Sequest) and that does work but it will expire soon.

Any suggestions on how to import the formats from any of the free search engines properly?

Erik de Graaf

PS. It would be great if you could integrate a search engine like MSAmanda to build a spectral library for targeted assay development!

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Spike of heavy peptides
(2 responses) nicolas pierre 2021-04-21


We develop a new SRM method with the help of crude heavy peptides. We started with 414 heavy peptides and after refinement we follow 244 heavy peptides. However, our mix of heavy peptides still contains 414 heavy peptides and we found that those peptides spiked at 500 fm each are intense (much more than the light) and decrease the peak quality of the light peptides (in general in create noise). We determined this effect by testing our method with and without the spike of heavy peptides. To ameliorate the quality of our signal, we want to do another mix of heavy peptides only with peptides that we follow (n=244) and we could reduce the quantity of peptides spiked. However, we do not know how to adapt the quantity of heavy peptides. We could try to reach an identical intensity for each heavy peptide (we are not dealing with absolute quantification) but in this case we do not know what is the ideal intensity. Alternatively, we could try to reach an intensity according to the light peptide (1/5, /10 ?). We do not want to have a ratio light/heavy of 1 since at this stage of development we still have some peptides with low intensity and bad quality of peak (refinement is not totally finished, for that we want the best signal quality).
Do you have some advises for this spike of heavy peptides ?

Thank your for your help,

Nicolas Pierre

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Slack support group
(3 responses) szymanski21071 2021-04-23

Hi all,
We are now during the Skyline Workshop and I had an idea, that was much appraised by others, that we could have a Slack channel for Skyline support. There are many experienced users, that could share their expertise with other people who have problems or questions. So my question is, if you have anything against, that I create a Slack Channel for this purpose?

All the best
Witold Szymanski

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How to open a spectra in skyline?
(5 responses) qwa227 2021-04-23

Hi, there:

I am new to skyline and have problem opening spectra that were collected elsewhere. Just wondering if you have a step-by-step instruction? Do I need any other file besides the spectrum RAW data file? I watched your educational online videos but still could not get it to work.



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Adding TMT ions to product ions in Skyline 20.2
(6 responses) juliane weisser22597 2021-04-21


first of all thanks for the great software, which I am using am almost on a daily basis.
I am currently facing an issue with adding TMT ions to the list of product ions for my peptides in the latest version of Skyline (20.2). It works, however, in an old version of Skyline (4.2), that we still had installed on an old computer.
Did anything change for the process to add TMT ions? If it makes any difference, Skyline 20.2 is run under Windows 10, whereas version 4.2 is still run under Windows 7.

I have attached a short document showing the respective transition setting dialogue from both software versions and a screenshot of the product ion pane that shows the TMT ions in v4.2 but not in v20.2

Thanks a lot for any insights!

On a related note, any chance that Skyline will be able to read MS3 spectra eventually? We are running several projects with PRM MS3 or TOMAHAQ style acquisition and this would be quite helpful. I understand if it's not a priority as it appears to be quite niche-y.

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MS Amanda Scores
Joerg 2021-04-23

Hi all,
is there a possibility to access the MS Amanda scores for the peptide identifications? (Yes, I got it to work :-))I would like to filter the peptides either before import or after.

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Does Skyline can evaluate digestion efficacy of enzymes?
(1 response) ref p 2021-04-15

In my assays, there's a need to monitor the trypsin digestion efficacy to improve the sequence coverage of the analyzed proteome.
In limited efforts, I didn't find useful materials for solving this. Does anyone can help me out?

Guihua Jia

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Isolation list export for Orbitrap Exploris 480
itv005 2021-04-21


I am currently setting up a PRM study on an Orbitrap Exploris 480, and I was wondering how Skyline relates to this instrument? E.g. the Isolation list does not come with any option to export to the Exploris.

Note: I have tried exporting the Q Exactive IL to use in the Exploris software, which did not work. Trying to nest up if my issues are related to the IL, or the setup in general.

Best regards,
Ingrid Oline Tveranger

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Exporting prm-PASEF method
(6 responses) meth 2021-04-16
Hi, im getting following error when trying to export a prm-PASEF method.
What am i missing out?

An error occurred attempting to export.
Query result unexpectedly empty: SELECT Value FROM PrmGlobalMethodInfo WHERE Key='SchemaType'
OK More Info
System.Reflection.TargetInvocationException: Query result unexpectedly empty: SELECT Value FROM PrmGlobalMethodInfo WHERE Key='SchemaType' ---> System.Exception: Query result unexpectedly empty: SELECT Value FROM PrmGlobalMethodInfo WHERE Key='SchemaType'
   at pwiz.CLI.Bruker.PrmScheduling.Scheduler..ctor(String scheduling_file_name)
   at pwiz.Skyline.Model.BrukerTimsTofMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor, TimeSegmentList& timeSegments, SchedulingEntryList& schedulingEntries, Boolean getMetrics) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Export.cs:line 3259
   at pwiz.Skyline.Model.ExportProperties.<>c__DisplayClass131_0.<ExportBrukerTimsTofMethod>b__0(IProgressMonitor m) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Export.cs:line 613
   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 1940
   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 204
   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 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\pwiz_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2111
Kind regards,
Mikkel Thomsen
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Installing/enabling external tool in Administrator install of Skyline
(10 responses) m j noga 2021-01-14

Dear Skyline Team,
We are using Skyline in a very restrictive environment where Administrator install is the only way to make it run for regular users. I am currently exploring the possibility to also use external tools to better integrate it in our workflows. I see I can install and run a tool with the Administrator account but this tool is not showing up in the Tools menu for regular users.
Is there any way to make Skyline discover tools installed by admin?
I see I can enable the tool manually by filling in the form through Tools->External Tools...->Add...->Custom and changing the command path for absolute path to the executable in Tools folder within Skyline installation directory. So while it allows me to achieve the goal, this is a rather complex workaround considering what my users need (it also requires manual installation of report and annotations). I wonder if there is a simpler way.
I see there was also a similar call in 2018: and I wonder what changed since then as Nicks suggestion was not very encouraging.
I will very much appreciate your help!

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(2 responses) maria hernandez-valladares 2021-04-15

Hi there,
just to be sure, can we analyze FAIMS PRM data directly in Skyline or do we have to work with split CV-raw files?

Many thanks

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SCIEX TripleQuad 7500 wiff2 import error
roosso 2021-04-19


We have recently bought a Sciex Triple Quad 7500, which only generates .wiff2 files. However, I am unable to import these files into Skyline. Whenever I try to do so, I get the following error message:

At 22:35:
Failed importing results file 'Filename'.
[Experiment2Impl::ctor()] Object reference not set to an instance of an object.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'Filename'.
[Experiment2Impl::ctor()] Object reference not set to an instance of an object. ---> System.Exception: [Experiment2Impl::ctor()] Object reference not set to an instance of an object.
at 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\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 194
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188
--- End of inner exception stack trace ---

Is there any way I would be able to solve this issue by changing a specific setting, or is Skyline not (yet) compatible with SCIEX Triple Quad 7500 wiff2 data? I am able to open the files using Sciex OS, so I am 100% sure the files are not corrupt.

Thanks for the help!

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Skyline occupies 100 % of CPU during DDA search
(4 responses) m kuppusamy 2021-04-19

When I perform a DDA search using MS Amanda in Skyline, it occupies almost 100 % of the CPU, causing the computer to terminate the program and eventually crash. Please suggest how to rectify this.

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Problem with error model from mProphet
(3 responses) Sangram 2021-02-16

Hello Team Skyline,

Thank you for this great application and wish you the best in the year ahead.

I am new to proteomics and trying it with toddler's step.

So basically I have SWATH data from sciex triple TOF of cell line knock out model. I am trying to quantitate differentially expressed proteins between wild type and knock-out cells. The steps followed were mostly from the following webinar and parameter suggestions from the following paper.

So my mProphet model looks like the fig attached. I think there is something wrong as the target and decoys they have huge overlap. What might have gone wrong ?
Secondly the Pval looks absurd (can be seen in the volcano plot attached) ??
Thirdly there were some proteins like ABL1 and MDM2 which was there in the search result (generated through PeptideShaker at 1% fdr) but was missing from spectral library ?? is there any default filtering that leads into this observation ??

Thanks and regards

 mProphet_model_report.png  p53_comparison_volcano.png 
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