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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  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
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Showing: limited to 100 requests
L/H Area Ratio Mismatch
(1 response) germolus 2021-05-17

Hi there.

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

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

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

8/17 = 0.4705

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

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

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

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


 Lib creationn issue_13May2021.pptx 
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Importing daughter scan data from a Xevo TQ-S
(2 responses) matthew daly-3 2021-05-11

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

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Waters SONAR data conversion for use in EncyclopeDIA
(6 responses) david schmidt 2021-05-11


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

Thank you very much,


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

Hi All,

May you please advice on the following:

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

  2. Is also GLP / GCP approved ?

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

Hi All,

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

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

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

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

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




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

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

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

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

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

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

Hello Support Team,

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

Please suggest.

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


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

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

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

Kind Regards,

 Skyline - CE optimisation troubleshooting.tiff 
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Skyline export to Chromeleon
(2 responses) Andrew 2021-05-12


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



view request
Running peptide search with MsAmanda from commandline
(2 responses) fcsigloch 2021-05-12

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

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

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

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

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

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

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

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

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

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

Hallo Nick,

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

Vielen Dank für Ihre Unterstützung

Alexandria Balac
LC-MS Service

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

Hi All,

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

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

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

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

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

Raw import:

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

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

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

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

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

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


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


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

Wanted to import .msp spectral libraries from:

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

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


 lipidblast error.png 
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differences in the volcano plot data generated from skyline
(7 responses) Linwei Li 2021-05-02

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


 data extract plot.png 
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comparison between groups with heavy peptides spiked
io 2021-05-06

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

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

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

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

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


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

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

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

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

Thanks in advance,

view request
Can a differential protein be validated by PRM measurement of a single peptide?
alima 2021-05-05

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

I would really appreciate your comments

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


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

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

David Pérez

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

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

The lowdown

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

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


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

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

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



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

Dear Skyline team,

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

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

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

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

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

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

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

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

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

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


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

See attached screen-capture for error message.

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

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

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

Dear Skyline team,

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

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

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


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

Dear Skyline Support team,

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

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

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

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

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

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

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

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

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

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

We will appreciate any help you might provide.


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

Hi all,

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

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

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

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

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

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


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

Hi MacCoss-members,

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

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

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

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

Erik de Graaf

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

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


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

Thank your for your help,

Nicolas Pierre

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

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

All the best
Witold Szymanski

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

Hi, there:

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



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


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

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

Thanks a lot for any insights!

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

view request
MS Amanda Scores
Joerg 2021-04-23

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

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

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

Guihua Jia

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


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

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

Best regards,
Ingrid Oline Tveranger

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

An error occurred attempting to export.
Query result unexpectedly empty: SELECT Value FROM PrmGlobalMethodInfo WHERE Key='SchemaType'
OK More Info
System.Reflection.TargetInvocationException: Query result unexpectedly empty: SELECT Value FROM PrmGlobalMethodInfo WHERE Key='SchemaType' ---> System.Exception: Query result unexpectedly empty: SELECT Value FROM PrmGlobalMethodInfo WHERE Key='SchemaType'
   at pwiz.CLI.Bruker.PrmScheduling.Scheduler..ctor(String scheduling_file_name)
   at pwiz.Skyline.Model.BrukerTimsTofMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor, TimeSegmentList& timeSegments, SchedulingEntryList& schedulingEntries, Boolean getMetrics) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Export.cs:line 3259
   at pwiz.Skyline.Model.ExportProperties.<>c__DisplayClass131_0.<ExportBrukerTimsTofMethod>b__0(IProgressMonitor m) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Export.cs:line 613
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\pwiz_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2111
Kind regards,
Mikkel Thomsen
view request
Installing/enabling external tool in Administrator install of Skyline
(10 responses) m j noga 2021-01-14

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

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

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

Many thanks

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


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

At 22:35:
Failed importing results file 'Filename'.
[Experiment2Impl::ctor()] Object reference not set to an instance of an object.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'Filename'.
[Experiment2Impl::ctor()] Object reference not set to an instance of an object. ---> System.Exception: [Experiment2Impl::ctor()] Object reference not set to an instance of an object.
at filename, MSData result, Int32 runIndex, ReaderConfig config)
at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 194
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188
--- End of inner exception stack trace ---

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

Thanks for the help!

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

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

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

Hello Team Skyline,

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

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

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

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

Thanks and regards

 mProphet_model_report.png  p53_comparison_volcano.png 
view request
Spectral Library generation using DIA raw workflow crashes
(1 response) alai 2021-04-18

Hi all,

I am following the DIA raw workflow using Skyline daily on some files collected using a TOF instrument. However, DIA umpire hangs at step 7/7 (widow something?) by using all the available memory (32 Gb).

I have previously tried using the same workflow on a sample set (ecoli/yeast/human) and it worked fine.


view request
two heavy labelled standards for one peptide
(2 responses) m teusel 2021-04-19

Dear Skyline Team,

I am using Skyline (64-bit) version
I have two different kinds of heavy isotope labelled standards: one 15N-labelled recombinantly produced protein and additionally a 15N13C-Arg synthetic peptide. Is there any option to have both heavy standards for my peptide together? At the moment I have an extra peptide for each sort of heavy label, which is fine to quantify light, but to compare the two heavy standards I have troubles as I would have to pick the same peak boundaries manually.
Optimally for this case I'd have one peptide with light and then two versions of heavy all displayed in one plot.

Thank you so much in advance.

view request
Skyline batch installation and use
(4 responses) j3 menzel 2021-04-18

Dear Skyline team,

I often run a small molecule analysis workflow using between 40000 up to 900000 transitions and exporting results with a report template. Both reading transition lists and datasets takes a long time, so I wanted to try Skyline batch to increase the speed of getting to the report file (as I have no need to look at or manipulate results manually in Skyline in this step).

When I downloaded Skyline batch I couldn't see all the parts of the menu (in Skyline batch configuration: Files, Refine) and I couldn't run the analysis of the dataset as the process exited before saving an output file.

Once I downloaded the latest version of Skyline Batch, installation (initialization) simply fails (on various computers), despite the latest Skyline version being installed.

Could someone help me or point me towards a site with more options?

Thanks for the great support,


view request
Determine sequence coverage for proteins
(7 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,

view request
Fine Isotope STructure
Will Thompson 2021-04-16

Hi Team, during Kendra's tutorial on isotope tracing today there was a discussion about, and a request for, Skyline to handle fine isotope structure for non-monoisotopic ions, above an instrument resolving power which the masses of 15N or 13C isotopes would be different (for instance). Currently, in order to calculate isotope incorporation for amino acids, Kendra is calculating A+1 and A+2 outside of Skyline because for most instances there are more than one ion in the fine stucture when the resolution is high enough. An example of a Skyline document with some data included is attached.


view request
Agilent PCDL database library transforming
(4 responses) emilia fornal 2021-04-16

Is there a way to transform Agilent PLCDL database/ libraries to library formats, which can be read by Skyline?

view request
Library Match - Prosit
(4 responses) blackmanbn 2021-04-16


I am unable to deselect (or remove) the Prosit library for library matching. It is not selected in the Molecule setting.

File attached. 
view request
How to group peptide sequences by peak quality icons
(7 responses) michel.petrovic 2012-09-12
Dear Brendan,

If I take for instance the tutorial 'Skyline Targeted Method Refinement' and go to the page 2, I can see the color explanation:

There are peak quality icons, and they indicate:
- Green – All transitions contribute a co‐eluting peak to the peak Skyline picked as the best.
- Yellow – At least half of the transitions contribute a co‐eluting peak.
- Red – Less than half of the transitions contribute a co‐eluting peak.

Our users would like to know if there is a way to view all information in a way that Skyline groups all peaks having a red icon together?
This will really simplify their manual control.

We will have much more protein sequences compared to your examples and this feature will be a must.

Best Regards,
view request
Scheduling window does not change
(2 responses) roberthardt 2021-04-16

Dear Skyline-team,

I am using the current version of Skyline Daily and noticed a bug when changing the scheduling window with an iRT predictor. No matter which value(2, 4, 5 min) we set the window to, Skyline keeps the window at 10 min. I first thought that this might be purely a visualization error, but the constant 10 min windows are also apparent in the inclusion lists we export. We also tried fixing the problem by exporting the whole project ("share") and opening it on a different computer, but the problem persisted.

Hope you can have a look at the attached project and help us with this issue.



view request
TIMS TOF DDA `mass errors unavailable' in histogramm
(3 responses) sarah lennon 2021-04-15

Dear Skyline team,

I have loaded some TIMS TOF PRO PASEF DDA data into Skyline Daily and have extracted the M, M+1 and M+2 precursor ions for a list of 43 000 peptides. I would like to display the mass accuracy histogram but Skyline is saying :'mass errors unavailable'. The mass error is well displayed on top of each chromatogram.
I had no problem with DIA PASEF data.
Please find enclosed all the parameters I have used to load the data and a screenshot of the problem I am encountering. I might have done something wrong.

Thank you very much for your help,

Kind regards,

 20210415_Skyline DDA PASEF_mass_error_histogram.pptx 
view request
Import MassLynx data into Skyline
(3 responses) blackmanbn 2021-04-13

I am collecting full scan data (Tof) in continuum mode with lock mass not applied. Because this generates very large data files, what it is best way to import into Skyline.

view request
FAIMS CV data analysis
(14 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!

view request
Issue importing QQQ data with more than one time segment
(4 responses) Alex Zhu 2016-12-05
Hi Brendan,
I have some runs on Agilent QQQ with two time segments and it seems skyline only can read the results from the first time segment. Is there any setting I need to change?
view request
PRM tutorial question
(5 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 
view request
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".

view request
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 
view request
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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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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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,


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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

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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 
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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!


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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,
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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.
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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.

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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.

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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?


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