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.

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Showing: limited to 100 requests
Collision Energy Optimisation on Agilent Ultivo QQQ
(2 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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PRM tutorial question
(4 responses) tun-li shen 2021-04-12

I am new to Skyline. Tried to follow PRM tutorial but missing the BSA library (see attached Word doc) when open the BSA example...

 Skyline questions_4.12.2021.docx 
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CCS measured vs. CCS explicit (or library)
(1 response) tim causon 2021-04-09

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

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

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

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

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Precursor Intensity from MS/MS spectrum
(2 responses) bseale 2021-04-09


I have a PRM skyline file for 100 proteins and 300 peptides acquired on a Bruker timsTOF. I have particular selections for all the various transitions and when I add my results in everything appears to be detected etc. correctly. However, I wish to compare the intensity of unfragmented precursor transitions to the other selected transitions (b, y ions). When I add the various areas or intensities to reports the precursor transitions always report the total area of the chromatogram while the fragment transitions appear to report the expected (lower) value.

My question is simple. From the screen shot, how to do I get a report that will tell me the intensity or area (either is fine) of the MS/MS precursor (red) so I can compare it to those of the other fragment transitions (blue)?

 Screenshot 2021-04-09 094849.png 
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Building a spectral library using msp
(5 responses) laura corveleyn 2021-02-02


I am trying to build a spectral library in Skyline using an msp file based on predicted spectra. Since we work with histones, we derivatize our peptides using propionylation, so all unmodified lysines and N-terms are blocked with a propionyl group. Therefore, Propionyl (K) and Propionyl (N-term) are fixed modifications, unless another variable modification is present. However, Skyline does not seem to recognize the modification "Propionyl" from the msp, although I also defined it as a modification in the peptide settings. All other modifications (e.g. Acetyl, Dimethyl,..) that I defined, are used correctly in the library. After some research, we figured out that Skyline recalculates the precursor m/z for each peptide (based on its sequence) and doesn't use the one that is stated in the msp. So for a peptide that carries a propionyl group, the m/z is recalculated as if the peptide would be unmodified because Skyline does not recognize the propionyl.

In the attachment you can find a short example of a predicted msp and the Skyline document with the corresponding library I built with this msp. In the msp you can see that the Propionyl group in the header is stated in the exact same way other modifications are, so I don't understand where the problem is.

Thank you in advance for helping me out!



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Missing retention time when importing msp file to build SL
(2 responses) laura corveleyn 2021-04-08


First of all I would like to thank the entire team for the features that were added in the last Skyline daily update!

I still encounter some problems when importing an msp file to build a spectral library. Although the retention times are stated in the headers of the msp, they are not included in the SL. One of my colleagues, Bart Van Puyvelde, submitted a request on this a while ago and it turned out Skyline wasn't able to read the retention time in the header. Would it be possible to fix this?

You can find an example of an msp file I am using in the attachment.

Many thanks!

Kind regards,

Laura Corveleyn

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Feature Request: Background Colors
(2 responses) jdevitt 2021-04-07

I am using the Skyline version 64-bit I was wondering if in the future an option could be added to change the background color of the Skyline chromatography profile and peak area/retention time graphs from white to another color, especially a black or gray option. When I use Skyline for hours at a time it would be nice to have a background color that is a little less harsh on the eyes. Not urgent, just something that would be nice to have!


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Error in Accuracy Calculation when Requiring Sample Dilution Factor
(1 response) Will Thompson 2021-04-07

Dear Skyline friends. Reporting a bug in Skyline calibration, wherein the accuracy is calculated incorrectly. Specifically, if linear regression against any external calibration data is performed and a 'sample dilution factor' is required for a quality control sample which may use a different volume than the standard curve, the calculated concentration is updated correctly, but the accuracy is not correct. This causes problems when a sample or QC volume (for instance) is different than a calibrator volume, and a sample dilution factor is needed. This hasn't been noticed before on my end because accuracy isn't usually calculated for unknowns, however i recently had a case where a different prep volume was needed for some samples in a batch, and therefore i prepared an additional set of quality control samples using the same approach as the samples needing dilution, to make sure the backcalculated concentration was accurate using the different sample volume. In the skyline document attached, you will see four QC runs which are called "QC1_45uL_01" (and others with 45 uL in the name) where (for example) the target concentration is 1 nM, the calculated concentration is 1.006 nM, but the accuracy is calculated as 110.7%. A sample dilution factor of 1.1 was used. Accuracy is calculated correctly for all other QCs which did not require a sample dilution factor. It seems like the accuracy calculation within skyline may be using some method other than comparing the "calculated concentration" field to the "Analyte Concentration*Concentration Multiplier" (which seems like would be the correct approach)

Thanks for looking into this.


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Determine sequence coverage for proteins
(4 responses) simon jaag 2021-03-23

I am using Skyline (64-bit) and want to determine the sequence coverage for all my proteins.
If I hover over the protein name in the Targets pane I can see which peptides were found for each protein but is it possible to get the % coverage of the peptide and export a list of the covered peptides from Skyline?

For this workflow I used IDA from a SCIEX QTOF 5600 system which were converted to mzXML format and as search engine Comet (pep.xml) was used (both out of the Trans-Proteomic pipline software suite).

Best regards,

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Question: How is the background calculated?
(1 response) hjl 2021-04-06

Hi, I am using Skyline version 64bit

Within the process of calculating LOD for standard, I used Area and background value from the transition result report.

Based on the exported result, the S/N value was less than 3 but I could still clearly see the peaks with no significant noise between it.

I'll attach the chromatogram which had 2.11 as the S/N value .

If you don't mind, can I know how the background acquired based on some other values or the algorithm to obtain it.

Thank you for your support as always.

 LOD chromatogram.PNG 
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(2 responses) rozas 2021-03-31

IMS filter rozas EDIT 2021-03-31

Hello Skyline team!

I would like to ask you something related to the drift time filter. I have made up a transition list including drift times but anyways, I should add an ion mobility library through "Transition settings-ion mobility- add" in order to filter my results taking into consideration the drift times. Is this correct?

On the other hand, I have seen that when I create an ion mobility library using drift times, the CCS is calculated automatically but, How could add the data of CCS calibration that I obtained experimentally in order to correct these values?

Thank you so much,

Kind regards

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Build spectral library from MSfragger SpecLib workflow
(1 response) af1234 2021-04-04


I am trying to build a spectral library from the output of MSfragger (SpecLib workflow). I do have the following files

  1. pepXML for all files (both individuals and combined)
  2. speclib format output
  3. osw library

However, when I try to build a DDA-Pasef spectral library using the pepXMLs there is an error because Skyline is looking for an mgf/mzXML.
When I try to use speclib the library builds but is empty and finally when I use the .tsv (which works in openswath) seems a lot of sequences are not recognized (see screenshot).
I was wondering which one would be the easiest way to build a library from these files and if any changes/conversion needs to be done to the files prior to importing into skyline.

 Screen Shot 2021-04-04 at 10.06.41 AM.png 
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Retention times - Scheduling
(1 response) Gao 2021-04-02

I am just wondering whether you can add back the concurrent transitions vs scheduled time plot for "Retention times - Scheduling". I think the concurrent precursors vs scheduled time plot is great for PRM, but the concurrent transitions vs scheduled time plot will be the best choice for SRM, especially when the number of transitions per precursors are not the same across all the peptides. Maybe we can have both in Skyline Daily?

Thank you!

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FAIMS CV data analysis
(7 responses) Keri 2021-03-31

Hopefully a very straightforward question. I'd like to pick the best CV for each peptide identified and have been trying to follow this prior post However, when I go to peptide settings>prediction there isn't a ion mobility prediction button apparent. Is there another place to find this feature or a way to add it? Thanks!

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fail to recognize spectral library from NIST
(6 responses) 851813663 2020-10-13

Dear nick
I have downloaded the Rat's spectral library from NIST, it was in msp format, and when I wanted to use the skyline to build the library, I found that the software could not recognize this format, and I was very eager for your help.
Thank you sincerely

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Error from importing Peptide Shaker mzid
(11 responses) lauh2 2021-03-30

Hello Skyline team,

I am trying to build a library using the mzid from Peptide Shaker. However, Skyline (64-bit) (a7a9e8c4f) is giving me the following error,

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

Command-line: C:\Users\HoTak.Lau\AppData\Local\Apps\2.0\8MPRNX2K.0X7\9EA9LC5J.M7P\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0.99 -i testShakerOut -S "C:\Users\HoTak.Lau\AppData\Local\Temp\tmp4C0F.tmp" "C:\Users\HoTak.Lau\Desktop\MS_tools\PeptideShaker-2.0.16\resources\example_dataset\data\testShakerOut.redundant.blib"
Working directory: C:\Users\HoTak.Lau\Desktop\MS_tools\PeptideShaker-2.0.16\resources\example_dataset\data
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156

I generate the mzid using PeptideShaker 2.0.16 (2.0.14 and 2.0.18 gave the same error) -> Open Example -> Export -> Follow Up Analysis -> Skyline Export -> Export as mzid, to the PeptideShaker example folder, as mzid v1.1.gzip -> extract the .mzid using 7zip -> import to Skyline.

Is there anyway to get around this?

Thank you

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(12 responses) jmr 2021-03-31

I would like to input a sequence that has 2 AAs linked (G1 to D) by loss of water making a loop: GNWWHHSSSPDWFHWWNTSPHTF
I tried to do this by going to modify and link the 2 peptides, but then I cannot reopen the file. Not sure what I am doing wrong.
I then want to see all the possible b and y fragments and then import a *.raw file from Thermo LTQXL-Orbi to identify the actual fragments. This file was acquired using DDA method with MS1 collected in orbi and MS2 and MS3 in LTQ trap.

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The values of idopt and dopt
(1 response) liudan 2021-04-01

Dear Skyline Team:

Can I export the values of idopt and dopt? And how will I do?

Thank you!

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No enzyme option
(3 responses) Jason Held 2012-02-25
Hi Brendan,

I’ve got some data from samples that were digested with less-specific enzymes such as chymotrypsin, pepsin, thermoylsin etc that I’d like to do MS1 filtering analysis on and keep intact the amino acid numbering for each peptide in a protein. No enzyme is completely specific and i'd like to assess the reproducibility of peptide generation, and I think your MS1 platform is ideal for testing these less-specific enzymes with a targeted proteomics approach. For example, chymotrypsin cuts at far more than the 5-6 putative 'specific' cleavage sites that most people use under typical digestion conditions. At the end of the day, targeted proteomics is more about reproducibility than specificity.

I did my mascot searches with 'no enzyme' cleavage and would like to move all my peptides over to Skyline. However, I hadn't realized beforehand that there is no ‘no enzyme’ option in Skyline. I tried putting all amino acids cleavages sites, but then I’m limited to 9mers since I can only have 9 missed cleavages. I then tried making a background proteome and adding spectra from the library explorer, but that requires an enzyme to keep the amino acid info intact. I could make peptide lists and paste them in – however, this loses the amino acid # info and would be a nice feature to have. I'd suggest including a 'no enzyme' option in Skyline.

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DDA and PRM analysis
(3 responses) dms73 2021-03-29

Hi -

I am new to Skyline and would like some help getting started. I have number of DDA experiments acquired on a Thermo Eclipse that I searched using Mascot and have created .dat files. I would like to create a library with data such that I can probe my PRM experiment acquired on a 480 Exploris which included targets ID'd from the previous DDA results such that I can determine if I evaluated these targerts in my PRM analysis. Can anyone provide a workflow or point me to the appropriate tutorial ?

Thanks !!


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Ion mobility value not displayed in Spectral Library Explorer
(7 responses) isabel marquardt 2021-03-24

Dear team,

I am a very beginner using Skyline. I tried to build a new spectral library with an istotopically-labeled peptide standard measured via IMS-MS/MS on a TimsToF machine. Afterwards I did a PEAKS DB Search and exported the Spectral Library for Third Party (Skyline). The library in Skyline was successfully built but I miss the ion mobility information.

I've checked some tutorials but couldn't find a helpful information. Could you please help me?



 Screenshot_Spectral Library Explorer.png 
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y2 and b2 ions for quantification
(2 responses) nicolas pierre 2021-03-26


What do you think of b2 and y2 in SRM for quantification ? I follow some b2 and y2 ions which sometimes are intense et show a good co-elution.
Is that a problem to use these ions for quantification ?
Thank you for your answer,


view request
Problem with precursor selection in MSconvert
(3 responses) Liyan 2021-03-25

Hello Skyline developers,

I have a project where the DDA spectra libraries and quantitative DIA runs were acquired some time ago. Recently I thought of using PECAN workflow to improve library coverage and re-process the DIA files. I thought having an inclusion for iRT peptides into PECAN GPF runs would help to improve RT alignment in case of LC drift (true enough we saw a delay of 4-5 min). I did these on an Orbitrap Fusion:
Experiment 1: DDA, triggering MS2 if iRT peptides are detected (max 5 scans loop)
Experiment 2: MS1 on GPF range
Experiment 3: overlapping 4 m/z GPF isolation windows

I thought to then selectively demultiplex GPF windows in MScovert while preserving iRT MS2 scans, and I'm running into problems with this. Here are some of the parameters I tested on the 900-1000 GPF run.

--filter "mzPrecursors [487.3,644.8,683.8,547.3,669.8,683.9,699.3,726.8,622.9,636.9,776.9]mode=exclude" --filter "demultiplex optimization="overlap_only"

--filter "mzPrecursors [487.3,644.8,683.8,547.3,669.8,683.9,699.3,726.8,622.9,636.9,776.9]mode=exclude" --filter "demultiplex minWindowSize=2"

These gave the same error:
Error writing run 1 in "20210318_NAFLD_Depleted_3_30k_Pecan_900_1000.raw":
SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.

Then I thought to have the GPF windows as mzPrecursor inclusion instead:
--filter "peakPicking [vendor msLevel=1-2]" --filter "mzPrecursors[902.6602,906.6620,910.6638,914.6656,918.6675,922.6693,926.6711,930.6729,934.6747,938.6766,942.6784,946.6802,950.6820,954.6838,958.6857,962.6875,966.6893,970.6911,974.6929,978.6947,982.6966,986.6984,990.7002,994.7020,998.7038,1002.7057,900.6593,904.6611,908.6629,912.6647,916.6666,920.6684,924.6702,928.6720,932.6738,936.6756,940.6775,944.6793,948.6811,952.6829,956.6847,960.6866,964.6884,968.6902,972.6920,976.6938,980.6957,984.6975,988.6993,992.7011,996.7029,1000.7048]mode=include" --filter "demultiplex optimization=overlap_only" --filter "demultiplex minWindowSize=2"

resulting in the error:
[SpectrumListFactory] Unknown wrapper: mzPrecursors[902.6602,906.6620,910.6638,914.6656,918.6675,922.6693,926.6711,930.6729,934.6747,938.6766,942.6784,946.6802,950.6820,954.6838,958.6857,962.6875,966.6893,970.6911,974.6929,978.6947,982.6966,986.6984,990.7002,994.7020,998.7038,1002.7057,900.6593,904.6611,908.6629,912.6647,916.6666,920.6684,924.6702,928.6720,932.6738,936.6756,940.6775,944.6793,948.6811,952.6829,956.6847,960.6866,964.6884,968.6902,972.6920,976.6938,980.6957,984.6975,988.6993,992.7011,996.7029,1000.7048]mode=include

Removing demultiplex optimization and keeping only min window size appeared to work:
--filter "peakPicking [vendor msLevel=1-2]"--filter mzPrecursors[902.6602,906.6620,910.6638,914.6656,918.6675,922.6693,926.6711,930.6729,934.6747,938.6766,942.6784,946.6802,950.6820,954.6838,958.6857,962.6875,966.6893,970.6911,974.6929,978.6947,982.6966,986.6984,990.7002,994.7020,998.7038,1002.7057,900.6593,904.6611,908.6629,912.6647,916.6666,920.6684,924.6702,928.6720,932.6738,936.6756,940.6775,944.6793,948.6811,952.6829,956.6847,960.6866,964.6884,968.6902,972.6920,976.6938,980.6957,984.6975,988.6993,992.7011,996.7029,1000.7048]mode=include" --filter "demultiplex minWindowSize=2"

... though his produced an output mzML file that was twice the size of the original .raw file (1.02GB vs 478 MB). Importing the mzML into Walnut gets stuck at "reading standard format FASTA database" on the console with no progress in the bar showing "Converting files" on the right over the afternoon. I recall from a previous exercise on Walnut should display a xxxx/xxxxx spectra converted, and feel like the conversion didn't really proceed correctly. Is there a way to salvage my GPF files?

Is there any way to save my GPF runs?

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Collision Energy warnings when exporting a Bruker tims-ToF MS method
(1 response) Susan Abbatiello 2021-03-25

Hello !

I just realized that when I try to export a Bruker tims-ToF MS method, I don't get an error if a different Collision Energy prediction has been indicated in the "Settings, transition settings, prediction" tab. The Skyline file I was using had a TSQ Vantage selected for the CE prediction, but I didn't get any message when I was exporting the method for a Bruker tims-ToF MS format. It would be super awesome if a warning came up to tell me I'm being stupid! I'm pretty sure the CE prediction should be listed as "none" for the tims-ToF MS, since the CE is populated by the instrument control software in the method.

Many thanks!

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Ion mobility libraries
(10 responses) Susan Abbatiello 2021-03-23

Hi There!
I have a couple requests when making ion mobility libraries. Some of these features may already in there, just haven't found them yet!

  • Can the m/z of the peptide please be listed along with the sequence and the m/z in the library, please?
    -- Is it possible to select a single data file from which to "use results" for pulling CCS and 1/k0, instead of using all files (or an option to select one or use all?


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Error: No matching mod
(3 responses) valerie huhle 2021-03-25

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

 msms.txt  Error-iodo.txt  modifications.local.xml  modifications.xml 
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any idea about the transitions of MFDCTNMPSPCHTK
(5 responses) xinqun wu 2021-03-25

i want to use MFDCTNMPSPCHTK as a surrogate peptide to develop a quantitative LCMS method. get transitions (2+ or 3+ )from skyline suggested. but none of them showed the peak. MFDCTNMPSPCHTK has two cysteines and two Prolines, what modifications do i need to add into the structure setting?

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Suggestion to Filtering Document Grid and Results Grid
alejandro.cohen 2021-03-25

Hi Skyline team,

It would be very helpful if the Document Grid and Results grid included within the filters, a dropdown menu with the populated items already contained in each of the columns (very much like Excel has when you choose to sort columns).

For example, when you choose to sort the Molecule Name columns, have a dropdown menu of the Molecules already populated in the Skyline file with their corresponding checkboxes: Eg: Adrenaline, Noradrenaline, etc.

That would save substantial time instead of having to filter by: "Starts with": Adren... etc.

This would be great since preparing to reports for customers takes substantial time. Maybe this could be added to the wishlist? Not sure if this is easy to implement... All I've coded was BASIC in 1997 ;-P


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Skyline not starting
(1 response) m kuppusamy 2021-03-25

I am unable to open skyline, nothing happens when I click on the icon in the start menu. I am using Windows 10 OS Enterprise LTSC, intel i5-9500 CPU @ 3 GHz, RAM - 8 GB and it is a 64 bit OS. Though I never do a full analysis on this computer, skyline used to at least work. Even when I click run as admin nothing is happening. I already tried uninstalling-reinstalling and restarting the computer. Neither switching the antivirus off nor installing the admin version helped. Skyline Unplugged seems to be working but when I click the setup that is in the folder I get an error message saying that an application with the same identity is already installed but cannot find any skyline file in the program folders and the control panel is also not showing any skyline version is installed. What could be the reason for this problem? It just happened out of nowhere.

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IMS filtering on monoisotopic mass is not working
(13 responses) Felina H. 2021-03-24

Hi skyline team!

I have a question regarding IMS filtering. In my skyline file I extract the two highest isotopologes. When comparing the Total Area export from before IMS filtering and after IMS filtering, it is getting smaller as expected. But when looking at the Area export for the monoisotopic mass and [M+1], only the area for [M+1] is changing but not for the monoisotopic mass. I attached an exemplary screenshot for one compound. As you can see for both the monoisotopic mass and the [M+1] there are signals outside the IMS filtering windows. Shouldn't the area change for both m/z after filtering?

Best, Felina

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CE optimization _transition list export
(6 responses) Sandy_MRM 2021-03-23

I am trying to optimize the collision energy for my peptide standards, but after exporting the transition list, I am getting very low values as attached in the file. I am also attaching skyline file 
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Retention Time shifts using deuterated internal standards.
(7 responses) alejandro.cohen 2021-03-23
Hi Skyline people,

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


 Screen shot2.docx 
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Ion Mobility Library generation error
(2 responses) Susan Abbatiello 2021-03-16

Hi There!

I'm trying to use Skyline to optimize FAIMS CV values (using CV optimization, similar to CE optimization, from Skyline). After importing the results and observing which CV steps generate the best transmission, I am trying to pull the best FAIMS CV into an Ion Mobility library. When I click "use results", I get the following error. Any advice?


System.Reflection.TargetInvocationException: mismatch in precursor values ---> pwiz.Skyline.Util.AssumptionException: mismatch in precursor values
at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 2055
at pwiz.Skyline.Model.Results.IonMobilityFinder.ProcessChromInfo(ChromFileInfo fileInfo, ChromatogramGroupInfo chromInfo, PeptidePrecursorPair pair, TransitionGroupDocNode nodeGroup, Single tolerance, LibKey libKey) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\IonMobilityFinder.cs:line 248
at pwiz.Skyline.Model.Results.IonMobilityFinder.ProcessFile(ChromFileInfo fileInfo) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\IonMobilityFinder.cs:line 218
at pwiz.Skyline.Model.Results.IonMobilityFinder.FindIonMobilityPeaks() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\IonMobilityFinder.cs:line 124
at pwiz.Skyline.Model.IonMobility.IonMobilityLibrary.CreateFromResults(SrmDocument document, String documentFilePath, Boolean useHighEnergyOffset, IProgressMonitor progressMonitor) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\IonMobility\IonMobilityLibrary.cs:line 322
at pwiz.Skyline.SettingsUI.IonMobility.EditIonMobilityLibraryDlg.<>c__DisplayClass49_1.<GetIonMobilitiesFromResults>b__0(IProgressMonitor broker) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\IonMobility\EditIonMobilityLibraryDlg.cs:line 582
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 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, Action1 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.SettingsUI.IonMobility.EditIonMobilityLibraryDlg.GetIonMobilitiesFromResults() in C:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\IonMobility\EditIonMobilityLibraryDlg.cs:line 586

view request
Normalize area values?
(2 responses) alejandro.cohen 2021-03-19

Hi Skyline people,

I'm performing an small molecule targeted analysis. Quantification done using calibration curve and spiked in internal Heavy standards. I'm a little confused about the Document Grid results. Why do I get a Normalized Area Values for the Heavy standards? I understand the normalized values for each molecule are total area divided the corresponding internal standard signal for each replicate?



 Screen shot.docx 
view request
Using SIS-only calibration curve to quantify light or light/SIS ratio
(3 responses) david schmidt 2021-03-18


I'd like to use a calibration curve generated with heavy (SIS) peptides only (no light peptides present) for the quantification of either light peptides or light/heavy ratios (SIS peptides as internal standards). Is that possible?

Thank you!


view request
water loss not shown for all peptides
(1 response) sophia foehr 2021-03-18

we are looking at MHC class I peptides and have often water loss (once or even twice) in the transitions. We can see the loss for some of the peptides but not for all of them e.g. DLLM(ox)GTLGIV (see attached skyline file). Do you have a solution to our problem?
Thank you,
Kind Regards,
view request
rdotp meaning
(3 responses) hwang2 2017-04-26
I'm trying to explain the rdopt value in a manuscript for publication. is the following statement correctly explain the rdopt?

The result dot product (rdotp) ( i.e. the dot-product between the analyte precursor and the isotope labeled standard precursor) that determines the spectral similarity between the native and the labeled peptide for these two peptide markers was 0.94 and 0.99 respectively.
view request
Transition list export for optimizing compensation voltage not limiting to transition limit set in export window
(2 responses) Susan Abbatiello 2021-03-12

I'm using the CV optimization tool and I'm trying to export transition lists that don't exceed 50 concurrent transitions (rough tune, a total of 7 CV steps). Skyline is not limiting the number of concurrent transitions for the CV optimization. Skyline file attached. I'm using a TSQ Altis with FAIMS.

Also, it would be awesome of the number of concurrent transitions in the scheduling graph provided in the transition export window would update to include the number when doing CV optimization.

view request
Integrator Options
(1 response) justyna zakaria 2021-03-15

For certain samples I run, I have extremely long tailing. This is not due to a contaminating peak. Typically I include the whole peak from baseline to baseline (Skyline usually finds this and I don't often need to manually adjust peaks), but in some samples when the tailing takes a very long time to come to baseline this ends up seemingly increasing the noise in the sample. Does Skyline have integrator options like a threshold setting than can be adjusted which is computed from the 2nd derivative which approaches zero as the tail becomes horizontal?
Attached is a photo of such a troublesome sample showing the 3 precursors, but if you look at individual transitions, the same tailing trend is evident across the board.

view request
unsupported score in MZid
(2 responses) af1234 2021-03-14

As previous posts in the forum I am trying to build a library from mzID files.
Specifically from this publication (mzIDs accessible here
The files have been searched with by COMET and/or X!Tandem, and filtered with an iProphet score of ≥0.9.
Build library fails with unsupported score and when looking one of the files seems there are 'normal' comet scores like xcorr and so on.
Should I convert it to another format?


view request
tims method export clarification
(3 responses) Susan Abbatiello 2021-03-12

Hi there!
I'm not sure what the "MS1 repitition time" means when trying to export a method for the Bruker tims-ToF. Is it possible to add details to this?


view request
Select samples in results table
(1 response) io 2021-03-12

Dear all,
Is it possible to select which samples I want to analyze in the results table?
I ususally go to manage results and save with a different name but it would be more useful for me to select the samples in the results table
Thank you in advance

view request
AvG global refinement: missing temporary file
(1 response) willems533 2021-03-09

Dear Skyline team,

I was recently trying to run the AvantGarde external tool (latest version and ran into some errors.
I have nine samples loaded and initially ran the Export Report -> AvantGardeDIA_Export. However, when trying to create the MySQL database from this via 'From_CSV_file' it could not load in the CSV due to incorrect headers. Headers in the CSV were for instance 'Fragment Ion' while it was anticipating without a space I think 'FragmentIon'.

As the exported file was 27 GB omitting the spaces in the header was quite annoying. However, I managed to now generate the MySQL using the 'From_Current_Skyline_File' option that is normally recommended for smaller analysis. When now trying to run the GlobalRefinement option however it gives me :

Warning messages:
1: In connection_release(conn@ptr) :
[1] "Parallel: Done!"
There are 1 result in use. The connection will be released when they are closed
2: In connection_release(conn@ptr) : Already disconnected
Error in file(file, "rt") : cannot open the connection
Calls: Read_AndFormatResults -> read.csv -> read.table -> file
In addition: Warning message:
In file(file, "rt") :
cannot open file 'C:/Users/pawil/Documents/Manuscripts/2021 - DIA2/DIA/MQ_lib/AvantGardeDIA/DataFormatting/TempFiles/Report_GR_ColNamesName_Tag.csv': No such file or directory
Execution halted

I noticed a very similar support topic here '' where the solution was to have multiple files, which is already the case for my data.

Thanks in advance for your help,

view request
Signal/Lärm für die Nachweisgrenze
(3 responses) alexandria balac 2021-02-26

Hallo , ich brauche bitte eine Unterstützung um das Signal/Noise zu ermitteln bei der Peptidmessungen . Wollte meine Nachweisgrenze darüber ermitteln.

Wo finde ich den Wert bzw. die Anzeige?

Vielen Dank für die Unterstützung Alexandria Balac

view request
Error in Turtorial DdaSearchMS1Filtering
(4 responses) Irene_G 2021-03-03

I want to learn how to analyse our DDA files from Waters SynologyG2Si and have therefore run tutorial Skyline DDA Search for MS1 Filtering. When run the tutorial I got error; "Unable to open file QE_140221_02_UPS1_300fmolspiked.mzid (file does not exist)". I use Skyline-daily (64-bit) (32d27b598). The computer 64-bit, 64.0 GB RAM with Windows 10 Pro. I got the same error when I try with my own files.

Please, can someone help me to solve this?


 Spectral Library Import Peptide Search.PNG 
view request
Import Peptide Library from PEAKS Online from TIMS TOF data
(14 responses) ed3 2020-10-09


I have been trying to import a peptide search from PEAKS online into Skyline but I keep getting an error and I was hoping somebody could please help me understand what is going on. The spectrum was acquired with a Bruker TIMS TOF, and when I try to import the pep.XML file in the "Peptide Search" option, I get an error saying "The .pep.xml file is not from one of the recognized sources." I have the IMS-TOF spectrum in .mzXML format in the same folder as the pep.xml file, and I am not sure why I get this error.

Thank you very much for your help.


view request
Older Skyline versions
(1 response) n-ochiai 2021-03-03

Hello, Skyline support,

I would like to install older versions of Skyline, but I cannot find any download links. Is it still possible to get them?

When I tried install Skyline 20.2, an error occurred installing .NET Framework 4.72 (attached error massage).

My company laptop runs an older version of Windows 10 (build 10240) which I am unable to upgrade.

Since this version does not support .NET Framework 4.72, I would like to install an older version of Skyline on my computer.

Is there a link for installation of older version of Skyline versions in your web site?


 Skyline setup error.png 
view request
Prosit unavalaible
(4 responses) laure bastide 2021-02-26
Hello Skyline team !

Thank you very much for your wonderful work !
When I am trying to do a Prosit Library this message appears and the server is unavailable when I am going to Tools > Options > Prosit.
Is there anything that you can do ? (It seems like a repetitive issue...)
Maybe it is not a problem with skyline and I create an issue too in the Github of Prosit but no answer so far.

Thanks a lot for your help and I wish you a good week-end !

Status(StatusCode=Unavailable, Detail="failed to connect to all addresses")
OK More Info
pwiz.Skyline.Model.Prosit.PrositException: Status(StatusCode=Unavailable, Detail="failed to connect to all addresses") ---> Grpc.Core.RpcException: Status(StatusCode=Unavailable, Detail="failed to connect to all addresses")
   à System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   à System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   à Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   à Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   à Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   à Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   à Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   à Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:ligne 96
   à Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:ligne 86
   à pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:ligne 230
   --- Fin de la trace de la pile d'exception interne ---
   à pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:ligne 234
   à pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:ligne 314
   à pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:ligne 112
   à pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:ligne 68
view request
Calibration curve - including information from replicates
(1 response) Kuba 2021-03-02

Hi Skyline Support,

I have a question related to how the calibration curve is presented.

I am trying to create a calibration curve for my heavy peptides at different injected concentrations. The injections were done in triplicates for each concentration of the peptide.

At the moment I can see calibration curves with concentration data points individually for each replicate. What I would like to see instead is a single point as the average of the three runs and the standard deviation. Is it possible in skyline? or do I need to export peak intensities for each peptide concentration individually and plot that externally?

Many thanks


view request
Lipodomics: How to apply single calibration curve (done with one representative molecule) to all members of a lipide class??
(4 responses) StefanW 2021-02-15

Because of the diversity of lipids they are normally separated in group, which share a common structural motive, like head groups etc. Typically these groups shares a common MS2 fragment.

The LipidCreator tool is very helpful in creating the corresponding transition lists.

For quantification it is usually to generate a calibration curve with one of the group member (i.e. PS (18:1, 16:0) ). Subsequently these calibration curve is used to quantify all group members. For this purpose the "slope" of PS(18:1,16:0) has to be assigned to all PS species (i.e. PS(20:0,16:0) or PS(16:1, 18:1) etc etc).
To my knowledge, such a assignment is unfortunately not possible in Skyline. Or ???

Inclusion this feature will procreate skyline to a unique and outstanding lipid my mind....

Thanks a lot for a comment on this support case.


view request
Strange looking chromatograms
(2 responses) Kuba 2021-02-28

Dear Skyline Support,

During the analysis of my PRM runs, I observed that for some of the peptides the chromatograms are strongly distorted - shattered.
Have you seen such data in the past? What could be a reason for it.
I attached screenshots of few examples.
If needed I can provide more technical info about the experiment.

Many thanks for your input


 1.png  2_heavyvslight.png 
view request
Parent-ion scanning
(11 responses) r bagshaw 2015-07-22
Will Skyline be able to interpret a RAW file from a parent-ion scanning experiment? In the experiment I specifically want to see parents of a product that is 85. Setting a transition consisting of a parent mass of something I expect to see in the sample with a fragment ion of 85 does not produce a chromatogram in skyline. (this is small molecule work)
Can you comment on this?
view request
Protein level FDR in Skyline?
Chinmaya k 2021-02-28


Does Skyline calculates protein level FDR?


view request
How to create library (BiblioSpec spectral library), background Proteome (Background Proteome file:- human, yeast, BSA, and others).
(9 responses) shelkarmicrobio23591 2021-02-25

I need all primary files to prepare the library (BiblioSpec spectral library), background Proteome (Background Proteome file:- human, yeast, BSA, and others).

So in your first tutorial that you have mentioned the interact-prob.pep.xml file which is not getting.

Please help to design the SRM experiment.


view request
Collision Energy Optimization Thermo Fusion Lumos
(3 responses) anne sanner 2021-02-25

Dear Skyline team,

I am currently setting up a PRM assay on the Thermo Fusion Lumos and would like to use Skyline's collision energy optimization function. In the default settings "Transition Settings -> Prediction -> Collision energy", there are only linear equations given for triple quads. Can you tell me what the regresison parameters for the Fusion Lumos are? Thank you!


view request
No chromatograms for transition ions
(1 response) michael-e-wright 2021-02-26

I wonder why transition ion chromatograms are not available for some peptides? This peptide was quantifiable using TMT across eight different samples. I'm not familiar with MaxQuant pipeline for scoring and quantifying TMT-labeled peptides. But this must be a lousy ID, correct? A lot of sketchy MS/MS spectra filter through .90 to 1 probability cut-off scores using MaxQuant search results. Do MaxQuant peptide scoring filters correlate with PeptideProphet scores? Am I looking at this the wrong way?

view request
Enable Peptide settings / Digestion / Enzyme == None for top-down
v delcourt 2021-02-26

Hi Brendan,

I think this is rather an improvement than an issue since it's still possible to get the result by another trick. However, it'd be convenient to have the protease choice to be set to "none" for MS1/MS2 quantification of intact proteins / peptides that were not subjected to proteolysis.

Let me know what you think.
Thanks once again for the magnificent tool you bring to the community.


view request
settings for importing timsTOF files in Skyline
kirti pandey 2021-02-25


I wanted to know if there are any specific settings say for e.g. in peptide transition whilst importing timsTOF files onto Skyline to visualise MS1 data.

Thanks in advance

view request
" don't know how to read "when uploading dia data in raw format
(3 responses) 1010419931 2021-02-24

Dear skyline development team, when using skyline's Data independent acquisition function, I encountered the " don't know how to read " prompt as shown in the figure when uploading dia data in raw format .I used Thermo Scientific's Q exictive HF to get the above data in raw format.I updated the skyline software, but it didn't solve the problem effectively.
What is the reason for this? How can I solve it?I hope I'm the first one to ask this question. I'm looking forward to your reply. Thank you.

view request
Can Skyline generate iRT assay library file used in Mobi-DIK script?
(1 response) Z. Jiang 2021-02-24

Dear Skyline team,

I am recently trying to use Mobi-DIK to analyze diaPASEF data. I was wondering if there is any way using Skyline to create the iRT assay library file that is used in the script "python --pasefdata data.d --mqout path/to/maxquant_data --irt irt_file.tsv"?


view request
How to use CIRT to predict retention time?
(5 responses) caixue 2020-05-20

I am trying to use the peptides selected by myself as CiRT to predict the retention time. The irt value of each CiRT peptide has been calculated and saved as a new Retention time predictor. Please see the attachment. However, when I chose the predictor, the document could not import the cirt peptides I selected, so I could not import the result. What should I do?
Looking forward to a reply. Thanks!

 Skyline cirt problem.pdf 
view request
Errors creating a spectral library and MS1 filtering
(2 responses) michael-e-wright 2021-02-23

I keep getting errors that RAW files are missing when I build a spectral library or carry out MS1 filtering. I'm processing MS data from an external source and I want to make sure the missing MS files are truly the source of the processing errors. Thanks.

view request
Generation of proteotypic peptides
(1 response) hogas aurel 2021-02-24

I have a suggestion regarding the Skyline analysis software. I am working with it for 1-2 years now, and I am having difficulties establishing the best peptides to analyze because I have to keep searching for softwares that help me generate the list of proteotypic peptides for my proteins of interest. Do you think you could integrate some form of software (in Skyline) which can help in generating the protetotypic peptides for the proteins of interest?

Thank you for your patience in reading my suggestion,
Kind regards!

view request
Group Comparison with multiple parameter
(1 response) david hoi 2021-02-24

Hello everybody,

I am trying to set up a group comparison were I want to compare a treatment vs a control at several time points in triplicates which I annotated . However, the group comparison only allows for annotation of 2 parameters such as treatment vs control and replicate, leaving me no option to also select the time points. Is there a way how I could overcome this and combine the analysis or will I have to do this separately?

Many thanks in advance,

view request
Import all peptides option using SkylineCmd
(1 response) jpaezpae 2021-02-23

Hello everyone,

I am trying to import a spectral library and add to the document all peptides in such library.

I am aware that using the GUI, this task can be accomplished by going to View > Spectral Libraries > Library > "<Add...>" > {add the library} > {click on the Add All... button} and it "just works" but I trying to use the command line interface for skyline and don't know exactly how to do this and cant seem to find any CLI flag for it.

As a workaround I think I could generate a transition list from my .sptxt by parsing all peptide names, duplicating precursor mass as a column and filtering the peaks that match y and b ions ... But I really feel like there should be a easier version.

Highly appreciating all of your help
Kindest wishes,

view request
Missing peaks MS1 quant centroid data
(14 responses) mstokes 2021-02-17

Hello everyone! We routinely use Skyline for MS1 quant with profile mode data and it works well. I have some centroid data that I'm trying to push through and I keep seeing missing peaks in certain files. When I do an XIC I can see the peak is there, the RT is good, the MError is good, but nothing in Skyline. I have tried running quant with both "Orbitrap" at a variety of resolutions and RT windows as well as "Centroid" with various ppm ranges and RT ranges, but to no avail. Many times it even looks as though there was a good MS/MS in the run that shows up as blank.

I have attached a screenshot of one example, peak is missing in Skyline in 2nd file but present in the XIC.

Has anyone else seen this issue or has ideas for a fix? Thanks!

 478.9123_Skyline_10ppm1min.png  478.9123_XIC.png 
view request
SYFT Data support
(4 responses) Christina lucas 2021-02-22

Hi there,

I was wondering whether anyone has every tried to implement data of a SIFT MS of syft technologies for small molecules (mainly VOC's). Datafiles are *.csv and *.xml, unfortunatly no mzML or mzXML available.

Data is quite simple you select a reaction ion (First Q), Reaction, product ion (Second Q), supported are SIM and Fullscan in the Second Q.

I attached some Datafiles in csv /XML, there no proprietary data format.

It would be great to get it into Skyline :)

Best, Christina

 Terpene_AromaticCitrus-BO 4.0 1h-20210122-131019.csv  Terpene_AromaticCitrus-BO 4.0 1h-20210122-131019.xml  Full scan 200 AMU pos only-Fullscan 5.0-20210122-134542.csv  Full scan 200 AMU pos only-Fullscan 5.0-20210122-134542.xml 
view request
Problems saving mprophet features
(5 responses) rmagni 2021-02-19
Hello Skyline Team,

I am having troubles saving the mprophet features in csv. When I attempt to do so I see this message:

Failed attempting to save mProphet features to C:\Users\rmagni\Desktop\GMU_projects\Tuberculosis_DIA\TB_DIA_dilution_curve\try.csv.
Access to the path is denied.
OK More Info
System.Reflection.TargetInvocationException: Access to the path is denied. ---> System.UnauthorizedAccessException: Access to the path is denied.
   at System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
   at System.IO.File.InternalMove(String sourceFileName, String destFileName, Boolean checkHost)
   at pwiz.Skyline.Util.FileStreamManager.<>c__DisplayClass24_0.<Commit>b__1() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\UtilIO.cs:line 713
   at pwiz.Skyline.Util.Helpers.TryTwice(Action action, Int32 loopCount, Int32 milliseconds) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1892
   at pwiz.Skyline.Util.FileStreamManager.Commit(String pathTemp, String pathDestination) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\UtilIO.cs:line 715
   at pwiz.Skyline.Util.FileSaver.Commit(IPooledStream streamDest) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\UtilIO.cs:line 1217
   at pwiz.Skyline.FileUI.MProphetFeaturesDlg.WriteFeatures(String filePath, MProphetResultsHandler resultsHandler, IList`1 calcs, CultureInfo cultureInfo, Boolean bestOnly, Boolean includeDecoys, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\MProphetFeaturesDlg.cs:line 146
   at pwiz.Skyline.FileUI.MProphetFeaturesDlg.<>c__DisplayClass13_1.<OkDialog>b__1(IProgressMonitor b) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\MProphetFeaturesDlg.cs:line 111
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_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_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1944
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_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_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.MProphetFeaturesDlg.OkDialog() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\MProphetFeaturesDlg.cs:line 118

My goal is to have a final list of peptides and a q-value associated with them so that I don't have to refine manually. Am I doing the right thing trying to export the mprophet features or should I look for some other report?

Thank you very much and keep up the awesome work!

view request
Duplicate transitions
(1 response) hober 2021-02-22

I regularly perform screening of peptides where I look for all potential +1 and +2 product ions from the different precursors I have selected. However, I often end up with cases where there are multiple identical precursor ion-product ion pairs, e.g. a b3 ion with the same m/z as a y3 ion and so forth.
This is unfortunately not limited to identical m/z, but also includes product ions with small differences in m/z that after rounding by the MS control software end up with the same m/z.

Would it be possible to implement something similar to the "Remove Repeated Peptides", but for transitions in Skyline?

view request
All-ions fragmentation data workflow
(8 responses) dmesasan 2021-02-18


I'm having some issues with importing results and exporting chromatograms of Agilent data that has been acquired using an all ions fragmentations mode. Maybe I need to adjust my settings somehow to accommodate this type of acquisition? I've attached an example data file. Thanks for the help!

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Feature request: Monitoring [M-1] ion for small molecules
(6 responses) Chris Ashwood 2018-08-08

Hi Skyline Team,

I'm working in the small molecule space and have noticed that sometimes, when using a wide extraction window (30ppm), my "new" targets are off-by-one errors of existing targets (e.g the monoisotopic peak for one target is within 10ppm of the first C13 isotope of another small molecule). This occurs due to my library-building DDA run performing MS2 on the 1st C13 isotope, and despite attempts at correcting this using Proteowizard's precursorRefine filter, there are still some uncorrected precursor masses that could mistaken for real targets.

In this case, the ability to integrate or include the [M-1] ion in peak integration would provide a way to quickly evaluate the off-by-one error with the peak area window in Skyline, rather than inspecting the target in full-scan view for each suspect peak. I've observed that idotp with a count of 3 is not a sufficient parameter for mitigating the off-by-one error (0.91 dotp value for both the target and off-by-one target, attached picture). I don't know if anyone else would value this feature, as this issue could be significantly reduced at the hardware/method level by running at higher-resolution or centroiding the data prior to analysis with Skyline but thought I would suggest it, in case it was seen to be a useful feature.


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Can I run without .NET 3.5?
(1 response) DWheatcraft 2021-02-11

I'm unable to install Win 10 Enterprise new workstations that are being setup, but it keeps trying to install .net 3.5 which is quite outdated. I can't get further and my IT doesn't want it on the workstation since it's so old. Is there a version of Skyline that can avoid this install?

Thanks, Dorothy

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Lipid Peak Integration Boundaries
(2 responses) dpeake 2021-02-17

Hi everyone,

I’m investigating using Skyline for integrating lipid annotation and QA/QC.
I have been very impressed with Skyline’s ease-of-use for quantitation.

I have several questions regarding adjusting integration parameters, in particular, the integration window that is displayed. When integrating a batch of lipids, some windows for the same species are displayed in a wider window than others. This window appears to change when the program sets different peak boundaries. This results in missed areas or integration of the another isomer, the largest peak in the window [see attached].

I would prefer to set a single integration window and peak boundary for all samples for a given lipid, thereby reducing the need for manual intervention.
After looking through this forum, I can't say that I have found any method that works. Adjusting the peak boundaries and "Apply to All" does not work.
Can anyone suggest a workaround other than manually changing peak boundaries one file at a time?


 Skyline 20.2 Lipid Integration.pdf 
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Q-TOF .raw data search stops after certain retention time
(10 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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Problem with error model from mProphet
(2 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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Peak boundaries for small molecule shotgun lipidomics
(7 responses) bharat 2017-07-19

I am interested in using skyline instead of Analyst for processing data from shotgun lipidomics. These runs are generated without a column, so all we're interested is an area under the curve from a start time to an end time. After these raw areas are generated, all other work on deconvolution, quantitation, etc are done externally. So, all we would need is for the integration to be done somewhat robotically from start time t1 to end time t2. I've been very successful, thanks to the good documentation, in getting all the transitions in (>140) and the integrations done. But, I can' get to the final step, which is this automatic peak boundary picking. So, you can see from the attachment that sometimes we get the good, and sometimes the needs adjustment. I've tried the "import peak boundaries" option, but that doesn't seem to work for me for small molecules. Anything I'm missing here?


 GoodIntegration.PNG  NeedsAdjustmentIntegration.PNG 
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Feature Request - Cycle Time
(6 responses) Tobi 2021-02-15

Hi Skyline Team,

could you please think about adding a calculated average cycle time to the report features? It is a really important feature to check in PRM and DIA runs repeatedly. While it can be easily calculated from RT start, RT end and Points Across the Peak, having it in Skyline directly would be a big plus.

With best wishes,

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Unknown error when opening LC-IMS-MS chromatograms
(9 responses) rozas 2021-01-28

Hello SkyLine Team,

I would like to ask you about a problem that I have using the software ( Skyline version 20.2). The data that I want to treat was was generated with a Synapt G2-Si.

I can open the MS files when IMS was turn off but it is not possible to open the data when Ion mobility was working. The following error appears:

At 12:17 PM:
Failed importing results file 'G:\My Drive\30-REC-20-ERGO.PRO\Data\20201204-004.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error

if I click in more info:

At 12:17 PM:
Failed importing results file 'G:\My Drive\30-REC-20-ERGO.PRO\Data\20201204-004.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'G:\My Drive\30-REC-20-ERGO.PRO\Data\20201204-004.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error ---> System.Exception: [pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
at pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1064
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 538
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasCombinedIonMobilitySpectra() in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 599
at pwiz.Skyline.Model.Results.FileBuildInfo..ctor(MsDataFileUri msDataFileUri, MsDataFileImpl file) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1430
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 216
--- End of inner exception stack trace ---

I would appreciate if you can help me.

Thank you,

have a nice day.


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QuaSAR and SProCop and AVG-DIA Problem
(2 responses) hatice akkulak 2021-02-15

Dear Skyline team,

I am trying to analyse my data through QuaSAR and SProCop tools; however, I am getting an error message all the time. According to the immediate window, there is a problem about some of the packages needed to use that tool. I tried to install these packages through R studio but I still couldn't get to use these tools. I tried to use these tools through other computers but I got the same error message. I don't know what to do. I'll be glad if you could help me. Thank you.

Best regards.

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Setting Transitions to Non-quantitative is not synchronized across light/heavy precursors
Tobi 2021-02-15

Hi Skyline Team,

hope you are doing well.

When working with isotope-labeled peptides I noticed that setting a specific fragment to non-quantitative is not synchronized between the light and heavy form, even if synchro was explicitly checked and when deletions of fragments were synchronized. Observed in Skyline and daily.
Please find an attached file.

With best wishes,
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parse rules MS Amanda
(5 responses) kguehrs 2021-01-05

Hi Skyline team,

First of all: Happy New year with good health and ongoing success in all things of your personal and professional life.

I have tried to generate a spectral libraries from DDA runs with MS Amanda integrated in the newest Skyline version. In my first approach, I have used a limited but refined (adapted to UniProt headers) database. MS Amanda performed well and created a library as expected. In my second approach, I used the same DDA files but a larger but not refined in-house generated database. MS Amanda did all the searches and created the mzID files but failed to create the library due the failure to parse the database entries. The database was generated in our institute and has a header format that is obviously not recognized by MS Amanda in its default state of integration into Skyline. Unfortunately, the standalone version of MS Amanda is not very handy for generating results of multiple searches and I actually have no idea how to change parse rules in MS Amanda.

I there any documentation about the parse rules MS Amanda applies and which type of database headers are supported. This would be helpful to adapt the databases if necessary. The other way would be to have a possibility to adapt/change the parse rules when using MS Amanda in Skyline to generate libraries.

Best Karl-Heinz

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Error when importing .speclib spectral library from DIA-NN
(3 responses) levasseurmaxence 2021-01-11

Hi Skyline Team,

I am trying to import a DIA-NN spectral library (.speclib) but get the following error message:

ERROR: Unable to read peaks for redundant library spectrum 1, sequence , charge 2.

Command-line: C:\Users\maxen\AppData\Local\Apps\2.0\WXXH5JG8.6M9\H2BPMA06.WGC\skyl..tion_e4141a2a22107248_0014.0002_5cab26a4bb7d0a9e\BlibFilter -b true "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\test.redundant.blib" "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\~SK41BE.tmp"
Working directory: C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test
OK More Info
System.IO.IOException: ERROR: Unable to read peaks for redundant library spectrum 1, sequence , charge 2.

Command-line: C:\Users\maxen\AppData\Local\Apps\2.0\WXXH5JG8.6M9\H2BPMA06.WGC\skyl..tion_e4141a2a22107248_0014.0002_5cab26a4bb7d0a9e\BlibFilter -b true "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\test.redundant.blib" "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\~SK41BE.tmp"
Working directory: C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibFilter.Filter(String sourceFile, String destinationFile, IProgressMonitor progressMonitor, IProgressStatus& status) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibFilter.cs:line 52
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 211

After reading that post I am pretty sure Skyline supports .speclib files but I nonetheless tried to convert the .speclib library to a .blib library using BiblioSpec. However, I get the same error message when I tried to import that .blib file instead (I assume that is how Skyline imports .speclib files in the background?).

Am I doing something wrong here? I am using the latest version of Skyline (

Thanks for your help!


 20210105_bnpagemsms_O7_speclib.tsv.speclib  20210105_bnpagemsms_O7_speclib.tsv 
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Triple Dimethyl Modification not imported from MaxQuantSearch
(8 responses) ida suppanz 2021-02-02

as I have now been struggeling for three days on this problem, I hope that someone can advise me. I am using Skyline regularly for MS1 filtering of ICAT data which works very fine. Now I have a dataset with triple dimethyl labelling, that means light +28, medium +32 and heavy +36, respectively on each N-terminus and Lysine residue . I do manage to set the modifications in the peptide settings in way that the correct m/z values are present in my test fasta (see screenshot). However, every time I try to use the "import pepide search" function with the msms.txt generated by MaxQuant, these settings vanish. If I try to do the "import peptide search" first (without adding the fasta first, only with the settings), Skyline tells me that there are no transitions, although I clearly have the m/z values corresponding to the Light and Heavy label in my msms.txt (see screenshot). If I try to import the whole msms.txt (not just the peptide I am interested in) with settings that worked for my ICAT data, Skyline correctly informs me that I do have Carbamidomethyl [+57] on C, and and Dimethyl [+28] on K, but that the following modifications cannot be interpreted: K [+32] and K[+36]. When I try to add, eg C'2H'6 - C2H6 as a heavy modification on K, Skyline tells me that this modification already exists and I cannot add it.
How can I get the chromatograms in Skyline?

 Settings.png  msmsScreenshot.png 
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small molecule integration filtering
(1 response) sufyan pandor 2021-02-11

For a large scale screening experiment (ion mobility 6560) i want to be able to have skyline firstly not integrate a peak if the mass accuracy is outside a specified range and secondly not integrate a peak if an M+1 M+2 is missing and not co-eluting or better yet not at the correct ratio from the theoretical fit

Is any of this possible to get through hundreds of data files/compounds?


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Routine Absolute Quant Method
(3 responses) alaine garrett 2021-02-11


I would like to set up a routine absolute quant method for peptides using PRM. Is there a way to save the set up in Skyline that would allow me to save all the setting and add data files as needed without going through the set-up as outlined in the Absolute Quant Tutorial?

Thank you

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Number format bug
(3 responses) jrenders 2021-02-10

Hi all,
Apologies if this is the wrong place to report a bug - I looked and was not authorized to add a new "issue" to the /home/issues page.

The issue is regarding number formats in the document grid. Previously, these number format designations would remain active until you changed the selected report. However, recently skyline began to reset all number formats every time the current report was filtered in any way.

As an example, I often change the "Calculated Concentration" from scientific to "#,###.##" to show commas at the thousands place and two digits after the decimal. Staying on this same report, if I search for a molecule by typing into the "Find:" text box above the table, the number formats all reset.

This is a new reaction and (I'm pretty sure) was not an intentional change since it is pretty inconvenient for the user. I have attached my .zip so you can see. I am running the most recent version of skyline daily.

Let me know if you need more info or if I should log this somewhere else. Thanks! 
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A fixed and variable modification on the same amino acid
(17 responses) laura declerck 2016-07-13

I study modified histone peptides. Because of the derivatisations every N-terminal AA contains a propionylation on the N-terminus. This modification was put fixed in Mascot. Sometimes the N-terminal AA is a Lysine which almost always contains a PTM.
I know skyline cannot recognize 2 different modifications on the same AA, so i have build new modifications for all the PTMs with the mass of propionyl added. However, Skyline cannot recognize any of these self-made modifications.

I think this is because Mascot reports the fixed and the variable modifications as separate delta masses and not as one.

Do you know how I can ensure that these peptides still get into my library?

Kind regards,
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Detection issues
(3 responses) luc camoin 2021-02-05

Dear Skyline Team,

I think it's probably a trivial question but I can't find the solution. I want to follow the signal of a peptide using Skyline. I know this peptide is present in my RAW file (see attached file). When I load the raw file in Skyline, I can see the beginning of the signals of the peptide (M, M-1, M-2 and ions fragments) but the signal is suddenly cut off (see the attached file). Apparently, Skyline did not load the rest of the signal from the raw file. Can you help me to solve this problem?


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Detection Q Value shows #N/A for solid peptides
(1 response) Z. Jiang 2021-02-05

Dear Skyline Team,

I am currently working on analyzing DIA data on Skyline. One thing I noticed is that quite a lot peptides with very good XICs and dotp scores have "Detection Q Value" shown as #N/A. For example, attached figure (Detection Q Value NA.png) shows the XICs of one of the iRT peptides. It is definitely in my sample and has very well-aligned XICs, intensities and dotp scores. However, the detection Q value is #N/A. Actually, all my iRT's detection Q values are #N/A. Does #N/A mean that the peptide is not significant?

I performed "Refine - Reintegrate" and trained "mPhrophet" model with target-decoy strategy (see figure "Skyline_Reintegrate.png"). After that, I selected "Only integrate significant q values" with a cutoff of 0.01. Did I do anything wrong?

Thank you,

 Detection Q Value NA.png  Skyline_Reintegrate.png 
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Display of protein abundances in PRM experiment
(3 responses) bpfister 2021-02-05

Dear Skyline team,

I was very excited about the recent implementation to show the actual protein abundances of each replicate within the Group Comparison grid, but am having problems with displaying them correctly. As my experiment included spiked-in heavy labelled peptides, I used "ratio to heavy" as the normalization method for the group comparison (see screenshot). I understood that I should also apply this normalization method in Peptide Settings --> Quantification (screenshot), but when I do that, it will only display me N/A for all replicates and proteins in the Group Comparison grid (why?).
When using "Equalize medians" in Peptide Settings --> Quantification, the protein abundances are displayed but they don't give me values that would lead to the fold changes calculated by Skyline (also not if I change the normalization to "Equalize medians" for the group comparison). Which settings do I need to use to get exactly the data that underlies the fold change calculated by Skyline?

I am using Skyline version

Thanks a lot for your help!

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How to use explicit retention time in proteomics interface?
(3 responses) yoneyamat 2021-02-03

Dear skyline team,

I am trying to measure peptides in SRM mode, and would like to use an explicit retention time on the chromatogram view. The small molecule interface has a column to paste the explicit retention time. Edit>Inset>Transition List. However, there is no column in proteomics interface to paste the explicit retention time for each peptides.
I would like to know how to use explicit retention time in proteomics interface.

Thank you in advance for your help.

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How to develop a MRM Method from scratch for known proteins (protocol/tutorial)
(2 responses) AWY 2021-02-03

I am interested in developing MRM biomarkers for specific plant proteins (Arabidopsis and Maize)

May I know if anyone could share any practical tutorial on how to develop a QqQ method from interested proteins.

Doesn't have to bein plant example protocol from any organism is OK to understand the workflow.

From theoretical digestion, how to identify unique peptides, to develop QqQ method.

Thank you.

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proteotypic peptides
io 2021-02-03

Dear all,
In order to choose proteotypic peptides, do I have to go first to peptide Atlas to select peptides or Skyline have this in consideration?
I have tried to install the proteotypic peptide viewer tool but my PC have problems in the installation.
Best regards

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Simple precursor ratios
(3 responses) chiva cristina 2021-02-02

Dear Skyline team,
I hope you are all doing well!
I have a question regarding the last version of Skyline daily. I have noticed there is a new checkbox (or maybe it is not so new but I have just discover it) in the Quantification tab under Peptide Settings called "Simple precursor ratios". Could you tell me what does it does and when can be helpful?
Thanks a lot

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Exporting Skyline document as report to include modified amino acid position on protein
(4 responses) paul.derbyshire 2021-01-29

Hi Skyline Team,

I have a question regarding document export as a report, i.e., File > Export > Report. Is it possible to extract the modified position of an amino acid within a protein sequence? In the jpg example attached, this protein is phosphorylated at position S134. Can such information (S134) be included in the report and if so which of the various option(s) should be selected?

Thanks in advance.


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copy/paste error from excel
(4 responses) Will Thompson 2021-01-29

Hi Skyline Team
On Windows 10 machines it seems when i copy/paste from excel into the document grid (for instance when pasting a small set of values for calibration standards), the 'receiving' column in Skyline document grid skips a row between all the values. So it pastes all the values, but it will leave an empty row between every value, so only the first one is in the right place and the rest are in the incorrect location. Its really annoying to not be able to copy and paste a list of values. :) Verified on two different machines. Can you verify?

Thank you


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MS1 filtering
(2 responses) paul.derbyshire 2021-01-28

Hi Skyline team,

I am having a go at MS1 filtering in Skyline and seems to be working quite well. If I click on the chromatogram peaks a new window opens up showing the precursor and it's isotopes (M, M+1, M+2 and so on). If I zoom in to say, the precursor (see attaches image file) I see that this peak is made up of other peaks. I am assuming these are subtle isotopic variants around this m/z and reflect their natural occurrence. Is this interpretation correct? Look forward to your reply.



 MS1 precursor zoom.jpg 
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Response of CE optimization (attached files)
(7 responses) hjl 2021-01-27

Dear Skyline team,

As you were asking a sample files of Skyline on my question of CE optimization, I prepared the files.

My workflow of CE optimization is described as below.

1.Specified 1 transition per compound

2.Based on specified transition, set methods for 1st CE optimization( step size 6, step count 3) = 1st analysis
Before) I used 4 step size and 6 count, I thought many step counts may draw some confusion to measure the best transition, I shorted it as 3 instead increasing step size( 4 to 6)

3.Analyzed and clarified the best CE for each transition

4.On the basis of 1st result, set methods for 2nd CE optimization( step size 2, step count 3) = 2nd analysis

5.Analyzed and acquired the final optimized CE for every transition each

6.Compared two methods( one = original method ; the other = obtained by CE optimization)

  • in the process of optimization, I specified each compound's RT as dragging the peak on the chromatogram, but in the sample file I attached, some of compounds might not be assigned for its peak. Please consider that flaws of those files.

My question is on the 1st optimization, I could clearly see that the CE optimization is needed for every compounds
then, after setting the optimized method( on the comparison step) Original data showed far better results than the other.
If the original data is better than modified one, what made the optimization result (especially that of 1st optimization) different ?

Thank you for your help as always.

 routine_1transition_1st(sample).sky  routine_1transition_2nd(sample).sky  routine_1transition_3rd_comparision_result(sample).sky  routine_1transition_1st(sample)  routine_1transition_2nd(sample)  routine_1transition_3rd_comparision_result(sample) 
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chromatogram files disappearing
(4 responses) Christian F 2021-01-25

Opening previous experiments doesn't open chromatogram for processed data, and results in the file for chromatographic data being erased completely. The file is not in the recycling bin (indicating it wasn't deleted), but seems to have been wiped from the computer all together. This happened for three experiments in a row.

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FDR control for DIA data
(3 responses) Z. Jiang 2021-01-13

Hi Skyline team,

I am currently using Skyline to analyze my DIA data. I was wondering if there is any way for Skyline to calculate protein and peptide FDR rate? I found there is a "Reintegratie..." function under the "Refine" menu. I was wondering if this one does the FDR calculations? If so, how does it return the results of 1% FDR? If not, what's the function of this "Reintegrate"?

Thank you,

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