support

Welcome to the Skyline support forum. If you have a question about using Skyline, or if you encounter a problem, you can post your questions here.

It is likely that your question has already been asked and answered.  Please use the search box in the upper right corner of this screen before posting a new question.

Support is provided by the creators of the software, as time allows, though we hope others will share their experience as the user community is now quite large.

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

In order to post to the forum, you'll need to sign-in or if you don't yet have an account sign up. Forgot your password? You can reset it using the "(forgot password)" link on the sign-in page.

You can also follow the Skyline support board through email updates after you sign up.

When you post a question, please include the following information:

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

If you are including text output from a tool, please attach files to your message, rather than pasting in long text.

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that .sky.zip file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
updating protein annotations
(12 responses) jdemeter 2022-07-08

Hi,

I am trying to update the gene field for proteins. I was following this posting #25357, clicked 'populate' and 'genes' and then followed the steps described. After the procedure finished, I got a new name - instead of the original e.g. NP_000012, now it is PSEN1. I'd like to keep the original name but add PSEN1 to the gene field. Is there a way to do this?

Thank you,
Janos

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Number of MRM method channels for peptides containing two or more amino acids modified with MassLynx
(6 responses) ryuta kobayashi 2022-07-04

I tried to export the method to MassLynx using Skyline, but the number of channels for peptides containing two or more modified amino acids was limited to five.
In such a case, is there a way to add 5 or more channels?

Kind regards,
Ryuta

 Skyline_error.pptx 
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SureQuant with two FAIMS CV voltages on Orbitrap Fusion Lumos - ZigZag shaped peaks on Skyline
(3 responses) aheinone 2022-08-11

Dear Skyline Team,

We are starting to do SureQuant on Orbitrap Fusion Lumos with FAIMS by using two CV voltages (-50 and -70). We are dealing in total with about 2000 peptides, but < 500 peptides at a time in one Skyline document. We have imported the raw files to Skyline for further processing. We have built Ion Mobility Libraries by using the results. Some peaks are drawn correctly, but in many cases product ion peaks are jagged (zigzag shaped in MS2 level). But still precursor peaks (MS1 level) look ok. It seems that this happens with CV -50 peaks. Probably Skyline takes data points from the both CV values for some peaks? We have tried many different settings for MS/MS filtering, but we haven't found any solution. We tested to change the CV values manually in the Ion Mobility library, but it doesn't solve the problem. And the CV values seem to be correct in the library. If we split the original raw files to two separate files according to the CV voltage, and then import them to Skyline, the peaks are fine. But if we build the Ion Mobility Library by using the two separately imported files, it didn't solve the problem. Is there any other way to handle multiple CV values than split the files according to the CV voltage. We would appreciate for any help with this, thanks.

Regards,
Arttu

 Skyline_issue_FAIMS_two_CV_voltages.pdf 
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Edit Group Comparison Scope protein
(1 response) io 2022-08-10

Dear all,
Why in some cases when I do the group comparison with scope protein some proteins are not display while in the scope peptide yes?
Best regards

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ID marker
(2 responses) bobxiong 2022-08-09

Hi Skyline Team,

Is there a way to manually set the ID marker in the chromatogram? I would like to explore the exported peak area data based on the manually "identified" peaks rather than what Skyline picked out. Screenshot attached.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address: bobxiong@joinn-lab.com

 screenshot_20220809_bx.pdf 
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MSstats installation error
(6 responses) hwang2 2022-08-08

I'm trying to install MSstats tool. I installed R-3.6.1 in Documents\R\R-3.6.1, then added the tool of MSstats, it automatically installed R-4.1.2 in Documents\R\R-4.1.2, then it installed some files and stopped with error message of "Unknown Error installing packages. Output logged to the immediate Window".

Immediate Window:
A version of this package for your version of R might be available elsewhere,
see the ideas at
https://cran.r-project.org/doc/manuals/r-patched/R-admin.html#Installing-packages
Error in loadNamespace(x) : there is no package called 'BiocManager'
Calls: loadNamespace -> withRestarts -> withOneRestart -> doWithOneRestart
Execution halted

It looks like "BiocManager" is missing. Could you help?

Thanks,

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"ERROR: No spectra were found for the new library" When making a simple DIA document
(4 responses) ehubb004 2022-08-08

My lab is just starting to collect DIA data on a Lumos and we've run into an issue while testing. After we do a DIA run and attempt to build a Skyline document there is an error (ERROR: No spectra were found for the new library) at the end of the DDA Search step of the import peptide search process. Our assumption is that there is some issue with the instrument's DIA settings, but we can't find an actual issue. I've attached the error message, the instrument settings, FASTA file used, the .raw file (collected on a sample of BSA tryptic digest), and the .mz5 file that Skyline made.

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Display/integration error when using small molecule iRT calculators
(4 responses) Chris Ashwood 2022-08-04

Dear Skyline team,

I hope you are doing well. When I have set up the small molecule iRT calculator and begin integrating peaks using the "Show XXXX Score" display, I'm encountering a few issues after successful calibration:

  1. The windows are not updated from "Retention time" to "XXXX Score" for both the file-specific chromatogram window and the "Retention Times - Replicate Comparison" window. This appears to happen the first time I click on each molecule, but after I click back and forth, Skyline then displays the right window.
  2. The first time I integrate inside the file-specific chromatogram window when it is set to "XXXX Score", the display bugs out and the integration appears to be different from what I selected in the window file-specific chromatogram window. I think it is setting the iRT value used for integration as the retention time instead of the iRT score (e.g. integration from 1 to 10 score instead integrates 1 to 10 minutes in the retention time view).

The Skyline file used for this is attached. Happy to give more detail.

Cheers,
Chris

 E4HN 69 Wk F_M_120k_Final_iRTwithAuditLog.sky.zip 
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Failure opening error
(1 response) shankha 2022-08-03

Hello,
I am trying to open .sky or .skyd on Skyline v21.2.0.568 and it throws an error - see attached. This same file would open on someone else's PC. Are there dependencies that I need to install to be able to open Skyd file? I am having hard time understanding the error message? I have the most updated skyline version and the person who made these files also has the most updated version, so in theory the file should be compatible.
Please let me know,
Shankha

 Capture.PNG 
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Import peptides AND charge states
(21 responses) gabe 2017-09-22
Is there a way to import a list of peptides AND charge-states into Skyline? I know I can import peptide sequences and have the charge-states auto-populated based on spectra in the reference library, but I can't figure-out a way to import specific charge-states into a document.

Thanks

Gabe
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Export Isolation List (DIA) for Bruker timsTOF
(4 responses) Premy 2022-08-02

Hello,

I use the Bruker timsTOF Pro, and wanted to export an isolation scheme through Skyline. I created a scheme under Transition settings and did the following:

File --> Export --> Isolation list --> selected Bruker timsTOF and set the run duration

After saving the .csv file, no file seems to be created. I'm not sure if I missed something on my end. Any help would be appreciated!

Best,
Premy

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PRM or DIA analysis for large number of proteins
asamaras 2022-08-02

Hi all,

I am during an experiment with target proteomics with non labelled peptides. My targets are fungal proteins in infected plant tissue. The goal of my study is to detect and then to quantify the proteins. I am trying to find the most sensitive way to detect them. I try to determine the retention time of the proteins of interest (350), but as I expecten the range of the retention time is really big, so I don't know if something like this can load it in the instruments. So, my question is , do you beleive that there is a limit of proteins when you want to predict retention time using PRM?? The other alternative is DIA running method. Does anyone has the same issue?

Thank you in advance for your response,
Best
Tasos

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Unexpected tab rearranging behavior
(5 responses) aboatman 2022-07-29

Hi there,

I'm on Skyline-daily version 22.1.9.208, using the small molecule interface and ion mobility filtering. I am trying to rearrange the tab order of my chromatograms to match the order of the results. However, when I click and drag a tab, it does not stay in the spot it is dropped. Instead it jumps two tabs to the left of where I tried to move it. This is really just a minor inconvenience, but it would be nice if the click and drag rearranging worked as expected.

It would be even nicer if there were an upgrade so that when you rearrange your results in Edit > Manage Results, the tab order automatically changes to match.

Thanks!
Anna

 2022-07-29 11_50_45-Skyline-daily - 20220727 gators first set.sky.png 
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Group comparison bar graph error bars
(4 responses) cmwats2 2022-08-01

Hi. A group comparison bar graph plots the Log 2 fold change with error bars. If I right-click to "Copy Data" from the bar graph and paste in to excel, I get column headers Protein, Log 2 Fold Change, and StdErr. I have multiple comparisons so I made a custom report to export the information I want. I added a column to export "Standard Error"; however, "Standard Error" does not equal "StdErr."

What is the difference between the 2 standard error values?

Thanks in advance,
Caroline

"Copy Data" results:
Protein Log 2 Fold Change StdErr
ALDOA 0.317333532 0.097111714

Report export results:
Protein Log2 fold standard error
ALDOA 0.3173 0.049317949

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Showing TIC with labeled peaks
(2 responses) arjun sharma6251 2022-08-01

Hello!

I was wondering if there was a way in Skyline to display the TIC with labeled peptides. I'm new to the software, so as I'm progressing through the tutorials (specifically, the Full-Scan Filtering one), I'm trying to see if I can label each peak with the peptide that it matches to and then display the entire chromatogram (instead of clicking on each peptide and seeing a portion of the chromatogram).

Thank you so much!

view request
GC-MS SIM data import
(8 responses) fiammetta dimarco 2022-07-27
Hello,
I have a problem in importing GC-MS data acquired in SIM mode. I followed the guideline "GCMS Data Analysis" and I was able to import GC-MS of Shimadzu coverted in mzXML acquired in scan mode but when I try to import the SIM GC-MS data it doesn´t work and it gives the following error:

At 22:07:
Failed importing results file 'C:\Users\b1064562\Desktop\GC-MS steroids\raw file\270725_FDM_mix std overnight_5ugmL_50MO_SIM_1.mzXML'.
Unable to determine isolation width for the scan targeted at 268
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\b1064562\Desktop\GC-MS steroids\raw file\270725_FDM_mix std overnight_5ugmL_50MO_SIM_1.mzXML'.
Unable to determine isolation width for the scan targeted at 268 ---> System.IO.InvalidDataException: Unable to determine isolation width for the scan targeted at 268
   at pwiz.Skyline.Model.Results.SpectrumFilter.CalcDiaIsolationValues(SignedMz& isolationTargetMz, Nullable`1& isolationWidth) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 1211
   at pwiz.Skyline.Model.Results.SpectrumFilter.FindFilterPairs(IsolationWindowFilter isoWin, FullScanAcquisitionMethod acquisitionMethod, Boolean ignoreIsolationScheme) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 1093
   at pwiz.Skyline.Model.Results.SpectrumFilter.<Extract>d__105.MoveNext() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 820
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ProcessSpectrumList(MsDataSpectrum[] spectra, ChromDataCollectorSet chromMap, Single rt, SpectrumFilter filter, String scanId) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 511
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 353
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 239
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 594
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 384
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
   --- End of inner exception stack trace ---

Do you have an idea of what is wrong? Shall I change the transition settings (they are set as described in the guideline "GCMS Data Analysis")?

Thank you for the help!
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Small Molecule Peak Integration
(8 responses) tony considine 2022-07-18

Hello,

I am using the molecule interface of Skyline in conjunction with the ALIS system to perform RNA/protein screens looking for novel chemical matter. Since I will be screening for novel binders, I need to have a peak integration method that identifies the correct peak. I cannot go through each chromatogram to perform a manual integration as that will not be feasible due to the sizes of the screens. Instead, I have to rely on applying different filters in the exported CVS file to filter out nonspecific binders from specific binders and trust that the initial peaks identified are the correct ones. I have tried to optimize the peak integration method by spiking in known "weak" binders into pooled compound wells of 250-300 compounds per well and seeing if the correct peak is integrated. I am doing this as we don't expect to identify very strong binders in the initial screening process.

However, in some cases, it identifies the wrong peak and a peak with an idotp score much lower than the correct peak with the much higher idotp score. I have tried to look under the "refine" tab and the advanced section and modify the parameters, especially the minimum idotp value. However, changing the minimum idotp value seems in mess up my method and results in the wrong compounds being identified as the peak in the respective chromatograms. I will typically know the chemical formula of all the compounds in the well and would expect an idotp score of 0.80 or greater. I have also tried to set the amount of transitions to 3 (M, M+1, M+2) and would expect to see at least main precursor and the C13 isotope. I also don't see a feature to isolate peaks based on the mass error (typically less than 10ppm) and in general, changing the parameters under the "refine" tab and the advanced section does not seem to have much of an effect. It would be super helpful if people who have experience could point me in the right direction and ways to optimize the peak integration methods so that only peaks with high idotp scores, low mass error, etc etc are identified from the chromatogram.

Thank You,
Tony Considine

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Prosit Server down after 21.2.0.565 update
(1 response) ocs4 2022-07-21

I have just updated to version 21.2.0.565 and now can't access the prosit server.
Server:
131.159.152.7:8500 Server unavailable

Then in the library match window it says:
Status(StatusCode=Unavailable, Detail="failed to connect to all addresses").

I have seen another error similar when skyline 21.1 was released in 2020.

view request
High LOD calculation due to baseline
(9 responses) brianna garcia 2022-07-12

Hello,

I am using Skyline (64-bit) 21.1.0.536 for small molecules where I have isotopically labeled and natural abundance molecules. I have noticed for many of my Blanks the Light/Heavy ratio is very high due to differences in the baseline, however no actual peak is present. This large difference in baseline intensity results in a LOD larger than most of my actual standards (where a peak is visibly present & integrated).

The only potential solution I've come up with is to exclude the blanks, however I cannot calculate an LOD without them in this case. Are there any potential solutions to this problem? I'm also, worried how this will affect samples (rather than blanks) where there no peak present, but a larger baseline difference between the Light/Heavy resulting in a high calculated concentration when there is really no peak present at all.

I have attached an example image here.

Thank you,
Brianna Garcia

 amMP_baseline_image.PNG 
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I wonder if Skyline will join MS-based RNomic and DNomic studies?
(2 responses) licky lck 2022-07-18
Dear Skyline team,

I really appreciate the massive contribution of Skyline to the proteomics community, and it is one of the most important software I used during all of my proteomic studies. Recently, researchers from epigenetics, biology and pharmacy are trying to "borrow" the bottom-up concept from proteomics to study modifications in oligonucleotides (from DNA and RNA) using MS-based methods. So I wonder if the Skyline team would consider joining the party?

I have been using Skyline to study site-specific protein modifications on peptides using the manual peptide insert function, and it would be absolutely a game changer if a similar function is available for nucleotide study in Skyline for epigenetics-related studies as there is still a blank in the study of DNA (or RNA) modifications site-specifically. I will be working in this field in the coming years, and I would love to hear if Skyline is also interested in working with DNA and/or RNA. Thank you very much for your time and patience.

Best regards,
Chengkang
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Peptide format from IMS library not recognized
(8 responses) Juan C. Rojas E. 2022-06-27

Hi all,

I tried creating an IMS library of cross-linked peptides with the "Use Results" option. After creating the library and re-importing the results I noticed that no IMS filtration was applied.

Closer inspection of the library shows that the name in the library does not include the appropriate notation to be recognized as a cross-linked peptide or the site of the cross-link and is only taking the "left peptide" sequence. Are there any extra steps I need to take or is this a bug?

I have provided some example files (raw data and Skyline project) in our private Panorama "MLU - AGSinz - Data Sharing" folder for testing in case it is a bug.

As always, thank you for your time and support.
Sincerely,
Juan C.

 IMS_library_error.PNG 
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Bruker files can't be import to Skyline 21.2.0.536
(4 responses) Le Hung 2022-07-18
Dear MacCoss lab, It seems the problem with Bruker files (for Solarix 12T FT-ICR) happen again with the new version of Skyline. The error is: "Failed importing results file 'C:\Data\2022\Proteomics\20220714-LCpepmap\BSA_1uM_AutoMSMS_Acc-0p3s_000001.d'. unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7 pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Data\2022\Proteomics\20220714-LCpepmap\BSA_1uM_AutoMSMS_Acc-0p3s_000001.d'. unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7 ---> System.Exception: unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7 at pwiz.CLI.msdata.ReaderList.read(String 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_21_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 197 at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291 at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188 --- End of inner exception stack trace --- " I attached the file I am used below. Could you please take a look when you have time? Thank you very much! All the best, Hung
 analysis.baf 
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How to improve BlibBuild CPU usage
(7 responses) nibarrola 2022-06-22

Dear Skyline Team,

We are trying to create a Skyline library using a .pep.xml file (3.31MB) generated with PEAKS, the related Spectrum file .mzXML has a size of 928MB. It takes more than 1h to create the .blib file. In case of bigger files it takes all the night. We have noticed that BlibBuild is using only one CPU, so we want to know if it is possible to improve processing speed incrementing the number of CPUs used by BlibBuild.exe which is the bottleneck.

We have installed SkylineDaily in two computers and we have the same problem in both:
-Computer 1: processor: Intel(R) Core(TM) i9-9900K CPU @ 3.60GHz 3.60 GHz, RAM: 64,0 GB (63,9 GB usable), Windows 10 Pro 64-bit operating system, sockets: 1, cores:8, logical processors: 16
-Computer 2: Intel(R) Xeon(R) Silver 4214 CPU @ 2.20GHz 2.20 GHz (2 processors), RAM: 512 GB (511 GB usable), Windows 10 Pro 64-bit operating, system, sockets: 2, cores:24, logical processors: 48

Thanks in advance for your help

Nieves Ibarrola

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Protein name too long for upload to Panorama
rj8 2022-07-14

I'm in the habit of using the Associate Proteins... feature of skyline, which correctly combines protein names that are grouped together. A problem seems to arise when I then upload the Skyline file to Panorama. Panorama seems to have an upper limit of 500 characters for a protein name, but when many proteins are grouped together I can get very large names (from large numbers of proteins in the same group). I can manually delete some of the characters to bring the size below 500, but maybe Panorama should set an upper limit of several thousand characters. Here's an example of the error message from within Panorama:

14 Jul 2022 11:46:18,743 ERROR: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
org.labkey.api.pipeline.PipelineJobException: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:275)
at org.labkey.targetedms.pipeline.TargetedMSImportTask.run(TargetedMSImportTask.java:63)
at org.labkey.api.pipeline.PipelineJob.runActiveTask(PipelineJob.java:841)
at org.labkey.api.pipeline.PipelineJob.run(PipelineJob.java:1077)
at org.labkey.pipeline.mule.PipelineJobRunner.run(PipelineJobRunner.java:40)
at java.base/jdk.internal.reflect.DirectMethodHandleAccessor.invoke(DirectMethodHandleAccessor.java:104)
at java.base/java.lang.reflect.Method.invoke(Method.java:577)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invokeMethod(DynamicEntryPoint.java:312)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invoke(DynamicEntryPoint.java:259)
at org.mule.impl.DefaultLifecycleAdapter.intercept(DefaultLifecycleAdapter.java:193)
at org.mule.impl.InterceptorsInvoker.execute(InterceptorsInvoker.java:47)
at org.mule.impl.model.DefaultMuleProxy.run(DefaultMuleProxy.java:470)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$CallerRunsPolicy.rejectedExecution(ThreadPoolExecutor.java:1486)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.reject(ThreadPoolExecutor.java:391)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.execute(ThreadPoolExecutor.java:865)
at org.mule.impl.work.ScheduleWorkExecutor.doExecute(ScheduleWorkExecutor.java:39)
at org.mule.impl.work.MuleWorkManager.executeWork(MuleWorkManager.java:277)
at org.mule.impl.work.MuleWorkManager.scheduleWork(MuleWorkManager.java:244)
at org.mule.impl.model.seda.SedaComponent.run(SedaComponent.java:483)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:650)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:675)
at java.base/java.lang.Thread.run(Thread.java:833)
Caused by: org.labkey.api.query.RuntimeValidationException: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
at org.labkey.api.data.Table.insert(Table.java:751)
at org.labkey.targetedms.SkylineDocImporter.insertPeptideGroup(SkylineDocImporter.java:1319)
at org.labkey.targetedms.SkylineDocImporter.importSkylineDoc(SkylineDocImporter.java:405)
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:248)
... 23 more
Caused by: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
at org.labkey.api.query.RuntimeValidationException.<init>(RuntimeValidationException.java:36)
... 27 more

view request
Error when creating spectral library from .mzID and .mgf export from PEAKS
(4 responses) Juan C. Rojas E. 2022-06-30

Hi team,

The most recent version of Skyline-daily (21.2.1.53) is now preventing me to create a spectral library from a PTM search from the export created for Scaffold (.mzID and .mgf files). I have been using this since creating a spectral library with the export for Skyline (.pep.xml and .mzxml) simply takes too long for timsTOF data.

As Matt pointed out (https://skyline.ms/announcements/home/support/thread.view?rowId=56180), it seems to be an issue with PTMs. I can create a spectral library when only Cys carbamidomethylation and Met oxidation are considered (PEAKS DB attached search). But when I try to create it with the results of all default PTMs withing PEAKS (PEAKS PTM attached search) then I receive a warning that does not allow me to finish.

What could be the problem? Let me know if I can help in any other way.
Sincerely,
Juan C.

 Cytochrome_C_PASEF-DDA_PEAKS_PTM_20logP_mgf.zip  mzid_error.pptx 
view request
computer resource configuration
(4 responses) bobxiong 2022-07-12

Hi Skyline Team,

I have an odd question about computer configuration in support of Skyline usage. I tested a manually prepared transition list of 4000 transitions on a 3G mzML DDA data file using the Molecule interface. While importing the transition list and the result mzML data seemed slow, it was not responding to any kind of clicking to view the data afterwards. The computer hung up. Please see the attached for my current computer properties.

Assume that I have generous funding, what kind of computer resource configuration should I go for? I do not want to be bottlenecked by a computer to perform Skyline work. For a disulfide linked tri-peptide molecule, the transition list can easily reach 50K transitions to consider a combination of multiply charged precursor and product ions. I expect it to become even more demonding if multiple replicates of mzML data are loaded in Skyline.

I would greatly appreciate any suggestion/recommendation.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address: bobxiong@joinn-lab.com

 screenshot_20220712_bx.pdf 
view request
Unusual behaviour in limiting number of peptides per protein
(1 response) Liyan Chen 2022-07-12

Hi Skyline developers,

I am using Skyline version 21.2.0.425 for DIA files and typically add all library peptides into the document. Recently we have decided that our data analysis works just fine with maximum of 10-20 peptides per protein, and additional peptides only work to slow down file import. Thus I setup a blank document with these settings update before adding library peptides to document:
Peptide Settings>Library: Limit 20 peptides per protein
Peptide Settings>Filter: Auto select all matching peptides

I still get more than 20 peptides for some proteins whether I select "Pick peptides matching: Library" or "Pick peptides matching library and filter". The only way I can get the limit of nPeptide/protein enforced is to go to "Refine>Advanced" and tick "Auto-select peptides". This refinement takes some time to run and results in repeating sequences within proteins (but still unique to their respective proteins) being introduced as duplicate peptides, and the rest of the peptides within the top 20 rank going missing.

In this example using rank 1 GTYSTTVTGR in the protein APOA_Human, only the first instance of this sequence is present in the document when no limit on nPeptides/protein is applied. Ranks 2-20 are occupied by other peptide sequences, with many more other peptides. At this point I had checked for duplicate/repeated peptides in the document and found none. However, when the limit of 20 peptides is enforced using "Refine", each occurrence of GTYSTTVTGR in the protein is re-ranked and counted as separate peptides.

How do I get Skyline to only keep one instance of GTYSTTVTGR as rank 1 and the other peptides ranked 2-20 in the document? Is there also a way to limit the peptides when the library peptides are being added to document, instead of adding all and then removing excess peptides?

I'm uploading the document "20220712_template.sky.zip" on the filebox.

Best regards,
Liyan

 Skyline library rank peptide limit issue.pptx 
view request
MSX SIM QExactive MS1 data missing scan data
(4 responses) simondbourque 2022-07-08

Hello,

I'm running a method which combines scheduled MSX MS1 scans with scheduled non-MSX PRM scans and the MS1 scans don't seem to import properly. In the attached image the MS1 data cuts out partway through the peak, this corresponds to where the scheduled acquisition window of one target ends and so the scan filter in the raw data changes. The data is there as extraction works fine in Xcalibur.

This is running on a QE+ with xCalibur 4.0. Skyline 21.2.0.536. Let me know if there is s private location I can upload the raw and project file to.

Thanks,
Simon

 MSX_Example.png 
view request
Using OD(Optical Densisty)for Normalization
(2 responses) ka79422 2022-07-11

Hello,
I am working on bacterial samples and want to use skyline to process my data.I am trying to normalize my samples using the optical density readings.Is there a way I can import these OD readings into skyline for normalizing my data before processing?

Thanks in Advance,
Keerthi.

view request
is it possible to insert iRT values from the library into iRT calculator?
(1 response) pavel shliaha 2022-07-11

I have iRT values predicted for several standard peptides by prosit in my spectral library. I would like to add them as iRT standards with iRT values predicted by prosit (see attached screenshot). My questions are:

  1. is there a way to do this in 1 click, i.e. to copy iRT values from the library?
  2. if there a way to export the iRT values from the library with corresponding peptide sequences, so I can copy and paste the table?
 Capture.PNG 
view request
Chromatogram Graph Properties
(4 responses) bobxiong 2022-06-10

Hi Skyline Team,

I am using Skyline as a visualization tool to display chromatograms for verification of data analysis results produced by vendors' DDA data analysis software. While going about this, I discovered that the chroma graph does not show any peak when my transition list contained say 1000 entries. Is there any way to not show the ion names in the chroma graph? My guess is that the ion names took all the space leaving no room for showing peaks. I have attached a screen shot on 40 entries in the transition list to help explain my experience in Skyline.

Thank you so much,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
E-mail address: bobxiong@joinn-lab.com

 screenshot_bx.pdf 
view request
can i use skyline to process product ion MS2 data to select one product ion to do quantitation
(2 responses) xue shi 2022-07-06

Dear customer service,
I am running product ion(MS2) experiment on thermo orbitrap. I want to process data with skyline. However, I can only process the SIM and MRM data but don't know how to process MS2 data for quantitation. Could i view spectrum by skyline and pick the strongest product ion to quantitation with skyline? Is there any video or manual can help with this?
Thank you a lot.
Xue(Katie) Shi

view request
Sciex TripleQuad7500 .Wiff2 support
(3 responses) roosso 2022-07-05

Hi there,

We've been using the Sciex TripleQuad7500 in our lab, which generally generates .wiff and .wiff2 files. However, during more complex analyses (e.g. positive and negative mode combined) only .wiff2 files are generated.

So far, I have not been able to import these .wiff2 files into Skyline. Is there a way to fix this, or could you possibly include this in a new update?

Thank you!

view request
Is Skyline compatible with chymotrypsin search for DIA?
perezpay 2022-07-05

Hello all,

We have some DIA data generated from digestions with chymotrypsin. We were wondering if Skyline supports chymotrypsin search for DIA.

Best,

Yadira

view request
Generating a mobility library
(7 responses) alan mckenzie-coe 2022-06-06

I’m trying to generate a mobility library, so far I have curated my target peptides and transitions. I have also made the following changes shown in the screenshots attached.

 Skyline_Error.docx 
view request
Early-run m/z + IM calibration (timsTOF / any Q-TOF ?)
(6 responses) v delcourt 2022-06-30

Hi,

Coming from nanoflow QE area and recently adapting to a timsTOF, one thing that is pretty handy is the early-run calibration. Briefly, a loop is filled with calibrant (e.g. sodium acetate/formate + agilent tunemix) and is pushed into the MS while in the dead volume (e.g. 0.1-0.3 min window). This allows through the Bruker software "dataanalysis" to perform a post acquisition calibration for m/z and IMS (tims), which is really nice to reach sub 2 ppm mass and ~ 1 % CCS precisions. This is also implemented for nanoflow with different procedures.
Are you considering adding this kind module to Skyline which would be awesome for both proteomics and small molecule analysis ?

Thanks again for developing Skyline.
With kindest regards,
Vivian

view request
Command line for importing peak boundaries and analyte concentration
(4 responses) jianqiaoshen 2022-06-29

Hi, I am using SkylineRunner to automate the processing of different dataset. I am wondering are there any commands to import a peak boundaries file and analyte concentration for each replicate?

view request
feature request, predicted 'score' for predicted migration time for CE in Skyline
Will Thompson 2022-06-29

Hi Brendan,

During a conversation recently you mentioned that there were 'buried' some calculations in Skyline related to predicting CE migration time based on solution charge and size. I'm just placing this message here as a formal feature request to discuss exposing those predicted migration times.

Cheers

Will

view request
Library search result "score"
(1 response) mmr6q 2022-06-29

How does Skyline calculate the "score" that appears on library match MS2 spectra, and why are many scores 0.000000 when the score of the MS2-sequence match is not zero? Is the Skyline "score" used in any way? Thank you.

 Library Match example.JPG 
view request
Mass error
(1 response) mmr6q 2022-06-29

Why are the displayed mass errors incorrect? Please an example in the attached PowerPoint file. Thank you.

 Mass error example.pptx 
view request
Adding library peptides to document giving impossibly large number of proteins
(13 responses) Liyan Chen 2022-05-24

Hi Skyline developers,

We run DIA on plasma and serum samples in our lab. We have an in-house library generated from fractionated DDA data that gets updated several times a year when we have a new cohort. This has been done on Skyline 19.1 for past two years. The library searches were done on Proteome Discover 2.3 or 2.4.

We are now setting up Skyline 21.2.0.425 on a server and we also have a new cohort to add on to the library. We know that the library PD search on each of our cohorts has around mid 1000+ proteins, and the concatenated library would have 2000+ proteins at most. However one of the library files is giving me an additional 5000 proteins when I add it to the document. This happens whether I append .blib files or select multiple libraries under peptide settings.

Could I send you the problematic library file to take a look?

view request
Skyline Tuning optimisation vs instrument software Tuning optimisation Shimadzu 8060?
(1 response) jxg918 2022-06-27

Hey all!

Does anyone have any experience comparing an instrument-automatic optimisation process to select the best voltage settings vs Skyline's CE optimisation? Does Skyline also optimise other voltage settings (such as interface voltage, DL bias, Qarray RF, Q1 and Q3 pre-rod bias)?
I'm using a Shimadzu LC-MS/MS 8060NX (QQQ).

I've been using skyline for a proteomics project to quantify very-low abundant proteins in very rare samples.. So I'd need to have my instrument settings as optimal as can be. Just don't know if it's best to use Skyline or the vendor-specific optimisation tool.
I already have my MRM transitions selected and I know the retention times of my peptides, so it's just the final step of finetuning the triple quad before running all samples, but I'm running low on heavy standard..

If anyone with experience on a Shimadzu 8060 could help me with this, would be much appreciated!

Many thanks,
Jan

view request
Strange ppm mass error averaging
(10 responses) jrenders 2022-06-24
Hi there,
I am observing some strange ppm mass error averaging for a signal in my document. Basically, when I choose to integrate an entire peak, the average mass error is calculated as 0 ppm. I understand this to be an average of all precursor scans across the peak (from previous related questions). However, if I integrate any individual point in the peak, there is no section that has a ppm mass error under 8ppm. Additionally, when I browse the raw data in Freestyle (Thermo) there are no MS1 signals that are within 25 ppm of the mass in question.

This leads to three questions for me:
1. Why does averaging several signals that all have ppm mass error's greater than 8, lead to an average of 0?

2. How can two different software platforms have such a different interpretation of of what the ppm mass error is when the theoretical mass is the same in both cases. There is only one formula for ppm mass error right (delta mass/mass * 10^6)? I see this discrepancy between mass error calculations often in Skyline (vs Thermo's Freestyle) and in some cases it actually leads to quite a few false positives based on exact mass.

3.Is there any way to have skyline filter out MS1 signals that are above a certain ppm mass error threshold? For example, based on my instrumentation and mass accuracy I don't care to see signals that are greater than 5 ppm at all in the MS1 dimension.

I have attached screen shots and my skyline doc. Let me know if you need more info. Thanks.
 Picture1.png  Picture2.png  ppm mass error.sky.zip 
view request
Is there any way of setting a transition with an m/z >10,000?
lam5672 2022-06-23
view request
Error when Importing .raw Files: "Search failed: [RawFileImpl::ctor()] Instrument index not available for requested device"
(8 responses) ehubb004 2022-06-21

Dear Skyline Team,

I am trying to build a Skyline document using proteomic DDA data, but am running into an error at the DDA search step at the very end of the import peptide search process:

Search failed: [RawFileImpl::ctor()] Instrument index not available for requested device
Parameter name: instrumentIndex

It seems like an issue with the raw files themselves, since Xcalibur won't open them and MSConvert gives the same error as Skyline. I've seen a similar error in other support threads but none with "ctor()" as the designated cause of the problem. I've attached a screenshot of the error message. The raw file itself is too large to post, though I'd be happy to provide it any other way if you'd like. Is there any solution to this issue?

Best,
Evan

 error message.PNG 
view request
Re-align RT peaks in chromatograms
(2 responses) mhhur 2022-06-17

Hello Skyline team,

I have a question about the large RT shifting issue among chromatograms. I am trying to find peaks in each other chromatogram for the presence of each RT peak in a chromatogram. I have developed an automated isomer detection algorithm in Skyline and mostly, it works very well but in some cases, it doesn't work because of the large RT shifting issue. By any chance, are there any features in Skyline to realign chromatograms? Or do you know any solutions to fix the large RT shifting issue between chromatograms?

Thank you,
Manhoi

view request
Highlight fragment ion in peptide sequence
Nadzeya 2022-06-19

Dear Skyline Team,

I do targeted mass spectrometry for phosphopeptides. Since the same peptide can be phosphorylated at different positions, it is crucial to have a specific fragment ion for site localization. Is there a function in Skyline to somehow highlight a selected fragment ion in a peptide sequence? For example, highlighting amino acids with another colour or a rectangle (see attachment). This would allow a quick visual assessment of the role of the fragment ion for PTM site localization. Now I'm counting with my eyes and moving a pencil on the monitor, both suffer :)

I really appreciate any help you can provide.

Kind regards,
Nadzeya

 specific_fragments_phosphoisoforms.jpg 
view request
Export PTM site
Nadzeya 2022-06-19

Dear Skyline Team,

I started to use Skyline recently. How can I export information about amino acids with a particular modification and their position in the protein? For example, I want information about phosphosites in the following format: protein, AA in one letter code and position in the protein.
I found the First position and Last Position (number) of the peptide in the protein. I can calculate the phosphosite position in Excel: find AA before [+80] and then add its number to the "First position" reported in Skyline. Undoubtedly, it would be nice to get information about phosphosite from Skyline directly.

Many thanks for considering my request.

Kind regards,
Nadzeya

 PTM site.jpg  PTM site_calculated.jpg 
view request
Cys occupancy and peptide inclusion
(1 response) Stella 2022-06-17

Hi everyone!

I have a simple question: I am working with compounds that react covalently to Cys on targeted proteins and I would like to ask if there is a way to automatically selected peptides containing Cys, similarly to the "Exclude peptides containing" in the Peptide Settings.

Thank you very much!
Stella

view request
Can't install skyline daily to Crucios's PC
(1 response) rj8 2022-06-17

Skyline daily was not able to update, so I uninstalled it, downloaded the setup.exe file and double clicked. At some point a window said that skyline couldn't be installed and that I should contact the administrators of the software. There was a 'details' button that provided the following information:

PLATFORM VERSION INFO
Windows : 10.0.14393.0 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.8.4290.0 built by: NET48REL1LAST_B
clr.dll : 4.8.4526.0 built by: NET48REL1LAST_B
dfdll.dll : 4.8.4290.0 built by: NET48REL1LAST_B
dfshim.dll : 10.0.14393.0 (rs1_release.160715-1616)

SOURCES
Deployment url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application
Server : Apache

IDENTITIES
Deployment Identity : Skyline-daily.application, Version=21.2.1.514, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil

APPLICATION SUMMARY
* Installable application.

ERROR SUMMARY
Below is a summary of the errors, details of these errors are listed later in the log.
* Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application resulted in exception. Following failure messages were detected:
+ Value does not fall within the expected range.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
No transaction error was detected.

WARNINGS
There were no warnings during this operation.

OPERATION PROGRESS STATUS
* [6/17/2022 8:26:54 AM] : Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application has started.
* [6/17/2022 8:26:55 AM] : Processing of deployment manifest has successfully completed.

ERROR DETAILS
Following errors were detected during this operation.
* [6/17/2022 8:29:33 AM] System.ArgumentException
- Value does not fall within the expected range.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.NativeMethods.CorLaunchApplication(UInt32 hostType, String applicationFullName, Int32 manifestPathsCount, String[] manifestPaths, Int32 activationDataCount, String[] activationData, PROCESS_INFORMATION processInformation)
at System.Deployment.Application.ComponentStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.SubscriptionStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.ApplicationActivator.Activate(DefinitionAppId appId, AssemblyManifest appManifest, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivation(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl, Uri& deploymentUri)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
--- End of stack trace from previous location where exception was thrown ---
at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)

COMPONENT STORE TRANSACTION DETAILS
* Transaction at [6/17/2022 8:29:32 AM]
+ System.Deployment.Internal.Isolation.StoreOperationSetDeploymentMetadata
- Status: Set
- HRESULT: 0x0
+ System.Deployment.Internal.Isolation.StoreTransactionOperationType (27)
- HRESULT: 0x0

view request
Import drift time and CCS from transition list
(1 response) rebecca glaskin 2022-06-16

I created a transition list with the species name, formula, charge state, adduct, CCS, and drift time but not able to observe the drift time and CCS values when I load in a datafile. I can observed drift times from the transition settings and use results to build a CCS database but that does not include isomers found where multiple conformers are observed for a species. Any suggestions?

view request
Document grid looks empty reg...
(3 responses) amey shirolkar 2022-06-16

hello Team,

After generating a speclib. file (from DDA data) and comparing my .raw data files (DIA and DDA, in separate Skyline studies) with it on left panel I am getting a list of proteins (and associated peptides).
But while exporting through peptide ratio results I am getting blank sheets, even view > document grid looks empty.
Even after doing Refine > Associate proteins, it doesn't look promising.

I am interested in
Peptide
Protein
Replicate
Precursor m/z
Charge
Product m/z
Charge
Fragment ion
RT
Area
Background
Peak rank

these column heads for this analyzed data.

Please assist me with this.
Regards.

view request
Custom made modification [Phospho-18O (STY)] is 'filtered out' during import of peptide search results
(9 responses) jmvb885 2022-06-13

Hi!

I want to explore the MS1 signal of some samples in which proteins were phosphorylated with heavy ATP. I know that the gamma phosphate of my heavy ATP has two Oxygen-18 atoms. Hence when I type in the formula in both Maxquant and Skyline, I get a mass of 83.9748. I did my database search using Maxquant and now I am trying to import the results into Skyline to explore the MS1 signal.
For some reason, it seems that Skyline is not recognizing the Phospho-O18 (STY) modification I've created, since the wizard for importing the search results doesn't suggest its addition (in contrast to when I do the exact same thing using results from a sample with light ATP, meaning the regular Phospho STY (80) is added). But most importantly, after the wizard finishes importing the raw files, the 18O modified peptides are not detected.
To me it seems like Skyline is filtering these peptides out during the import of the database search results, hence they are not in the target list and the chromatograms are not extracted.
I have already confirmed that the 18O peptides are there (they are detected by Maxquant and I've seen their MS1 signal in Qualbrowser)
Any ideas on how to fix/solve this?
Thanks in advance for the help!
Best regards,

Juan M. Valverde

view request
Waters Synapt G2-Si data
(18 responses) diaz-galiano 2022-05-23

Dear Skyline developers,

We are currently working on a Waters Synapt G2-Si instrument in IM-MS mode. We have been searching for a way to analyze the samples using Skyline, including CCS/drift time values, but we haven't found a tutorial on that (perhaps we haven't looked well enough?)

Could you please direct us to a tutorial on converting the Waters .raw files with MS and Ion Mobility information for their use on the Skyline software?

Thank you in advance for your time.

Best regards,

F. J. Díaz-Galiano

view request
Is SkyLine filtering MS/MS peaks with low intensities?
(4 responses) klongnecker 2022-06-15

Hello,
Multiple members of our lab have started to use SkyLine and have posted questions here in the forum. Each time we appreciate the answers and the help you have provided. Thank you. Generally, we work on small molecules so the recent additions to SkyLine in that realm are helpful.

In a current project I have three sets data for each molecule: unlabeled, 13C labeled, and D5 labeled. I have 13C- and D5-labeled compounds for different standard curves (in the same sample). Hence, this is a use case not within the standard use of SkyLine and I have setup a specific set of molecule names so I can process peak areas that I export out of SkyLine (as raw peak areas, not normalized, no ratios, etc.). We do our quantification on MS1 peaks. However, we use the MS/MS peaks to verify the identity of our known compounds. Hence, even if there is only one MS2 scan with a transition, we find it useful. To make this work I have taken a few extra steps. Thanks to the help you have provided here on the forum (e.g. here, here, and here) I have set the transition settings/full scan to DIA and blanked out the explicit retention times when importing the transition settings.

This gets me the MS1/precursors for the unlabeled, 13C labeled, and D5 options. However, I am still missing some transitions. In the PowerPoint file I show an example with tryptophan where I only see one fragment (shown in MZmine and SkyLine) and a second example with tryptamine where I see the two fragments I am expecting. The missing MS2 peak is the lowest intensity, but it still appears in 6+ MS2 scans so I know it is there.

Any thoughts on additional settings I may try to have SkyLine show me all (or more) of the MS/MS peaks?

Thanks again for your help.
Krista Longnecker
Woods Hole Oceanographic Institution

One detail - I also uploaded a zip file (from File/Share) that is Longnecker_LowIntensityMS2.zip, and the two *raw files to the FTP site.

 Longnecker_SkyLine_question.pptx 
view request
Strange error when making Library from mzML files from TimsTOF
(11 responses) mnt 2022-06-07
Hi Skyline

I am attempting to use skyline on a dataset of mine, however in the process of creating a library, the following error is generated.

---------------------------
Skyline
---------------------------
ERROR: No spectra were found for the new library.

Command-line: C:\Users\au297068\AppData\Local\Apps\2.0\V13GT215.OHN\V48609XA.R6H\skyl..tion_e4141a2a22107248_0015.0002_38ac599f018d3163\BlibBuild -s -A -H -o -c 0.95 -i IMTest -K -S "C:\Users\au297068\AppData\Local\Temp\tmpC0B2.tmp" "C:\Users\au297068\Desktop\IMTest-xiSEARCH\SkylineOUT\Compressed\IMTest.redundant.blib"
Working directory: C:\Users\au297068\Desktop\IMTest-xiSEARCH\Data\Compressed
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\au297068\AppData\Local\Apps\2.0\V13GT215.OHN\V48609XA.R6H\skyl..tion_e4141a2a22107248_0015.0002_38ac599f018d3163\BlibBuild -s -A -H -o -c 0.95 -i IMTest -K -S "C:\Users\au297068\AppData\Local\Temp\tmpC0B2.tmp" "C:\Users\au297068\Desktop\IMTest-xiSEARCH\SkylineOUT\Compressed\IMTest.redundant.blib"
Working directory: C:\Users\au297068\Desktop\IMTest-xiSEARCH\Data\Compressed
   ved pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) i C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:linje 149
   ved pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) i C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:linje 201
   ved pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) i C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:linje 157
---------------------------

I am unsure of what exactly this means, however the purpose of the test is to investigate if we can use mzML files that are converted like this. The orriginal data is from a TimsTOF, the converted mzML file is with ScanSumming in order to collapse the IM. Thus my question is if this is an error, or if this approach is not compatible with Skyline.

Best
Martin
view request
Online Skyline Courses
ronen gabizon 2022-06-14

Hello

Are there any online skyline courses planned in the near future, or is everything going to be in-person from now on?

Thanks!

Ronen

view request
The document format version 22.12 is newer than the version 21.2 supported by Skyline (64-bit) .
(2 responses) aa16518 2022-06-13

Hi Skyline Team,

I am trying to open a Skyline file, but I am receiving this message as stated, "The document format version 22.12 is newer than the version 21.2 supported by Skyline (64-bit). 21.2.0.425 (e653b4c5e)." This is not my own file - I downloaded it through Dropbox.

I read a previous post with a similar issue, but this is different because I cannot seem to find a 22.12 version?

Thank you in advance for your help,
Audrey

 opening_file_log.txt 
view request
Required third party software
(2 responses) taperez 2022-06-09

Hello Skyline Team:

Our IT (Information Technology) department is evaluating our PC’s and software’s to determine what is pre-requisite for your instrument/test equipment product(s) to run properly. The goal of this exercise is to minimize our agencies security exposure. If possible, can you give us a list of pre-requisite software needed for your products to run properly? some examples of third party software: 7-zip, Dynamic Web TWAIN, Java, etc.

thanks!

Tatiana

view request
Observed transition mass
(3 responses) dan15060 2022-05-30

Dear Skyline support

I was wondering if it is possible to get the measured, observe transition mass from skyline for a specific scan in a specific sample/raw file. So something in analogy to "Raw Spectrum Ids" or "Raw Times". I'm after the transition mass(es) that I see when I look at a fragment spectrum in xcalibur/freestyle.
I'm was not able to find this in the Export Report options, I apologize if I just missed this option.

I'm running Skyline 21.2.0.369 on win10, the raw files were acquired on a Thermo Exploris 480 and the measured transition masses were accessed using Thermo Freestyle 1.6.

Thank you for your help

Dan

view request
Error when inserting cross-linked peptides into document
(2 responses) Juan C. Rojas E. 2022-06-09

Hi Skyline team,

So I am trying to insert a set of cross-linked peptides into Skyline through Edit -> Insert -> Peptides... by copying a list of crosslinked peptides that I parsed from a MeroX output into Skyline format. I loaded a .txt file (attached) with these sequences in Excel and attempted to copy-paste the whole list into the window in Skyline as I usually do for other "normal" peptides, but keep getting a "This peptide sequence contains invalid characters".

Strangely, when I click inside the cell of "Peptide Sequence" and paste a single sequence from the parsed list it accepts the entry. Any ideas what is going on?

Attached is also the Skyline document with the required mods and some pre-loaded peptides.

Hope ASMS was fun!
Sincerely,
Juan C.

 MeroX_skyline_identifiers.txt  ribosome_timsTOF_publ_share.sky.zip 
view request
Error when inserting cross-linked peptides into document
Juan C. Rojas E. 2022-06-09

Hi Skyline team,

So I am trying to insert a set of cross-linked peptides into Skyline through Edit -> Insert -> Peptides... by copying a list of crosslinked peptides that I parsed from a MeroX output into Skyline format. I loaded a .txt file (attached) with these sequences in Excel and attempted to copy-paste the whole list into the window in Skyline as I usually do for other "normal" peptides, but keep getting a "This peptide sequence contains invalid characters".

Strangely, when I click inside the cell of "Peptide Sequence" and paste a single sequence from the parsed list it accepts the entry. Any ideas what is going on?

Attached is also the Skyline document with the required mods and some pre-loaded peptides.

Hope ASMS was fun!
Sincerely,
Juan C.

view request
Windows upgrade-to windows 11
(3 responses) paul.derbyshire 2018-10-08

Dear Skyline support team,

I am running Skyline on Windows 7 operating system but we are due for upgrade to Windows 10. Will Skyline still perform the same after the upgrade and will my Skyline documents be unaffected?

Thanks

view request
Retention Time Black Arrow
(1 response) taperez 2022-06-01

Hello:

When we exported this data, there was low peak area, we believe its because Skyline is automatically selecting the wrong "peak". Could you explain how we can automatically have skyline select the "true peak" which I believe seems very obvious in the first top box. It seems that it does not recognize the true peak because the peak boundaries are off. When we manually moved the peak boundaries, the correct peak was selected. Is there a way to set these boundaries so that the right peak is recognized? We imported multiple runs into the same file, and if you see image attached, the correct boundaries are applied on some runs, but not all runs (i.e. first top left box). Is there a reason for the file not to universally use the same boundaries? Do we have something switched on/off incorrectly?

Thanks,

Tatiana & Team

 20220531.JPG 
view request
QuaSAR and AvantGardeDIA
(2 responses) stephenaw777 2022-05-26

Hello Skyline,
Are the plugins QuaSAR and AvantGardeDIA still being used and updated? I was unable to load either plugin into Skyline. I had no issue with all the other tools. I noticed the plugins were from 2012-13 time, so I am wondering if people no longer use them since our EIS specialist could not get either plugin to load? Better alternatives? Please advise.
Thank you,
Stephen

view request
Issues importing *.wiff files in 21.2.0.425
(6 responses) matt albertolle 2022-05-27

Running on Windows 10:
This is the error I encountered

Failed importing results file sample Blank3.
[ExperimentImpl::getSIC()] Could not load file or assembly 'OFX.Core.Contracts, Version=1.0.0.0, Culture=neutral, PublicKeyToken=50cfc49bfcc958d0' or one of its dependencies. The system cannot find the file specified.

view request
Are all peptides incl. iRTs used for normalization?
(1 response) roberthardt 2022-05-31

Dear Skyline-team,

I am just curious to know if Skyline actually uses all peptides/precursors present in the document when I select "normalize medians" in the quantification settings? Or does it only use the target peptides and omits the iRTs? I am asking because I currently rely on the CiRt peptides, of which not all are present in all my samples at the same amounts. Thus taking also this peptides into account for normalization could potentially skew my results.

Best

Robert

view request
Failed to check for an update
(3 responses) Joerg 2022-05-28

Hi,
since the latest update, my Skyline Daily version fails to check for an update. It does the checking automatically during starts, but the error occurs also when I try to update manually. Am I the only one experiencing this?
Best Regards,
Joerg

view request
ERROR: Failed to find required column named 'file'.
(6 responses) zzhu248 2022-05-26

Dear Developers and all researchers,

When I was trying to build the library via peptide setting, I upload my .ssl file and it pops up warning as title.

For the specific workflow, I am following a paper from nature protocol: https://doi.org/10.1038/s41596-018-0089-3
I was working on the crosslinking setting and stuck on the step 39.

I would be very appreciated for your help!

Best Regards,
Peter

 Screenshot 2022-05-26 143119.png 
view request
Crash when importing raw files
(5 responses) erin weisenhorn 2022-05-25

I am running Skyline Daily with version 21.2.1.485 on Windows 10 and am trying to import MRM raw files from a Xevo G2-XS. When I import the files Skyline crashes and gives no error. I have replicated this on multiple computers. Under transition settings I have the mass analyzer set to 'TOF' and not centroided as another user reported causes error. I would really appreciate help with this and would be happy to provide the raw files.

 TransitionSettings.PNG 
view request
Deconvoluted Spectra - Intact Proteins - timsTOF
Diego Assis 2022-05-24

Dear Skyline Developers,

I would like to quantify relative abundance of intact proteins isoforms from data acquired on timsTOF (mobility ON and a deconvoluted spectrum). Is possible to import this deconvoluted mass spectrum (or some file extension) and perform quantification on skyline? If yes, which Bruker's file format would be compatible with this workflow?

Thank you so much

Diego Assis

view request
DIA Analysis through command line tool.
(11 responses) jingyangzhang0222 2022-05-17

Hi Skyline Team,

I am trying to implement a Skyline workflow through the command line, referring to this document(https://skyline.ms/files/c90e7bac-8854-102e-9e41-e08d31b9c205/%40files/docs/Skyline Command-Line Interface-4_2.pdf?renderAs=DEFAULT&_docid=dav%3A%2F_webdav%2Fc90e7bac-8854-102e-9e41-e08d31b9c205%2F%40files%2Fdocs%2FSkyline Command-Line Interface-4_2.pdf).

It is very useful to open, modify and save the existing Skyline files through the command line, but there seems to be no "new Skyline Project" in the command-line options, and how can I set the work mode to "DIA"? If there is a general DIA workflow for Command Line Tool, it would be very helpful, thank you!

view request
SureQuant Method export
(3 responses) klemens froehlich 2022-05-19
Dear Skyline Developer Team,
I am currently trying to export a SureQuant method from Skyline for an Exploris system.

I get the following error message:

System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found. ---> System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found.
   at pwiz.Skyline.Model.ThermoMassListExporter.EnsureLibraries() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 1065
   at pwiz.Skyline.Model.ThermoSureQuantMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 1992
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_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_21_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1939
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_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_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2232

I checked the Thermo.TNG.MethodXMLFactory.dll and found it here:
C:\Thermo\Instruments\Exploris\3.1\System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll

Is Skyline looking for this dll somewhere else (where I could copy it to)? I have tried to copy the dll into older Exploris software version eg:
C:\Thermo\Instruments\Exploris\3.0\System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll
but it still gives me the same error.

Any help would be much appreciated, as the method export for sureQuant would be extremely convenient!!!

Best, Klemens
view request
Can I set up Skyline to exclude quantification results when scan numbers are too low?
(1 response) wangn2 2022-05-20
Hello there!
I got a question about the scan numbers (or also called "points across peak"?) in Skyline.
When the concentration of a target peptide is too low in a sample, usually the scan numbers of the chromatograph peak would be very small, say 3-6.
Can I exclude such peaks for quantification, meaning, ask Skyline to report intensity as N/A or 0, for peaks that have scan numbers less than a specific number, e.g. six?

Thanks a lot!
 skyline_scan_numbers.JPG 
view request
Relative quantification with surrogate standards
(3 responses) clay davis 2022-05-20

I am trying to quantify different lipid classes using several isotopically labeled surrogate standards. Once the labeled lipid internal standard is chosen as "surrogate standard" and set the "Normalization Method" to the respective standard for the other lipids, the normalized area reports n/a. I’ve looked at several ratio outputs in the report option but only the “Total Area Normalized” (Molecule list – Molecules – Precursors – Precursor Results and Area Normalize (Molecule list – Molecules – Precursors – Transition Results) seems to output a value as a percent and not necessarily the ratio or ratio x IS concentration.

  • Clay
 Skyline.JPG 
view request
Collision Energy Optimization Small Molecules
(2 responses) klemens froehlich 2022-05-18

Dear Skyline Developers,
First of all thank you very much for your continued support and an amazing software package!

We are currently starting to use Skyline for small molecule projects.
I know that Skyline can do collision energy optimizations for SRM type experiments using transition list method export as described in a tutorial.

However, we only have high resolution instruments (orbitraps) in our lab and would like to do PRM of small molecules.

Accoding to this thread:
https://skyline.ms/announcements/home/support/thread.view?rowId=55461
Skyline cannot do collision energy optimization for PRM data.

Now my question is:
Wouldn't it make sense to optimize the collision energy for small molecules even if we are using PRM? For example if I go to a publication where a compound was measured on a machine that uses a different type of fragmentation and I do not know in what range my collision energy would be optimal?
Or if only one transition is reported in a SRM experiment and I want to make sure to get the best sensitivity from my orbitrap machine for this transition?

Of course I can use more transitions in PRM but for small molecules I think that collision energy optimization is more important as you often only use one or two transitions as compared to pptide quantitation where you use more transitions.

Would it be possible to integrate PRM collision energy optimization somehow in Skyline?
Maybe by shifting the transition mass also by 0.0001 instead of 0.01 as for SRM experiments?

Best, Klemens

view request
Integration of single masses in a DIA experiment on Thermo Scientifi Orbitrap ID-X Tribrid Mass Spectrometer
(7 responses) harald schoeny 2022-05-12

Dear Skyline Team,
I am trying to integrate a single mass (184.07332 Da) on MS2 level of a DIA experiment, in which I selected a specific precursor mass range (670-900) and measured a specific product mass range (180-190). I want to obtain a sum value for a lipid class and I have already applied this method on a Thermo QExective HF (see attached publication- AIF method).
Now I have measured the same on an ID-X instrument and the MS2 was recorded in an ion trap instead of the orbitrap.
Unfortunately, I am not able to get anything in skyline although these settings worked for the HF experiment. I have tried to set the product mass analyzer in the transition settings to QIT (but also every other option) but nothing worked.
Do you have any idea, what I can try?

Thnak you very much! All the best,
Harald

 Schoeny 2022 Anayltical Chemistry.pdf  Capture.PNG 
view request
Can I specify the retention time when exporting quantification results?
(6 responses) wangn2 2022-05-18
I am using skyline 21.2.0.425.
I found that skyline doesn't always pick the right retention time peak for quantification, possibly due to ion interference from the background (I am using cell lysate as background). Since I have already got retention times of my peptide by running neat standards, I was wondering if I could set up skyline so that for a specific peptide, it only export the quantification result at a fixed R.T.
For example, by running neat standard sample of peptide A I found its RT to be 60.28 min, then by running a sample with peptide A in cell lysate, I would like skyline to export the quantification of peptide A only at the R.T. of 60.28 +/- 5 min, is this possible?

Thanks a lot for your help!
view request
Installation Skyline-daily
(1 response) dima kovalerchik 2022-05-17

Hi Skyline team,
I am trying to install Skyline-daily. I enter the link https://skyline.ms/project/home/software/Skyline/daily/begin.view? but it is written:

"Oops! An error has occurred.
User does not have permission to perform this operation.
Please contact this server's administrator to gain access."

How can I get the permission?
Thanks Dima

view request
Average Mass Error PPM
(4 responses) sarah.lennon 2013-11-27
Hi Brendan,

I'm currently using skyline Daily vr. 2.1.1.5169 to do MS1 quantification using Maxis Impact Bruker datas. In order to apply a second filter, I wanted to export the average mass error ppm. Here, it appears, that the number reporter in the .csv file is not exactly the same than the one on the top the peak indicated in the skyline file.
Do you have an explanation? Maybe I'm not exporting what I'm thinking.

Enclosed, you will find the skyline file as well as the .csv file.

Thanks for your help,

Regards,

Sarah
 20131028 6 bandes vr2rs 40000 br.csv  20131028 6 bandes vr2rs 40000 br.sky.view 
view request
Optimization of injection or fill time in Exploris 240
(1 response) danielacgranato 2022-05-16

Dear,
We are performing PRM in Exploris 240 and we would like to optimize the best injection time/fill time for each peptide, especially in the case of peptides that compete in the same scheduled window. Can this optimization be performed in Skyline? Can Skyline extract information of best injection time or acquisition time? Can Skyline help us evaluate what peptides need to improve injection time?

Thank you very much.
Best, Daniela

view request
DNA and mRNA oligonucleotide mapping using Skyline
Alok 2022-05-16

Hello Skyline Team,

In addition to protein/peptide therapeutics, there is a quite a lot going on with regards to DNA and mRNA therapeutic development. As far as I am aware, one can analyse a small number of DNA or mRNA using 'molecule interface' of Skyline. However, this is not possible when a long DNA (e.g. full length plasmid DNA) or mRNA need to be studied using mass spectrometry based workflows. Similar to bottom-up proteomics, the long DNA or mRNA needs to be digested by endonuclease(s) followed by either MS1 based mass fingerprinting or MS2 based sequencing analysis. I wonder if these kind of full length DNA or mRNA MS-based analysis features are available on Skyline. If not, then is there any plan to develop such features in future?

Thanks,
Alok

view request
Bad DIA Result, is something I set was wrong?
(12 responses) jingyangzhang0222 2022-05-03

Hello Skyline Team,

I am a university student trying to reproduce some results from this paper(https://pubs.acs.org/doi/10.1021/acs.jproteome.1c00490?ref=pdf). I have used the same setting(including Isolation Scheme, Instrument, etc.) as the paper did, but the result was not good enough to show the data's validity.

Only a few peaks show reasonable shapes, which can be observed in the official DIA tutorial. Besides, I was trying the acquire quantification data from the software, but it was not reliable, either. I have uploaded the project file(.zip) to the server, you can download it here(https://skyline.ms/_webdav/home/support/file sharing/%40files/blib_narrow_50fmol.sky.zip), is there any advice on how to adjust the settings, or to correct my operation? It would help a lot for my project if any guidance should be given.

Best,
Finn

view request
13C phenylalanine and second isotope abundance of unlabeled phenylalanine
(1 response) Sam110 2022-05-10

Hello, I have a dataset with phenylalanine and isotopically labeled 13C phenylalanine which is only one mass unit from the unlabeled phenylalanine. I wanted to know if Skyline can subtract the value for the second isotope abundance of the unlabeled phenylalanine from the 13C phenylalanine?

view request
Error message when trying to open Bruderer.bcfg on Skyline batch
(1 response) lutho 2022-05-10

Hi there,

I am trying to open the Bruderer.bcfg files in preparation for the online course currently running at the May Institute. I have installed Skyline batch and downloaded the Bruderer.bcfg file. I cannot open the file on Skyline batch and I have attached a screenshot of the error message that I am getting.

 Screenshot 2022-05-10 161435bruderer error.png 
view request
SureQuant Tutorial for Custom Method
(1 response) comtewal 2022-03-31

Dear Skyline Developers,

Would it be possible to create a generic tutorial to use when acquiring data on a Thermo instrument using a SureQuant custom method (as opposed to using the commercial kits of reagents/peptides) supplied by Thermo.

I have been trying to create a Skyline method to quantify our peptides of interest using Skyline v. 21.2.0.425 but the transitions for my peptides (heavy and light versions) are importing but I am not able to visualize them.

Thank you so much,
Susana Comte-Walters
MS Redox Core Facility
Medical University of SC
comtewal@musc.edu

view request
Support for SureQuant custom panel
(1 response) edf4n 2022-02-14

Dear Skyline,
I read in your release notes for Skyline 21.2 the announcement:
“New! Improved SureQuant support with "SureQuant" MS/MS acquisition method and method export for Exploris.
o Also added new PRM acquisition method (an improvement over the old "Targeted" method – now deprecated).”
I’m wondering if you can point me to using these features in your tutorials? I have been working my way through the skyline Targeted Method Editing tutorial. Eventually I would like to create a transition settings report for a custom SureQuant panel. I’m not sure that I am doing it correctly, especially for the export settings.
I have an Orbitrap Eclipse instrument and am using version 21.2.0.369 of skyline
Thank you!

view request
Prosit score export
(1 response) nifr 2022-05-04

Dear Skyline team,

is it possible to export the dot products from matching of my spectral library with Prosit via the document grid?

Best
Nicola

 Screenshot 2022-05-04 171832.png 
view request
Protein group, razor peptide and issue #861
ho-tak lau 2022-05-06

Hi Skyline Team,

I have been hoping to be able to remove repeated peptides in certain ways in Skyline for a long time. Recently, I saw the discussion in issue #861, and I would like to chime in.
I wonder if Skyline can integrate ProteinProphet or to read the prot.xml from ProteinProphet, and then have an option to remove/export the repeated peptides/empty proteins. Alternatively, Percolator also does protein inference now. Percolator wiki Maybe this can be an option for MSAmanda search.

Regards
Ho-Tak

view request
Which formula to choose for combining peptides intensity into protein intensity ?
(2 responses) nicolas pierre 2022-05-05

Hello Skyline Team,

I have measured endogenous peptides with their heavy counterpart by SRM. I have also spike a protein in my sample for normalisation.
I have some questions for translated results at protein level.
When I divide the intensity of endogenous peptide by their heavy counterpart it become a ratio and in many cases this ratio totally change the ponderation of each peptide for their contribution to the protein intensity. Typically, peptides representing the highest intensity (the one which would contribute the more to protein signal with the addition of peptide intensities) for the protein can, when divided by its heavy counterpart, be the one with the lowest ratio among the other peptide of the protein. In this case this will totally change the ponderation. I am not very confident with this phenomena because usually signal with the lowest intensity are those with the lowest quality (near LOD, lowest level of precision, lowest quality of the peak...). It means that with a division by the heavy peptides I will give more importance (not systematically) to signal with the lowest quality.
In fact their is different solution to combine the signal of peptide into protein signal. For instance:

-Signal of protein A= (Signal of endogenous peptides 1/ signal of heavy peptide 1) + (Signal of endogenous peptides 2/ signal of heavy peptide 2)
-Signal of protein A = (Signal of endogenous peptides 1 + Signal of endogenous peptides 2)/(signal of heavy peptide 1 + signal of heavy peptide 2)

As explained, the first formula can change the ponderation of the peptides contributing to the protein signal, the second one will not. We could imagine other solutions...Also, I have spiked a protein for normalising my signal and I do not know where I can put it in the formula, where would be the best place..
Do you have some advises for this problem ? In there some Skyline solutions ?
Thank you in advance for your help.

Nicolas Pierre

view request
Zeno MRM Wiff 2 file
(1 response) bkr 2022-05-04

Dear,

I am currently working on Zeno MRM data and I am not able to import wiff2 data into skyline.

I get the following error

"At 13:10:
Failed importing results file 'F:\20220504\GA_Zenooff_q1_open_50ms_MRMHR_01.wiff2'.
[WiffFile2Impl::ctor()] Object reference not set to an instance of an object."

Kindly help me with this.

It would be also great if you can suggest transition settings for this type of data

regards,
Bharath

view request
Collision Energy Optimisation on TimsTOF Bruker
(2 responses) karweens jean 2022-05-03

Dear Skyline Team,
I am using a Bruker TimsTOF for proteomic analysis in PRM-PASEF mode and I would like to use the skyline software to optimize the collision energies of the produced ions. I have created a file taking into account the different collision energies to test per ion.

The acquisition went well and I managed to import the file on skyline.

However, skyline refuses to display the full histogram for the different collision energies tested.

I would like to know if it is possible to optimize collision energies with a TimsTOF file on skyline.

Thank you very much for your help,
Kind regards,

Karweens

 Collision Energy Optimisation on TimsTOF Bruker.docx 
view request
Generating mass list
(1 response) tdpatsch 2022-05-03

Hi, I am quite new in the field of proteomics and therefore my request might be rather trivial. I have a database containing hypothetical proteins in FASTA format. I would need a program that imports these FASTA sequences, does the tryptic digest in silico and provides me with an MS list that I can use as an inclusion list on a Thermo QExactive Plus to search specifically for these proteins. Somebody told me that skyline can do the job. Would be great if someone could explain how to do it or refer to the proper tutorial.

view request
ERROR: No spectra were found for the new library - since updating to v 21.2
(10 responses) Francis Beaudry 2022-04-28
Hi,
 
Since I updated to 21.2, I have a very difficult time with Skyline. Sometimes it works, but most of the time it doesn’t work. Currently, we are trying to process DDA datasets and use the option DDA Raw to search and build the library. Attached, you will find the message we are getting 9 times out of 10 trials using the same raw files and search setup. Weird … Can you help ??
 
Regards,
Francis
 
---------------------------
Skyline
---------------------------
ERROR: No spectra were found for the new library.
 
Command-line: C:\Program Files\Skyline\BlibBuild -s -A -H -o -c 0.95 -i 07ADT_27avr_std_3 -S "C:\Users\francisb\AppData\Local\Temp\tmp8B1F.tmp" "C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22\Skyline\07ADT 27avr std 3.redundant.blib"
Working directory: C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.
 
Command-line: C:\Program Files\Skyline\BlibBuild -s -A -H -o -c 0.95 -i 07ADT_27avr_std_3 -S "C:\Users\francisb\AppData\Local\Temp\tmp8B1F.tmp" "C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22\Skyline\07ADT 27avr std 3.redundant.blib"
Working directory: C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22
   à pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) dans C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:ligne 149
   à pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) dans C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:ligne 201
   à pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) dans C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:ligne 157
---------------------------
view request
Normalizing Unknowns?
(5 responses) whale 2022-04-29

Hello:

I am running a peptide quant experiment and spiked the same amount of ISTD into each unknown. . However, according to the BCA assay, a few of the unknowns had a very small amount of protein (likely due to treatment) so I was unable to inject the same amount of total protein for each sample. Is there a way to normalize these small amounts (i.e. I injected 10x less material for unknown 1 vs. unknown 2)?

Many thanks!

view request
Mass Error Plots
(2 responses) philip remes 2022-04-30

When I attempt to view the Mass Error plots for PRM QIT data, no data are displayed. I don't know if this is because mass error plots are not supported for this type of data, or the errors are larger than what the tools are anticipating, or something else. It would be useful to have this feature working for the fragment ions. Ask Lilian if you need some data or a skyline document. It would let the user know what mass tolerance is feasible. A larger request would be if you could implement Jarrett's Fine Tune algorithm to calibrate the mass scale. https://pubs.acs.org/doi/abs/10.1007/s13361-012-0482-z.

Thanks
Philip

view request
Import DIA peptide search
(1 response) dilip singh 2022-04-28
Dear Team,

I am using tool "Import DIA peptide search" and getting error at last step, which is mentioned below. Kindly suggest the solution for it.

---------------------------
Skyline
---------------------------
Error
---------------------------
OK More Info
---------------------------
System.ComponentModel.Win32Exception (0x80004005): The system cannot find the file specified
   at System.Diagnostics.Process.StartWithCreateProcess(ProcessStartInfo startInfo)
   at System.Diagnostics.Process.Start(ProcessStartInfo startInfo)
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 73
   at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 162
---------------------------

Regards
Dilip Kumar Singh
view request
idotp filtering
(2 responses) lisa walrond 2022-04-26

Hello,
I am trying to filter my data based on idotp threshold (0.8). It appears when I apply this filter that peptides from all samples are deleted if peptides from any of the samples do not meet the idotp threshold. As opposed to just filtering out the specific samples that do not meet the threshold and leaving the good result. I am looking for a faster way to remove peaks from peptides that aren't real. I hope that is clear.
Thank you

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"Error getting score type for this file" (.mzid)
(3 responses) anita liu 2022-04-27

Hi,

I have been encountering errors whenever I try to build a spectral library by importing ".mzid" files generated from Byonic (Protein Metrics). The error message is as follows:

"Error getting score type for this file.
ERROR: Unknown exception
ERROR:
ERROR: reading file test.mzid"

This happens even when I try to import files that have previously been imported in the past (when my skyline version did not have the "score type" and "score threshold" columns). Could you please advise on how we currently import .mzid files to build spectral libraries?

Thank you!

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Import Sciex 7600 Zeno TOF data (wiff) - get error
(1 response) heather eastwood 2022-04-26

Hi,
I am trying to import Sciex 7600 Zeno TOF data (wiff file) but am getting the attached error. Do you know how I can fix this?

Thanks,
Heather

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