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.

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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.

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
How do I limit the RT window when use 'Apply Peak to All' function
(2 responses) qzhang31 2019-10-17

Hi there,
I am running MS1 filtering for a bunch of peptides, I have a standard with high concentration and every peptide's peak is properly picked. Then I am loading some data with much lower concentration of those peptides and I find that skyline will pick wrong peaks at a very different RT.

How do I tell skyline to just look at 1 min RT window within the peak in file A for all the other files?


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Absolute Quantification in Skyline for (Peptide/Protein)
(2 responses) iej7 2019-10-18

I'm working with skyline (v19.1) to do some peptide based absolute quantification. All experiments are from MRM files (Instrument: 6500 QTRAP). As a general example, let's say the skyline file contains 2 proteins that are in different concentrations in my cal curve :

Protein A
-Peptide ABCDE
=Peptide FGHIJ

Protein B
-Peptide KLMN
-Peptide OPQR

I am very likely missing something basic, but don't see how to specify unique concentrations for the calibrators if Proteins A and B are in my cal curve at different concentrations. It seems like I can only specify analyte concentrations for 1 protein only and those values are repeated for all other proteins in the skyline file (i.e. Calibrator concentrations for Protein A will also be the concentrations for B).

How do I specify different concentrations for the proteins in my skyline file so that cal curve data can be evaluated appropriately?

I'm sure I'm overlooking something simple. If this is true, just point me to the right tutorial.

As always, thank you.

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Real-time transfer of data to Skyline
(2 responses) benoit fatou 2019-10-18

Hi Skyline Support,

I was wondering if there is a possible way to perform an automatic transfer of data to Skyline just after the acquisition.
That would be very helpful especially for large number of samples.

Thank you very much for your help,

Benoit FATOU

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PRM Workflow with Spectral Matching
(1 response) sa825 2019-10-18

Good afternoon,

I have watched Webinars 3 and 4 and found it very helpful but I have a question regarding spectral matching.
I conduct PRM on the Thermo Q Exacitive and this is my current workflow:

  • I choose the precursor and product ions of interest and export an isolation list for the Q Exacitive
  • I get the Raw Data file from the Thermo and run it through PD (with Percolator) which produces a PD result file
  • I then import this PD result file as a PRM search (File -> Import -> Peptide Search) and match it with the same PRM Raw file by following the instructions in the Import Wizard
  • This produces what I would call really good spectra. The chosen product ions are clearly detected with Peptide ID annotations.

However, when I import the raw result file ( File -> Import -> Results), I don't get the same level of detection. In most cases, none of the product ions detected when I do the workflow above are seen in this result file approach.

Can you please explain why there is this discrepancy? And if my current workflow is correct?



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Not possible to extract chromatograms from MGF files
(3 responses) marc isaksson 2019-10-17


I tried to use Skyline(v.19.1) for MS1 filtering + spectra library building, by importing a mzIdentML file from PeptideShaker (v.1.16.42). While the spectral libraries were built, I got stuck in the peptide search import at the Extract Chromatograms part. The MGF files referred to in the mzIdentML file were missing. SEE ATTACHED.

When I tried to help Skyline locate them by clicking on the Find button, and navigating to the MGF file location, nothing was displayed. Apparently, Skyline does not support the MGF spectra files referred to in the mzIdentML file.

Will MGF files be supported for chromatogram extraction in a later release? Then I could do MS1 filtering on the PeptideShaker result, something which I think would be a nice feature.



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Standard deviation in quant experiments
(3 responses) julius fuersch 2019-10-16

Hi guys,

I have a very general question regarding error distribtion in quant proteomics experiments. Often the standard deviation and therefore the p-value is calculated using log values of the extracted ion chromatogramm areas. By using the log values instead of the original values you will get a completely different distribution and a much lower relative standard error! Why do people use the log areas instead of the original values? In my very small understanding of statistics, this artifically improves the test statistics and makes the results better than they appear! I would be very happy about an explanation because we are using an in-house developed cross-link quant software which also does it in that way and I would really like to understand the background!

Thanks a lot in advance


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issue_exporting sepctral lib.
(4 responses) Wael 2019-10-15

Dear Skyline Team,
I am getting an empty .blib file after exporting spectral lib from skyline. can you please help me?

I have generated the lib from PD2 msf file and everything was good, but when I try to export the lib. I am getting a 20kb empty file. I have tried to generate the lib again but that did not help. I have also tried to open the skyline file using skyline-daily and export the lib. but I am getting the same emtpy folder.

Best regards


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iRT error when adding Protein Prospector generated blib
(2 responses) anatoly.urisman 2019-10-16

Dear Skyline team,
I am getting an error while adding an “on-column” spectral library (blib generated directly from Protein Prospector) to an iRT calculator (Pierce iRT set supported by Skyline). The error is “1 run was not converted due to insufficient correlation.”

I’ve uploaded a dummy Skyline document to the Upload folder. It contains two libraries, both of which are generated from the same DDA data, just two different searches (one focused on identifying the iRT peptides and the other on other peptides in the samples).

Could you take a look at the error and let us know if it is something that needs to be fixed on the Protein Prospector end?

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Question regarding DIA Isolation Window Scheme output for Thermo Fusion DIA m/z list
dhardie 2019-10-16

Dear Skyline Team;

I have a question regarding the DIA Isolation Window Scheme output when using Skyline Daily 64-Bit the output table contains "Start and End m/z" with the ability to show "Margin" and "CE Range" and has the "optimized window placement" feature but there is no "Centre m/z" similar to the EncyclopeDIA Scheme Wizard? Older versions of Skyline and presentations show "Generate Target " toggle that would show a centered target m/z in the output list that could be exported as a csv into the Thermo Fusion MS2 targeted list. The current output is just start and end m/z. Do you suggest manually calculating the "centre mass" for the DIA acquisition as a work-around.

Also I have noticed the masses generated when using margin widths of 0.2 if using Skyline and EncyclopeDIA window generators give different values. I have seen Searle's bitbucket documents describing Skyline's settings for DIA windowing acquisition settings.

Is there a formal document explaining the windowing scheme settings MSX,Overlap, Overlap MSX, Fast Overlap Experimental and what these settings are? I see many presentations for Q-Exactives but not much for Lumos or Fusion.

Thanks for entertaining my questions


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The error with cdb file
(3 responses) shin 2019-10-09

Hello skyline team,
I'm trying to analysis proteome data with Mascot dat file.
Previously I succeeded to analyze other data.
However It doesn't work in this time.
Investigation of the reason of error clarified some files made "dat.cdb" file.
What is dat.cdb file and how do we analyze them?

Thanks for your assistance and effort.


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Missing MS2 from Mass Inclusion List.
(5 responses) mwmann 2019-10-10

Hi, much like everyone else here, I should probably mention how incredibly useful Skyline has been for me. I've been using it for the last 2.5 years, starting as a learning tool for MS and now using it for my own projects as a graduate student. That being said, I would probably still consider myself a beginner with this software. Right now I'm using Skyline to help me quantify changes in histone ptms, and it's not an understatement to say that I couldn't do it without this software. However, I'm running into a weird issue related to MS2 spectra not being recognized in Skyline.

I'm running Skyline Daily on Windows 10.

I'll start by detailing my workflow:

  1. I run my samples on a bruker QTOF using a DDA workflow. I've manually added certain masses to a mass preference list so that some ions would fragment at all points during their elution. This is to facilitate quantification of co-eluting isobaric ions. (I'll admit, it's possible that this step isn't working as intended, but it wouldn't explain all the behavior I'm seeing).
  2. I convert my files to centroided .mzml files and use Metamorpheus to calibrate and run a search for modified peptides. I import the resulting .mzid files into skyline as a spectral library with a cutoff of 0.99. My peptide settings allow a large number of PTMs (10), but restrict peptides to those found in the library.
  3. I import a fasta of selected histones (just H3 for now), and remove repeated peptides.
  4. I import my .mzml files and use the peptide identification times on each spectra to select peaks.

All of this seems to work well. However, when I look at MS2 spectra, I notice that I have relatively few MS2 peaks, even when peptide ids are shown on the spectra. I've attached an image demonstrating this. I can click on the IDs and see my fragment ions changing, but Skyline doesn't seem to have any MS2 at those timepoints. On the image, you can see the highest scoring ion as a straight line from practically zero abundance to its max, That same ion in the ID spectra increases, but not in a straight line manner.

Noting another poster had a similar issue which was resolved by changing the MS/MS filtering to a DDA strategy, I attempted that without success.

Any help would be appreciated. I've uploaded a minimal Skyline project that replicates this as a .zip to Uploads Page (File Name: M.Mann I imagine you'll need the raw files too, so I've uploaded those as well (File Name: M.Mann Raw

Thanks regardless! Hopefully this can be resolved easily.

Morgan Mann
University of Wisconsin-Madison
Brasier Lab

 M.Mann Bug.png 
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MSstats analysis using ratio light:heavy
(1 response) Taina Marques 2019-10-11


I was wondering if is possible to use the ratio light:heavy to perform group comparison analysis using the MSstats in skyline?
As far I could see, skyline only does with the area without any ratio light:heavy. Is there a way that I can switch to ratio?

Thank you!

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Small Molecule Quan using light:heavy ratio without deuterated (heavy) version of each analyte (light)
(1 response) kbrady 2019-10-08


The assay I am working with quantifies 5 metabolites of methotrexate (MTXPG1, MTXPG2, etc), but rather than using the deuterated version of each compound as internal standard for each metabolite, the method cheats a bit and quantifies MTXPG1 and MTXPG2 as the ratio of MTXPG1:MTXPG1-d3 and MTXPG2:MTXPG1-d3, respectively. MTXPG3-5 are quantified as the ratio of MTXPG3:MTXPG3-d3, MTXPG4:MTXPG3-d3, and MTXPG5:MTXPG3-d3, respectively. (Yes I realize this is less than ideal, but I did not make the method :) )

Following your advice here (, I formatted my transition list as shown in the attached translist.csv ('True Identity' and 'True Identity Purpose' columns are just for clairity, I'm not pasting those columns). Can skyline handle an experiement set up like this?

Also attached is the document grid generated by Skyline through Edit -> Insert -> Transition List. You can see Skyline has not kept the m/z as provided in the transition list, and has instead constrained 'heavy' MTXPG2, MTXPG4, and MTXPG5 to be similar in mass to the 'light' precursors. I am guessing you have some internal check along the lines 'A heavy transition must be heavier than the light transition it is associated with'? Is there a way around this? I would like to use light:heavy ratio for my calibration curve, but this currently only works for MTXPG1 and MTXPG3 (which are actually matched with a deuterated compound, unlike MTXPG2, MTXPG4, and MTXPG5).

 translist.csv  doc grid.csv 
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Differnt product ions not shown together in Skyline
(2 responses) FlorianBonn 2019-10-10

I have a strange problem with Skyline I want to quantify a large peptide in a SRM experiment with a triple quad. The precursor is m/z ~700 with +4. We measured 3 product ions: b3 (m/z 350 +1), y19 (m/z 1050 +2) and y21 (m/z 760 +3) with a Qtrap and loaded the data in Skyline.
If I select all three Transitions it shows me data for y19 and y21, but for b3 only a zero signal. If I remove either y19 or y21 from the list, only data for b3 is there and for y19 or y21 only the zero signal is there. If I again add y19/y21 to the transition list, the b3 trace is gone and the two from the large ions are both displayed. Can anyone help me out to get all three transitions displayed at once or at least b3 and y19 together?
Sorry if this questions was answered, but search terms I tried were either to specific (nothing was found) or to general.

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Cannot import iProphet pepXML file to build a spectral library
(5 responses) marc isaksson 2019-09-24


I tried to generate a library in Skyline from an iProphet pepXML file, but failed in the last step.

Long story short:

I tried to create it the library by first starting a blank document. Then I went to Settings>Peptide Settings and clicked on the Library tab.
I entered all info regarding file names, output path for library, cut-off score and what iRT peptides I used.

In the next step, I added the iProphet pepXML file, and then clicked on the Finish Button. After a few seconds, the software threw an error. It seems like Skyline/BlibBuild cannot find an attribute called SpectrumNativeID in the iProphet pepXML?


FYI: I made sure to copy the iProphet.pep.xml file into the same directory(Working directory: D:\Data_TPP\RAW_files) as the mzML files used for the database searching (combined both Comet and X!Tandem searches into a single iProphet file).

I used the TPP v.5.0.2, with X!Tandem (old: Jackhammer 2013.06.15.1) and Comet (v.2018.01 rev.4).

If you could help me solve this issue, I would be really happy.



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Problem installing Skyline Daily
(2 responses) joel federspiel 2019-10-09

Hello Skyline team,

I have one computer that is refusing to install Skyline Daily ( We have used daily on this computer routinely in the past, but at some point recently the software was removed. When I try to install it again, I get the attached error and am unable to complete the install. This computer is running Windows 7 Professional.

Any help would be greatly appreciated. I am happy to provide any other information that would be helpful.



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Skyline not recognizing Heavy Acetyl modification of lysine residues
(13 responses) dbade001 2019-09-20

I am trying to upload my maxquant files to skyline. It has worked for my other files but it seems to have an issue with the heavy acetyl modification on the lysines. I am uploading a pic of this. Have you seen this issue before?

Thanks for your assistance and effort :)


 skyline error heavy acetyl.png 
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Skyline cannot import iProphet pepXML files to build a spectral library - if X!Tandem was used as a search engine
(1 response) marc isaksson 2019-10-07


I have noticed that Skyline (v.19.1) cannot import a iProphet pepXML file, if X!Tandem was one of the search engines, or the only search engine. I have tried to combine Comet searches and X!Tandem searches into an iProphet pepXML. When importing into Skyline, I get a error message saying(see attached for full error message):

ERROR: comet_tandem.ipro.pep.xml(line 555966): Missing required attribute 'spectrumNativeID'.

Command-line: C:\Users\marc\AppData\Local\Apps\2.0\W8H7VL7Y.7R9\HMJZHTQA.5XR\skyl..tion_e4141a2a22107248_0013.0001_f6e2bff174ff9e23\BlibBuild -s -A -H -o -c 0.999677 -i H9D_6F_190913_lib1 -S "C:\Users\marc\AppData\Local\Temp\tmp17BF.tmp" "D:\Data_Skyline\H9D_6F_190913\H9D_6F_190913_lib1.redundant.blib"
Working directory: D:\Data_TPP\RAW_files

Apparently, Skyline/Blibbuild is looking for the term SpectrumNativeID. Problem is that only Comet results have such a reference. X!Tandem does not produce any column or reference saying SpectrumNativeID. I was using X!Tandem version Jackhammer 2013.06.15.1, as part of the TPP 5.0.2 distribution.

When I created an iProphet pepXML file from Comet searches only (v.2018.01 rev.4), it was no problem to import the pepXML file into Skyline.

I would be really happy if search results from Comet and X!Tandem could be combined into iProphet and then imported into Skyline! As I understand, BlibBuild is supposed to import X!Tandem results, given that the file format is xtan.xml.



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Q-TOF .raw data search stops after certain retention time
(9 responses) Yao Chen 2019-08-13

Dear Skyline Team,

I am importing Waters .raw data generated by Q-TOF instruments into Skyline. The whole file collects data for 120 min (pic 1), however, the searches in Skyline seems stopped at around 60-70 min for almost all the transitions I was monitoring (pic 2 shows four examples).
I could see a very clear signal of a spiked-in standard peptide in the raw file, which eluted at 77 min (pic 3), however, its retention time was not reached in the search of Skyline (pic 2, upper left). How should I change my settings so I can do a full chromatographic search, please?

I have my partial skyline processing file attached.



 pic 1 _ base-peak intensity chromatogram.PNG  pic 2.png  pic 3_raw chromatogram of standard peptide.png 
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How to normalize the peak area with b-gal peptide
(1 response) drrenugoel 2019-09-30

I would like to normalize the peak area with one peptide of b-gal which we had added in all teh samples

There is a variation in the peak area. How to normalize all other sample peptides by this peptide?


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Calibration Curve Errors for Light:Heavy Ratio
(1 response) bwidner 2019-10-04

Hello Again,

I have been using skyline for small molecules and prior to now I have made linear standard curves using the peak area. That has worked great. I just introduced isotopically labeled standards, and I am now having trouble getting the standard curves to appear for some molecules. Because it works for most molecules, I dont think it's a problem with my settings or Document Grid, etc. The problem is that the Calibration Curve plot will have data points on it, but it won't draw a line and lists NaN for Slope and R-Squared. I have tried everything I can think of to address this problem, and I can't solve it. It does seem to happen with the curve is fairly crappy.. When I just plot the peak area of the light compound for these, I get a curve as normal. Is it something to do with very low intensity heavy peaks?

I attached a photo of the problem, and I will upload the files to the file share thing momentarily.


 ectoine curve error.tif 
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Failed Importing Results... Failed to Centroid Scan
(4 responses) bwidner 2019-10-02

Hello Skyline Team!

I tried to import a batch of ~30 files and got the following error for three of them

At 2:35 PM:
Failed importing results file 'C:\Filepath\mtab_BCderiv_BC43_092619_69.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=3119": [RawFileThreadImpl::getMassList()] failed to centroid scan

I opened each file in XCalibur and looked at the scan indicated in the error message (1972 for the example given above). The scan is for an MS2, and it appears to be completely empty of data (see attachment). I guess this is why it could not be centroided! Is there any way around this problem so I can import the files, as it seems to be just one bad scan?


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Skyline-daily importing (extracting product ion data) from only light peptide even though heavy & light are both in RAW file
(3 responses) mlane 2019-10-02
Hi, Upon recently updating Skyline Daily version to, targeted MS raw data files (PRM data generated from Orbitrap Lumos) is not fully imported into Skyline documents. We went back to official Skyline release (most current v 19.1) with the same file, created new document and the problem was resolved. Also, on multiple PCs, we created new Skyline Daily version document template, and the problem repeated. The raw files are confidential, but if you need help with source files to recreate the issue, let us know and we will try to come up with a example that is not confidential. At minimum, can you send link or instructions to install previous version of Skyline Daily (we did not have issue in previous release)?
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Two-step normalization
roman sakson 2019-10-02

Dear all,

I have a more general question concerning data analysis. Is there any possibility to have a two-step data normalization workflow implemented in Skyline? We often use spike-in SILAC experiments and normalization to heavy very nicely removes technical variance from our sample preparation workflows etc. However, it would be great if we could then use these L/H ratios and still normalize them to some housekeeping peptides to address biological variance (I realize that this can be done via global or surrogate standards but I would very much like to have it in addition to heavy normalization and not instead of it). Is something like this possible in Skyline or should I rather try working with MSstats for this purpose?

Thanks for this great piece of software!


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Group comparison
(5 responses) Enzo 2015-08-11
Dear Brendan:

First, thanks for developing such a nice software for targeted proteomics.
Second, got some questions about the group comparison:
1. We are focusing on the protein level instead of peptide level, so how did Skyline define the intensity for the protein? Does it simply add up all the intensity of peptides from same protein?
2. Take the first protein from the attachment for example, in the "fold change result" what does 38.66(99%,CL:24.83 to 60.21) mean? I assume 38.66 is the mean of the fold change from 3 repeats (I have done the 3 biological repeats) and 99% means 99% confident level.
3. Can I get the ratio for the 3 different repeats instead of median?

Thanks a lot for your help.
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diaPASEF data analysis
(1 response) thsofia17681 2019-10-01

Hi I would like to ask you if you have a basic tutorial on how to import diaPASEF data and how to see the MS2 raw spectra of the files.
Please note I am pretty rookie on Skyline. I have taken courses but never used it extensively.

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

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


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problems during install
(3 responses) zhengki 2019-09-29

Please help me with this problem in installing.

Thanks very much!!!

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remove peptides that are not present in all replicates
(2 responses) contet 2019-09-27
I am working with Skyline files from a DIA experiment in which samples were processed in 2 batches, which each contain replicates from all experimental conditions and have their own reference for normalization. I want to merge the Skyline files from the 2 batches to analyze the data in MSstats. What I have done so far is open the Skyline file of batch 1 then File>Import>Document to import the file of batch 2. I selected Add new replicates and Merge matching peptides, such that peptides that have been detected in both batches show up under the same protein ID. My problem is that some peptides were detected in only 1 of the 2 batches, which causes problems with MSstats (the error message says that "the input dataset has incomplete rows"). I only want to keep peptides that have been detected in both batches. How can I remove peptides that are not present in all replicates (e.g., in the attached screenshot, keep the aqua peptide but exclude the green one)?
Thanks for your help!
 Screenshot (40).png 
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Raw files moved
(1 response) ingo.wohlgemuth 2019-09-28


thanks for the great software.
I analyzed a larger DDA dataset with MS1filtering. Unfortunately, the external disk with the RAW files crashed. I still have the skyline file on my computer and a backup of the maxquant search plus raw files of the corresponding analysis. However, Skyline expects the original location of the raw files on the broken external memory.

  1. Is it possible to reimport the data in the skyline files from a new folder?
    or 2. is there another way to reconnect skyline analysis and raw data (plus maxquant search)?

Thanks in advance!


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IS it possible to do PRM data analysis using skyline?
(9 responses) ekramh1982 2019-09-26

The format (column headings) for the inclusion list of PRM in Q Exactive plus is different from the example provided in "" file. And creating a file similar to the values gets this problem: when I import the raw files it says "no usable data". How do I quantify the PRM files from Q Exactive Plus using skyline? The message is as follows:

At 11:52 PM:
Failed importing results file 'D:\Ekram\2019\Sept_20\Swab_Recovery_PRM\Pos_SwabRecovery_PRM\Pos_PRM_P40_Tang_100uM_stock_A10.raw'.
The sample Pos_PRM_P40_Tang_100uM_stock_A10 contains no usable data.

But I can open that file in quail browser:

How do I solve this? Thank you.

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how to upload chromatogram
(1 response) drrenugoel 2019-09-27

Kindly see the snapshot. I have built the library. Now I want to import my result chromatogram. However, it is showing library raw files. How to import result chromatogram. these are HR-MRM runs files.

 upload _chromatogram.jpg 
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Facing problem in instaling
(1 response) mchsahu 2019-09-27

Dear Sir/Madam,
I tried many times to install skyline daily but unable to do and getting the attached error. Will you kindly plz help me regarding this.

Thanks and regards

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PEAKS export for library building
(1 response) mauro galli 2019-09-26

I am a new user and I have a question about the Cut-off score setting in the library building for a DIA analysis.

I processed the data on PEAKS applying 1% FDR and I exported the file for Skyline. In the ion library building process in Skyline, I set the Cut-off score equal to 0, not to apply a double filter on my peptides (I don't want to cut 1% of the file where I already cut 1%). Do you think it could be a problem?

Thanks in advance!

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No transitions visible since latest Skyline daily update
(6 responses) lena reimann 2019-09-25

Dear Skyline team,

I updated our Skyline daily version to yesterday. Since this time point, we are no longer able to see any transitions in our AutoQC pipeline or following manual data import into Skyline. If I check for the transitions manually with Xcalibur or use older Skyline daily versions e.g. all the transitions are visible.
Is there anything I can do to recover the visibility/import of the transitions? At the moment they are also not imported into PanoramaWeb, so we are lacking some valuable information about our instument performance.

Thanks for your help and providing this great software,

 Skylinedaily_19-1-1-248.JPG  Skylinedaily_19-0-0-190.JPG 
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LOD determination
(1 response) djlj1 2019-09-25


I have been trying to determine LOD and LOQ for a series of calibration lines. If I use a bilinear model I can get both LOD and LOQ. I only get LOQ if I choose a linear model. Am I choosing a wrong setting please.

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Skyline won't import PD result files
(1 response) sa825 2019-09-24

Good afternoon,

I am trying to import some DDA files that have been processed in Proteome Discoverer (PD) using the Import --> Peptide Search format.
However, when I do this, I get the following error: "This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives."

If I reduce the cut off score to 0, it works but then as it says, such results are unreliable.

I have attached a screenshot of the error.



 Skyline 1.png 
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Easier visual for Heavy and Light in
(1 response) waltteri hosia 2019-09-24


Is there an easier way distinguish heavy and light versions of the same peptide in document grid? Looks like that total area is the only column that distinguishes them. At least with my settings, (double calibration curve, normalize to heavy), skyline doubles the rows with same calculated values for both, (which is slightly odd, heavy normalizing heavy should give 1, or?) In other words for those peptides that I have heavy peptide to use as ISTD I see in normalized grid 4 calibration sequences, from two sequences actually injected. But two of them have identical values, although total area reveals they are from the heavy ISTD. Modifying the "normalized" grid from "peptide modified sequence" does not quite work either. Choosing "show monoisotopic mass" shows the plain sequence for both light and heavy.

BR, Waltteri

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Heavy peptide as global or surrogate standard
(4 responses) waltteri hosia 2019-09-20

Hi, Skyline keeps coming better with every update, thank you for keep improving it.

Question. Is there a way to set the heavy peptide as a global or surrogate standard? I have an quant experiment with few proteins and I only have one heavy peptide per protein. I see though that sensitivity would be better with other peptide than the one that I have the heavy for. The heavy is in as one fixed concentration.

view request
AutoQC- No existing files found
(3 responses) davis 2019-09-20

Hi Skyline team!

I am a new user to AUTOQC. AutoQC is unable to import/find my existing files although the appropriate data folder is provided in the Folder to watch: field in the AutoQC Configuration settings. Please help me.

Current log message.

9/20/2019 8:25:27 AM] Getting the acquisition date on the newest file imported into the Skyline document.
[9/20/2019 8:25:27 AM] Running SkylineRunner.exe with args:
[9/20/2019 8:25:27 AM] --in="D:\Masslynx_projects\20180830_Metabolomics_Lipidomics.PRO\Skyline Results\AutoQC\" --report-conflict-resolution=overwrite --report-add="C:\Users\Administrator\AppData\Local\Apps\2.0\MY2EMOE9.LK6\QNADK77V.1XC\auto..tion_e4141a2a22107248_0001.0001_6f67fb96aa7015e7\FileAcquisitionTime.skyr" --report-name="AcquisitionTimes" --report-file="D:\Masslynx_projects\20180830_Metabolomics_Lipidomics.PRO\Skyline Results\AutoQC\AcquisitionTimes.csv"
[9/20/2019 8:25:30 AM] The name 'AcquisitionTimes' already exists.
[9/20/2019 8:25:30 AM] Resolving conflicts by overwriting.
[9/20/2019 8:25:30 AM] Success! Imported Reports from FileAcquisitionTime
[9/20/2019 8:25:30 AM] Opening file...
[9/20/2019 8:25:30 AM] File opened.
[9/20/2019 8:25:30 AM] Exporting report AcquisitionTimes...
[9/20/2019 8:25:30 AM] Report AcquisitionTimes exported successfully to D:\Masslynx_projects\20180830_Metabolomics_Lipidomics.PRO\Skyline Results\AutoQC\AcquisitionTimes.csv.
[9/20/2019 8:25:30 AM] SkylineRunner.exe exited successfully.
[9/20/2019 8:25:30 AM] The most recent acquisition date in the Skyline document is 9/10/2018 5:46:08 AM
[9/20/2019 8:25:30 AM] Initializing Panorama pinger...
[9/20/2019 8:25:30 AM] Starting configuration...
[9/20/2019 8:25:30 AM] Importing existing files...
[9/20/2019 8:25:31 AM] No existing files found.

Best! Sonnet

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DDA --> PRM not working
(9 responses) sa825 2019-09-19

Good afternoon,

I am trying to conduct PRM on a Thermo Q Exacitive for a list of proteins/peptides. They are heavy labelled so I am really interested in picking up the heavy peptides.

To do this, I first conducted DDA using the sample of interest on the Thermo and detected the many of the proteins/peptides of interest (Skyline shows that their precursors M, M+1 and M+2 are present)

However, when I run a Standard PRM (Exported a single method, standard isolation list) on the same instrument, using the same sample, only ~2 product ions are detected.

Can you please explain to me why this is the case? Maybe its a setting I have missed out?

I have attached a zip file containing the situation on the File sharing Page and titled it: "DDA -> PRM Not working"

Thanks and I look forward to your reply,


view request
Metabolomic isotope data- can only view chromatogram for the nominal mass
(4 responses) sarahmacpherson676 2019-08-06

Hi, I am looking at metabolomics isotope data using GC-MS with a QIT mass analyzer. I was initially able to get the results for each isotope using Skyline Daily. However, when I opened a new document with the same data, I was only able to view the chromatograms for the nominal mass. Not sure if there is something wrong with the software, as I was able to analyze the data before with the same settings.
Attached is the .sky file that works and doesn't work.
Thanks for your help

 gc-ms analysis 
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Spectral library problem from MaxQuant search (no isotopic labels)
(3 responses) Steve S 2019-09-13

Dear Skyline Support,

I created a spectral library in Skyline from a large MaxQuant search. There were no errors. I can find the expected peptide IDs in the library.
However, when I choose "View Library Match", the wrong spectrum is shown. The raw-file name and scan time are correct, however I don't know where the spectrum is from- it is another MS2 spectrum, but it certainly is not the correct spectrum.
Do you have any suggestions for what could be causing this?

I read a few earlier problems related to heavy-labelled samples, however in this case the MaxQuant search is from an unlabelled sample (DDA data).

Thanks for your help.

All the best,


view request
Install Version 19.1.
(2 responses) sandra maass 2019-09-18

Dear All,

I'm trying to install the newest Version in 64-bit. Unfortunatley I'll get an installation error every time I try. Please find the install log attached.

Do you have any idea what I do wrong?

Thank you in advance

view request
Optimizing FAIMS on new Thermo instruments
Martijn van Duijn 2019-09-18


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

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


Martijn van Duijn

view request
Peak picking within a specified retention time range.
(10 responses) wangqingok 2019-09-11

I have manually selected peaks corresponding to my target peptides in several reference samples and I have figured out my peptides' relatively stable and reproducible retention times. And I have 200 more samples to analyze. I know I could export and import peak boundaries to my future analysis when I want to quantify these peptides, but I found that for some peptides where a significant amount of noise signals are very close-by the target peaks, the quantification results are far off and the rentention times are not that reproducibly where to specify a fixed window for peak boundaries will have a lot of false positive signals being integrated and counted into the abundance. Therefore, I do want to also have skyline pick the peaks, but only within those predefined retention time windows (~ 2 minute windows) that I could manually get. Is there a way of doing this? Thank you so much for your help!

view request
Export structural modification list
(1 response) io 2019-09-17

Dear all,
I would like to export the structural modification list of Skyline to send it to a colleague
He wants to study O-glycosylations but I am not sure of the modification we have to add.
Thanks a lot

view request
Sciex analyst .wiff files fails to import showing: Object reference not set to an instance of an object.
(2 responses) kamal 2019-09-02

Dear Skyline Team,

In my experiment, each replication’s retention time is 6 hour. However the *.wiff and *.wiff.scan files comes in 3 part for each run. For example, P100_Cold_30min (Cold stress for 30 min) comes in P100_Cold_30min-1_1, P100_Cold_30min-1_2, P100_Cold_30min-1_3

On the other hand, the *.group and *.group.xmlcomes in single file P100_Cold_30min-1 as the following screenshot.
The MGF files also come in a single part which I used to generate mascot DAT file as an alternative to the group.xml files for creating the spectral library.

I am using these *.group and *.group.xml or just mascot DAT files to create the spectral library for MS1 based DDA analysis. However, it seems like that when importing the final results the following problem is occurring-

At 5:42 PM:
Failed importing results file 'C:\Kamal RIKEN DATA\190614\P100_Cold_30min-1_3.wiff'.
Object reference not set to an instance of an object.

Inner exceptions:
Exception type: System.NullReferenceException
Error message: Object reference not set to an instance of an object.
Object reference not set to an instance of an object.
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.get_PercentComplete() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 805
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.UpdatePercentComplete() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 598
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 586
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 236
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 460
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 263

I am in desperate need of help because of my graduation deadline is approaching soon.

Best regards,

view request
Not able to see "library peak area" in peak areas-replicate comparison
Monica E 2019-09-15


I am trying to compare the peak areas of my different proteins among 16 different samples. I can now see the different peptides for each protein as well as the spectra corresponding to every replicate. However, I cannot see the "library" peak area, only the peak area for each replicate. I had a read through the forum and found a suggestion that said to "right click" on the replicate comparison panel and select to see the library peak area, however, this option does not come up when I "right click". Would you be able to help me? The library I am using in peptide settings is a reference pool sample of all the 16 replicates. I attach a screen shot as well in case it is any help.

Thanks in advance,
Monica Espinosa

 Skyline query.png 
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using external calibration curve?
(1 response) Cedric 2019-09-09


is it possible with Skyline to use the calibration curve acquired for a molecule X and apply it to another molecule Y? This will allow to do semi-quantitative analysis of molecules which do not have a specific calibration curve.

If it is not possible, do you have a way to export the calibration curve equation determined in Skyline? Like this, we could use it in excel to perform concentration calculation.

thanks for your support and your great software

Kind regards,


view request
(8 responses) Tobi 2019-08-06

Dear Skyline Team,

could you please take a look at an issue with Avant-Garde DIA? Sadly the forum for Avant-Garde DIA does not seem to be maintained.

When trying to run it the execution is halted. The full error message is attached, but the main point seems to be

" cannot open file 'S:/avant garde test/AvantGardeDIA/DataFormatting/TempFiles/Report_GR_ColNamesName_Tag.csv': No such file or directory Execution halted"

The named file does not seem to exist. The preperation was done according to the AVG Manual, but it might still be an issue. Did anyone else experience an error like this one?

Best regards,
tobi  AvantGardeDIA.rar  Error MSG.txt 
view request
i did not get standard deviation whiskers by doing --Transitions > Total. yes, I am looking for the standard deviation of the total.
(1 response) drrenugoel 2019-09-10

i did not get standard deviation whiskers by doing --Transitions > Total. yes, I am looking for the standard deviation of the total.

view request
Error building spectral libraries from Comet/percolator output
(14 responses) timothy acker 2018-05-23

Hi All,

I hope this message finds you well. I am having a recurring problem building spectral libraries or importing search results from Comet/percolator output.

In peptide settings when I try to create a spectral library or import results, I am given an error informing me that the pep.xml file is not from one of the recognized file format and it references the first line in the search results within the pep.xml file. I am running comet from the crux interface in Cygwin.

crux v, percolator v 3.02.0, Skyline v, Comet version "2017.01 rev. 4"

I cannot find another example in the support forums.

Thank you,


view request
Transiton Results Report
(1 response) Khizra 2019-09-13

Hello Skyline Team,

I would greatly appreciate if you could help provide some explanation to aid my understanding of the Transition Results Report. It includes multiple rows for the each fragment ion of each protein and each row has a different retention time. It is also omitting some fragment ions from the table. Can you please provide some information on firstly why it is omitting some ions from the report, and secondly why there are multiple rows each with a different RT for each ion displayed. Please review the attached file which shows as example.

Thank you,

 KE Export Result Skyline Forum.pptx 
view request
PRM Quantification of Croos-linked Peptides
(8 responses) julius fuersch 2019-09-09

I am doing quantification of cross-links in PRM mode. I generated my transition list by the plugin Xlink Transition Calculator. I am using the latest Skyline version. I recognized that I need to increase the ion match tolerance up to 0.5 m/z in the transition settings to get matched peptides. Nevertheless, after doing this, the upshowing best matched peptides have very low mass error (0.5-10 ppm, measurement was on an Oribitrap Fusion). As a consequence there are a lot of other matching peptides, often difficult to judge which one is the correct hit.
Additionally when using this plugin, it seems not to be possible to add decoys because skyline runs in the molecule mode. Changing to proteomics mode is not possible! So I can not use mProphet.

Do you have any suggestions how I can improve my pick picking? I know approximately the expected retention time, so now I am only trusting hits within a certain time range. Do you have any ideas to make a reliable pick picking?

In the PP file, I tried to illustrate the problem of more than one well matching peak with a red box and the other mentioned points!!

Thanks a lot

 PRM Cross-links Request to Support.pptx 
view request
Spectral library
(1 response) Nathan Manes 2019-09-12

After building a spectral library, I noticed that "Use explicit peak bounds" is checked. What does this do? Thanks.

view request
Batch import custom annotation settings and tracking chromatogram corrections.
(4 responses) velasqe2 2019-09-12

I had two questions, one regarding custom annotations and the other regarding peak editing.

  1. Is there a way to import a batch of custom annotations at a time. Currently the user has to add annotations to the protein environment one at a time through document settings but we were wondering if there is a way to pre-define custom annotations and import those settings into document settings.

  2. Is there a way to track the process of peak editing that a user might perform on certain chromatograms? and if so, can we export the editing process as a csv that holds all that information?

Any advice helps, thank you all!

view request
Exporting all peaks and mass accuracy for chromatograms
(3 responses) thomas hopf 2017-07-24
Dear Skyline developers,

I am trying to set up an automated evaluation scheme for peak picking outside of Skyline. For this, it would be very nice to have access to all candidate peaks and their associated mass errors. While I see that information in the GUI, I wasn't able to fully access the underlying data so far.

1) When exporting the chromatograms for all of the precursors, besides the intensities is it somehow possible to get the corresponding mass error/accuracy for each time point? In the reports, I only seem to be able to get the error for the one chosen peak per precursor.

2) For each precursor, is it possible to get a list of all candidate peaks chosen by the Skyline heuristics?

3) I seem to be able to extract some of the above information by exporting the mProphet peak features; however the associated mass errors for the mProphet candidate peaks seem to differ quite substantially from nearby Skyline peaks. How does this occur?

4) Can the mProphet features be automatically extracted using a command-line parameter?

Thanks for your help!
view request
Show Modified Sequence Missing
(2 responses) romeally 2019-09-11

I am trying to do quant on modified peptides and the one webinar says to treat them as proteins to do replicates and get statistics to work. I have done this and it works but for some reason I can't get my final report to show the modified sequence in the table (see attached). I would like the final report to show this since any name for the protein is less than descriptive. Am I not defining something correctly in my document grid?

view request
Unable add Panorama server in Skyline
(5 responses) it-admin-team 2019-09-11

Hello Skyline Support,

We are unable to add our Panorama server in the Tools / Options / Edit List in Skyline (
We are getting the following message "The server is not a Panorama Server".

We've also tried by hostname and with port number.
Please advise.

Thanks, Greg

 Screen Shot 2019-09-11 at 11.32.50 AM.png 
view request
Standard addition calibration for small molecule
(3 responses) J Larose 2019-09-09


I analyse several small molecules that are endogenous. I use the standard addition method to determine what is the initial concentration in the sample in which I spike my standard solutions to build my calibration curve. Then, I adjust the concentration of my curve points to take into account the actual concentration of the curve points, ie the endogenous concentration plus the added concentration. Is there a way to write this in the document grid? My compounds have different added concentrations so I use the concentration multiplier to correct that but I see nowhere where I can add a fix amount of each molecule to the concentration.

Thank you!

view request
Product Ion Selection Without Spectral Library
(4 responses) sa825 2019-08-29

Good afternoon,

I wanted to ask how I can select the best product ion for a peptide in Skyline?

I have been trying to detect heavy labelled peptides using PRM on a Thermo Q Exacitive. These peptides have already been detected using DDA on the same instrument but when I run PRM, I don't get detection of all the peptides and they are sometimes detected at different retention times to what is expected.

To overcome this issue, I considered that there were possibly too many precursors being viewed within the same time window on the Thermo so I have selected 1 precursor per peptide per protein. However, this has only yielded a slight improvement in detection.

I am now considering reducing the number of product ions per precursor to just 1 per precursor. What do you think of this and if so how do I select the best product ion? I have done the Method Editing Tutorial so I know I can rank the product ions using a spectral library but I didn't use one when I created my peptide list as I was trying to do a targeted approach so using one here doesn't seem the right option?

I really hope all of that makes sense?



view request
Saving file to older version of skyline
(7 responses) dsullivan 2019-09-09

Hi all,

I am currently using the docker image for running Proteowizard/Skyline developed (

It's great! Thanks!

Recently Skyline updated to version 19, and this has sadly broken the pipeline since skyline 4.2 cannot open the blank .sky files necessary for running a job in skyline. Is there any way to save a .sky file with an old version tag? (and/or update the docker image)


view request
Mass error and isobaric peaks analysis
(1 response) Vahid F 2019-09-06

Hi all,

I have acquired some MS/MS spectra at 120k resolution in PRM mode on QE-HF to be able to resolve small isobaric ions labeled by one 13C or 2H or 15N. Data files are imported into Skyline and I can see the MS/MS in Full Scan panel (picture attached).

The issue is that due to a ~5ppm mass error, m/z that Skyline is looking at is slightly off. Is there a way to force skyline to look at the peaks and not the theoretical m/z or somehow or set a m/z offset? The attached picture illustrates the issue well.


 mass error.pptx 
view request
How to get standard bar on peakarea view by choosing group by condition
(1 response) drrenugoel 2019-09-07

How to get a standard bar on peak area view by choosing group by condition

Should I normalize the retention time? Will it affect my analysis as there is not much difference?

view request
How to get peak in the upper right panel
(2 responses) drrenugoel 2019-09-07

Thanks for your help. I would like to know how to get peaks in the upper right quadrant.

view request
ion trap Bruker D
(1 response) pucci biagio 2019-09-06

I'm new in skyline. I want to analyse some data produced in ion trap low resolution. the file is in .d from buker . Is it possible to analyze this files with Skyline for label-free quantification?. do you give me please a manual or link where I can study this software?


view request
Calibration weighing error
(1 response) siwei chen 2019-09-06

Hi, I am encountering an calibration weighing issue here. Every time I am trying to apply weighing 1/(x^2), it shows there is an Error: Matrix must be positive definite. Does anyone have experience with this? Thanks.

 weighing error.PNG 
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Peptide ratios to standards
(10 responses) john.leszyk 2013-04-12
Hi Brendan,

  How would one go about getting peptide ratios against a standard peptide (not heavy) within the context of Skyline.


view request
Normalization DIA TIC
(3 responses) dkueltz 2015-03-16
Hi Brendan,
Thanks for the 3.1 release! I was wondering whether there is a way to normalize label-free data against the TICs of DIA MSMS runs (I get those using Bruker DataAnalysis). I have entered a separate column in the Skyline replicates table with the relative TIC integrals for each replicate. But I cannot select that column for normalization under group comparisons. It only shows up as an option for control group annotation and identity. It would be nice to have the option to select this column of DIA TIC values for normalization. I have been using the DIA TICs to normalize each transition in a sample/ replicate-specific fashion to account for small differences in loading or sample ionization after exporting the data to Excel. But with the new group comparison that includes statistics it would be great to be able to do it within skyline to save time.
Thanks for any suggestions,
view request
semi-automated removal of 'interfered' transitions?
(2 responses) Jason Held 2019-09-04


I've got a very large DIA analysis, used mProphet for peak picking, and am wondering if Skyline has any additional features I'm not aware of that help for (semi?)-automated removal of interfered or bad performing transitions such as the y10 in LPDGNGIELCR highlighted in your large scale DIA tutorial.


view request
Average the duplicates' XIC for quantitation
(4 responses) 97 2019-08-29

Hi Skyline team,

We're using Skyline to create standard curve and quantify unknowns for amino acid. We're running triplicates for each standard level and also triplicates for the samples. Is there a way to use the average XIC area for the duplicates when plotting the standard curve and use the average XIC area to quantify the unknowns?

Another question we have is whether we can specify a retention time for L and iso-L so the software can differentiate them.

Thank you!

view request
potential bug decoys
(1 response) Tobi 2019-08-30

Dear all,

i encountered this reproducable issue with decoy transitions.

When adding decoys to the target list they contain exactly the specified transitions (p,y,b) as in the settings. However if they are already added and one tries to change the setting, the change is applied only to targets but decoys remain as they are while they should change in the same way.

Trying to go with refine/advanced/auto-select transitions does not resolve the issue. The already set transitions remain and even change their symbol from red to green line (not sure if this has any further implication and I cannot really tell if skyline then sees the transitions as non-decoy ones).

Hope the attached file helps you.

Best regards,

view request
Can employee in R&D of a commercial company use Skyline?
(5 responses) george wang cal 2019-09-03

I could not find anywhere else to pose this question so if there is a person I should contact please let me know. I am an employee at a commercial biotech company and our team wishes to use Skyline as part of our R&D for proteomics development. Our legal team found two components that cannot be used commercially, thus right now it appears we can't legally use Skyline. These are part of the Apache license:

  1. Thermo-Scientific MSFileReader Library
  2. Waters Raw Data Access Component Library

I wanted to ask if there is any way to remove these components and use the remaining components of the Apache license?


view request
Create Background Proteome
(9 responses) Richard Lam 2019-08-13


I followed the tutorial document 'MethodEdit-3_7' (see attachment, step on pg 6) to create the 'Yeast' background proteome in the 'MethodEdit' skyline document (see attachment). After I clicked 'OK" to digest the background proteome, Skyline stalled and could not proceed with the digestion (see the screen shot in the attachment (Building Background Proteome in Skyline).
I tried several time and restarted Skyline and my computer, the issue did not go away. Please help.



 MethodEdit-3_7.pdf  Building Background Proteome in Skyline.docx 
view request
Could Skyline delete disqualified transitions during importation?
(2 responses) Yao Chen 2019-09-03

Dear Skyline Team,

I am doing exhaustive PTM search with Skyline. However, my computer RAM is too small and the data importation is very slow. Could Skyline have something like "real-time data refining criteria" that deletes disqualified transitions during raw data importing? For example, i don't need transitions with idotp < 0.95, transitions lower than that are deleted during the search.


view request
small molecule support for Agilent Chemstation data files
(6 responses) dgmccaskill 2019-08-08

Are there any plans to support working with Agilent Chemstation data files (not MassHunter) coming from an LC-MSD single quad system? This might be a small niche, but it would be very helpful (at least for me) in the small molecule arena to be able to work with SIM data from Chemstation using Skyline.

view request
Importing raw data to skylin
(2 responses) nfatoukane86 2019-08-31

Kindly I wanted to know if it is possible to import waters raw data into skyline, and if it is possible how to do it? I m using skyline 64bit version 19.1 in window 7.

view request
missing gene names, potential bug
(2 responses) Tobi 2019-08-30

Dear all,

when importing the attached fasta against the human proteome+isoforms with both filtering by gene or protein specificity, the filtering works as intended but the document grid shows empty fields for the Protein Gene (supposed to contain "HSPB1")

This is not a big deal, just so that you know.


 HSPB1.fasta  missing gene name in doc 
view request
Problems about install skyline 19 on Windows10?
(1 response) kaylee xu 2019-09-01

Hi skyline team,
I download a skyline19 package from skyline website, but I can not install it successfully. When I click the setup, It always occurred a problem saying that application download did not successed.

 skyline problem1.PNG  VGR1MU34.log 
view request
Drift time predictor training from SkylineRunner
(2 responses) Aivett Bilbao 2019-08-30

Dear Skyline team,

I'm using the SkylineRunner to extract data from Agilent (.d) ion mobility files, importing a transition list (small molecules format, with RT). Please I have 2 questions:

  1. Is it possible to execute the "Drift time predictor" (using results) from the SkylineRunner?
    I would like to import the ms files, train the Drift train predictor, then re-import the ms files (to have better signal by filtering with the drift time).
  2. Could you provide some details about what the "Drift train predictor" is doing? is it taking the drift time from the most intense ion mobility peak it finds?

Thank you in advance for your help and thank you so much for the great software!

view request
Specify retention time in command line transition list?
(2 responses) dsullivan 2019-08-26

Hi all,

I have an experiment where I know the expected retention time for some MS1 data. Is there a way to add this information to my precursor transition list that I'm importing? Currently I'm importing something like the attached file.

There may be a totally different and better way to do this too, so please let me know if the way I have set this up does not make sense.


view request
local FDR/PEP
(3 responses) Tobi 2019-08-19

Dear Skyline team,

could you imagine including a local FDR value to complement the Q-values in the results grid? (PEP) For me it seems nice beeing able to retrieve the actual chance of a specific peptide beeing a false discovery, but you might know more about limitations and relevance of this.

Looking forward to hear your opinion on this.

Best regards,

view request
Skylinedailyrunner.exe could not save .exp file
(2 responses) shridevi patil 2019-08-28

Hello Skyline Team,
I am working to save experimental file upon executing command on SkylineDailyRunner, but it do not save the .exp file.
below the command i use,
--dir="C: \Users\test\AppData\Local\Apps\2.0\LOETQMLY.BBK\W1GG8G3M.TXA\skyl...exe_e4141a2a22107248_0013.0001_none_ed856a301652adf5" --in="D:\TestData\HV1\docs\" --tran-product-ion-types="p" --exp-method-instrument="Waters Xevo TQ" --exp-template="D:\TestData\HV1\docs\template.exp" --exp-run-length=15 --exp-file="C:\Users\test\Desktop\tt\testAUG29\RT.exp " --out="C:\Users\test\Desktop\tt\testAUG29\" --exp-strategy=Buckets --exp-max-trans=100

The output is as follows:
Opening file...
File opened.
Transition Settings -- Filter > Ion types changed to "p"
Settings > Transition Settings -- Filter > Ion types changed from "p, y, b" to "
Removed: 148 tran
Saving file...
File saved.
Warning: The vendor Waters Xevo TQ does not match the vendor in either the CE or
DP prediction setting. Continuing exporting a transition list anyway...
Exporting method RT_0001.exp
Error: The file C:\Users\test\Desktop\tt\testAUG29\RT.exp could not be saved.
Check that the specified file directory exists and is writeable.
ERROR: Saving the method was unsuccessful.

Do let me know if anything is not supported on Skylinedailyrunner. Because if in given above path I could create new file and edit and save, then ideally runner should also be able to save file.
Please suggest.
Thanks & Regards

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Failed importing results file
(8 responses) sandberg 2019-01-22

Dear Skyline Team,

I am trying to import 4 wiff2 files into a Skyline peptide search but one of the files keep giving me the “Failed importing results file” message, picture attached (Pic1). All wiff2 files are fine when I open them in BioPharmaView so I don’t think that the wiff2 file is corrupt. It does not help to just import a FASTA file as my library and then import the wiff2 file directly.

Could you please help me to figure out what the problem is?

I also attached the problematic wiff2 file.

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overlap DIA
(3 responses) Tobi 2019-08-28

Dear Skyline team,

could you please give out some information regarding overlap DIA? You recommended to use MSConvert to demultiplex DIA data with overlap. However, MSConvert usually results in an error message:

Failed - System.Exception: SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.
at pwiz.CLI.msdata.MSDataFile.write(MSData msd, String filename, WriteConfig config, IterationListenerRegistry iterationListenerRegistry)
at MSConvertGUI.MainLogic.processFile(String filename, Config config, ReaderList readers, Map2 usedOutputFilenames) at MSConvertGUI.MainLogic.Go(Config config, Map2 usedOutputFilenames)

Is this because of acquiring the DIA with overlap and with margins? I also noticed when having overlap DIA with margins that those margin sizes can slightly differ in size after window placement optimization, could that be another potential issue?

I know that potential debugging MSConvert is not your job, but can you please tell me which variants of overlap SWATH you tested and what sould be working (margins, optimized placement...).

If you are interested please find the uploaded raw file for you to read the DIA scheme.

Best regards,

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Import transition list with modifications that have not been pre-specified
joshuasmith 2019-08-28

Hi, I am doing DIA to discover novel adducts on a specific residue of a certain peptide. We have previously done PRM experiments to search for pre-specified adducts to that residue (e.g., Cys adducts, or oxidation, or various electrophilic adducts), where I would manually enter each pre-specified adduct into the "structural modifications" list on the Modification tab in the Peptide Settings menu. However, now that we are doing discovery experiments, I have a lot of potentially novel adducts that i have not already entered into the structural modifications list a priori. When I try to import a (properly formatted, attached) transition list with novel adducts, I get an error message for each adduct that Skyline cannot automatically reassign to a current "structural modification" entry:

"Precursor m/z 903.4449 does not match the closest possible value 895.0976 (delta = 8.3473), peptide ALVLIAFAQYLQQCPFEDHVK. Check the Modification tab in the Peptide Settings, the m/z types on the Prediction tab, or the m/z match tolerance on the Instrument tab of the Transition Settings."

Is there a way to have Skyline automatically create new structural modification entries for novel adducts in an imported transition list?

Let me know if this isn't clear, or you need more info.


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Peak quality score
(2 responses) Zac 2019-08-27

I am looking at ways to automate the flagging of problem peaks for manual inspection. It would require some form of peak quality score. Wondering if anyone has tried to tackle this before? Or if there is way to get this indirectly in skyline, possibly from difference between a transformed and untransformed peak shape.

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is there possible to improve modification types of skyline?
(1 response) spklongz 2019-08-27

Hi all,

Recently, I studied modifications of vaccine including methylol adduct (+30.011 Da for lysine, arginine, histidine, tryptophan, cysteine, asparagine or glytamine) and Schiffbase (+12.000 Da for lysine). One relevant manuscript of these modifications is attached.

I want to predict modified peptide using Skyline, while there is no methylol adduct or Schiffbase modification choice in Skyline. Is there possible to take some improvements?

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Multiple precursors per peptide
(1 response) sa825 2019-08-27

Good morning,

I was wondering why there are 3 precursors of diff m/z’s for the peptide? And is it alright remove the ones with irank 2 & 3?

The reason I am asking about this is because when I do a DDA run using the same peptides on the Thermo Q Exacitive, my peptide is detected at the expected retention time but when I do a targeted run (PRM), the Thermo is unable to detect my peptide.

I fear its because the Thermo is looking for 3 precursors at the same retention time when it should only be looking for one?

I have attached an image to show what I mean.



 3 Precursors per peptide.png 
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Close all chromatograms
(3 responses) pier-luc plante 1 2019-08-26

Dear Skyline Team,
is there a keyboard shortcut to close all chromatograms or is it possible to add one? I am working with more than 1500 samples and the close all button in the menu is difficult to access since it's located at the end of the sample list. Also, is there a way to disable opening all chromatograms when opening a skyline project? I am working with small molecules.

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Accurate Values for peak intensity
(9 responses) sa825 2019-07-23

Good morning,

I am using Skyline on Windows 10
I wanted to ask how you would accurately read off the intensity of a peak/peptide in skyline?
I can read it off by eye but II feel as though there is probably a more accurate method?
I have attached an image so you can see what I mean.

Also, how do I view values for the area of the peak?



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Not able to see libray ion in the result file and how to do the quantitation of the peaks
(3 responses) drrenugoel 2019-08-23

Kindly see the attached file. I have built the library in the peptide setting tab. However, I am not able to see the library ions in the attached file (skyline_doubt). some setting has been changed as I used to get it. Kindly see the attached (skyline2).

I have run these samples on 5600 sciex in MRM-HR mode and now want to do the quantitation. Could you please let me know how to take it out peak area for the same.

 skyline2.JPG  skyline_doubt.jpg 
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CE method export for application in MassHunter controlling an Agilent 6490 instrument
(5 responses) azad 2019-08-22

Hello support team,

I've searched the forum and exhausted my wit to find a solution, other than brut force which is borderline "manual" method development.

-I am running Skyline-daily and MassHunter version B.07.01 build 7.1.7112.4 SP1 which is controlling an Agilent 6490 instrument.
-skyline file consists of 2458 transitions corresponding to 389 peptides
-transitions per peptide varies from 2 to 10
-My intention is CE optimization for every transition and I have only included heavy stable isotope labelled peptides in the file
-In the transition settings tab - collision energy tab - Agilent QQQ is selected
-Regression values exist for charge sates 2 and 3 but not 1 and 4, I do have the latter for a subset of the transitions in the file
-Step size is 3 and step count is 2
-When exporting methods the max concurrent transitions is set to 60 which results in 20 methods each containing ~600 total transitions
-When I attempt to load a method in MassHunter the Agilent6490 switches from online to offline
-To attempt to diagnose the problem I manually shortened the number of transitions for a method to 450 and was able to run the method. However, when I used the same approach for a subsequent method the instrument once again went offline.
-I manually looked over the transitions in excel to ensure the maximum concurrent transitions were actually 60 or less and appears they are.
-At this point I can't think of anything else expect to manually add transitions in the QQQ tab until the instrument goes offline and then scale it back and run the method. However, I would prefer to avoid this if there is a "smarter" solution.

Thanks in advance for your assistance.


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What's wrong with my Thermo RAW files
(1 response) solis 2019-08-19

Dear support team,

lately, some of my RAW files (independent of experiment or date) are not being imported by Skyline. This is the error message I always get:

Failed importing results file 'Ref_xxx_VS_090819.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=45461": [RawFileImpl::getMassList()] Object reference not set to an instance of an object.

Can you help me figure out what's wrong? If I convert the files to mzXML, Skyline does upload them successfully.


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Issue importing waters RAW files, Unhandled Exception: Invalid Function Type
(1 response) 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,
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Unable to process chromatograms for the molecule
(12 responses) chinmaya k 2019-08-11
Dear Team,

I was trying to analyse 2 DIA files (technical replicates) using Skyline (version under proteomics interface. When I start importing DIA raw files, skyline come up with an error (please find the attached text file for the same). In brief, the error says "Unable to process chromatograms for the molecule 'AGEEGGSVGSGVFLIGR' because one chromatogram ends at time '' and the other ends at time '48.8989682017979' ".

What does this mean? How can I rectify this issue?

Request you to resolve this issue.

Thank you,
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Order by document settings
(2 responses) mcqueen p 2019-08-19

I have been using Right-Click -> Order -> "Column Name" to order small molecule samples by specific variables within Skyline. The only options that seem to work are "Document" and "Acquired Time". I have previously been able to order by custom definitions that I made in "Document Settings" -> "Annotations" but they do not seem to work anymore ie when I select the custom options they do not affect the sample ordering. This seems to persist across different .sky files and I have tried starting from the beginning with brand new analysis files with no avail. I recently updated to Skyline v. (with Windows Pro 64-Bit) from the previous version, could this have something to do with it? I'm not sure what I'm doing incorrectly.

Thanks for any help,


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Using Skyline to monitor glycopeptides in DIA
(14 responses) rmeccariello 2019-08-13


I am a newbie Skyline user and need some help using Skyline. I am monitoring glycopeptides with a DIA method. I'm looking at 3 different peptide sequences, and monitoring the same 13 glycans on each of those peptides. In order to calculate relative abundance, I'm monitoring the Y1 ion (full peptide backbone + GlcNAc) and the Y1+Fuc ion (full peptide backbone + GlcNAc+Fuc).

I'm not sure how to define my peptide settings and transition settings. Where do I input the parent mass (full peptide backbone+full glycan), and where do I specify the mass of the Y1/Y1+Fuc ions that I want Skyline to look for?

Thank you!


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ssl search results not being imported
(3 responses) solis 2019-08-16

Dear support team,

I'm trying to create a spectral library from an ssl file. Everything runs normally until the upload of the fasta file, where it says that the number of matches is 0. Can you help me figure out the issue?

So I'm doing this via: File -> Import -> Peptide Search. Please find the necessary files attached.

Thanks for your help!

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