Hello,

I saw in an old thread, that comliance with CFR 21 part 11 is a long-term goal for Skyline development.
What is the current status of this endeavour?

Does Skyline provide an audit trail and user management functionality?

Best,
Tobias

Hello,

I did a Surequant run using synthetic heavy isotope-labeled peptides as triggers and for one peptide (HLVEALYL[+7]V), the product ion chromatogram was not found for the light peptide (attached) although there is a trace for the precursor. However, I could see the light peptide MS2 spectra at the correct retention time when I searched the Surequant data (attached). It seems like somehow this spectra information was not extracted by Skyline. I am using Skyline-daily 22.2.1.501

I am new to using Maxquant and Skyline for MS data analysis.
I have kept the Maxquant setting as default and tried to build the library using the msms.txt file.
The combined folder, mqpar file are all kept in the same place after the Maxquant run finished.
However, when reading the msms.txt, skyline showed the error attached, indicating that there is an error in finding the "Raw File" column in the msms.txt.
Does that mean that skyline could not find some key files because the directory might be wrong or the files are misplaced?
How might I solve the issue?
Thank you for helping!

Dear Skyline team,

Is Top-down analysis can be done on skyline, if yes could you provide tutorial/webinar link.

Thank you

I want to build an Arabidopsis library, which file(s) should I download and import into Skyline from PeptideAtlas? Thank you!

Dear Skyline Team,
Is it possible to directly import Bruker .tsf files into skyline (as possible with .tdf and .baf files)?
Thank you in advance and best regards,
Karin

Hi all,
I want to build a peptide spectral library by using .raw file,and then when I use the DDA Search for MS1 Filtering of the Skyline, the results show that the search failed,so I convert the format of my fasta files to .mz5,because I find the file format of tutorials is .mz5,then the results show “No spectra were found for the new library”.
Can anyone tell me how to fix this issue?

Hi Skyline team,

I've been optimizing peptides and finding that when I import the collected optimized declustering potential data back into the skyline file that it is missing a portion of the transition data.

I'm using Daily and running on a Sciex 6500+. When I go into the wiff files in Analyst I can see that the data was actually collected. I've found starting with a fresh skyline file helps sometimes but not always.

Thanks!
Jess Becker

Hi!
I'm working on a new label similar to TMT(tandem mass tag) applied to proteomics. Now I need to extract the quantitative information of the label (with a specific mass/charge ratio) from the secondary spectrum of the peptide. I wonder if skyline can do that. If it can be done, please tell me how to do it.
Be grateful!

Hello,
Hopefully this request makes sense..
I have imported PRM data into several different Skyline sessions successfully many times. Today when I attempted to import PRM data into a new Skyline session, the software does not appear to be trying to import the data. No status bar or window appears indicating the progress of the import and in fact nothing ever happens given longer times to work. I can import the same data into older Skyline sessions, but not the ones I created today (I've checked and all the full scan settings are correct). When I try to import data I previously imported successfully to other Skyline sessions into the newly created Skyline sessions from today, the same lack of activity issue occurs. Can anyone provide any insight or advice on this issue?
Thanks,
Sarah

Hi, I am new to the skyline and I am trying to build a spectral library using mouse raw peptide raw files.
but after the files are processed I am facing error where it shows "No spectra were found for the new library"

I am also attaching the screenshot.

Thanks

Dear support,
Uploaded a file 'MS12_error.zip' to file sharing.
The attached file is a skyline template and two raw data files from a Bruker QTOF.
I have been using this skyline template before and it always worked without issues.
It contains molecule lists of different lipid classes and their respective fragmentation precursors.
Now, chromatograms are shown during data import but are not visible afterwards anymore.
For the negative file at least a viel EICs are presented but in a seemingly random way throughout the lists.
Do you have an idea what the issue might be?
Best, Dennis

Hello, I got trouble to install the software on my computer. I saw error when the "setup.exe" tried to download the installer. Is there a link or measurement that I can manually download the installer to install, rather than rely on setup.exe to download? Many thanks.

Hi,
I am new to the Skyline and below is the error I am facing while trying build a spectral library.

System.IO.IOException: ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=11022"
ERROR: reading file OR4_110128_EDG_11EDEG002_smallSCX_26w_fr18.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=20469"
ERROR: reading file OR4_110129_EDG_11EDEG002_smallSCX_26w_fr11.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=7522"
ERROR: reading file OR4_110204_EDG_11EDEG002_smallSCX_16w_fr18.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=11924"
ERROR: reading file OR4_110204_EDG_11EDEG002_smallSCX_16w_fr20.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=7562"
ERROR: reading file OR4_110206_EDG_11EDEG002_smallSCX_16w_fr13.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=23734"
ERROR: reading file OR5_20100802_MA_EDG_ERCC1_26w_small_f20.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=16277"
ERROR: reading file OR5_201023_EDG_ERCC1_8w_small_f18.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=7780"
ERROR: reading file OR5_201023_EDG_ERCC1_8w_small_f9_2.mzid.gz
ERROR: [SpectrumList_Thermo::spectrum()] Unknown exception retrieving spectrum "controllerType=0 controllerNumber=1 scan=12722"
ERROR: reading file OR5_201029_EDG_ERCC1_16w_small_f7.mzid.gz

Command-line: C:\Windows\system32\BlibBuild -s -A -H -o -c 0.95 -i spectral_library_PXD000046 -S "C:\Users\USER\AppData\Local\Temp\tmpA768.tmp" "Z:\MS_raw\PeptideAtlas\Mouse_2016-01-JP\PXD000046\spectral_library_PXD000046.redundant.blib"
Working directory: Z:\MS_raw\PeptideAtlas\Mouse_2016-01-JP\PXD000046
위치: pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) 파일 C:\proj\skyline_22_2\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:줄 161
위치: pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) 파일 C:\proj\skyline_22_2\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:줄 412
위치: pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) 파일 C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:줄 161

Please help at the earliest.

Hi,
I want to work with human and tuberculosis proteins in the same study. How could I combine the 2 background proteomes?
Thank you
irene

Dear Skyline Support,
I'm analyzing the extract of a mussel for phytoplankton toxins that have accumulated in its tissues. I'm trying to detect dozen most prominent toxins, but would also like to record presence of hundreds of rarely occurring analogs or even some unrelated environmental pollutants. I'm collecting data using LC QTOF instrument, Sciex 6600+ in MRMHR, DDA and DIA (SWATH) modes. It seems that the most promising pipeline for SWATH data analysis, as suggested in the article in Pharmateuticals 2022, 15, 901. https://doi.org/10.3390/ph15070901, is to create a transition list using MS-DIAL, which is then exported to a Skyline for analysis of DIA data. Only document exported in .txt or .msp formats cannot be imported directly as a transition list. It requires a lot of manual intervention to get an acceptable document that can be used. This only works for a small list, not for an all-encompassing list that I'd like to have for untargeted analysis of SWATH data. Is there any better way to create a transition list/assay library, presumably from my DDA or MRM/PRM data. How big can it be? I've consulted all tutorials for small molecules, they all start with transition lists already prepared. Sorry for a long question and thank you in advance,
Stjepan Orhanović
Faculty of Science
University of Split, Croatia

Hello,
when importing a .dat file to create a library I get the following error:
"Cannot get quantitation method Butyl_double_KN_fix in file F008150.dat"

The datfile was created with the newest mascot server and daemon. I suspect this is why it is not working anymore.
I used the exact same quantitation methods before we upgraded the mascot server and then this was not a problem.

Could you have a look at the dat file for me please? Or give me a hint on which things to check in order to get the import working?

Where can i upload the files (dat file and skyline file)
?

Hi Skyline team,
I got stuck on a step and I hope someone can help me.
When I build the library in Skyline-daily I realize that I'm missing some information when it is loaded. For example, in a random protein, the DDA (msms.txt) shows it has about 200 peptides found, when I build the library in Skyline using that DDA, Skyline shows me around 100 peptides. The same goes for some modifications that I'm interested in seeing, they are not appearing in the same number that I see in the msms.txt. Does anyone know why I'm losing information when setting up the library? Is it a setting issue? I have tried several setting changes.

Thank you!

Liti

Is it possible to add support for importing search results from Sage into Skyline? Sage is an open-source, free, and faster implementation of the fragment indexing approach popularized by MSFragger.

Sage writes a simple TSV based format - a couple notes (happy to provide more info if you decide to add support):

• filename column may contain ".gz" or ".gzip" at the end - Sage can directly search mzMLs packaged in gzip, as exported by MSConvert, etc. ("b1906_293T_proteinID_01A_QE3_122212.mzML", "b1906_293T_proteinID_01A_QE3_122212.mzML.gz", etc). Sage only supports the mzML format at this time.
• Search results may be concatenated from multiple files
• Peptide sequences follow the ProForma notation: N-terminal modifications are specified as "[+42.0]-PEPT[+57.0]IDE", and C-terminal as "PEPTIDE-[+42.0]", etc.
• Unless supplied with a fasta file already containing decoy entries, Sage will generate decoys by reversing the interior amino acids (SAMPLER becomes SELPMAR)
• Currently, Sage is outputting unfiltered results, so decoys will be present (label = -1), and uses a built-in linear discriminant analysis PSM rescorer (with spectrum_q, peptide_q, protein_q corresponding to Q-values at each aggregation level and "posterior_error" corresponding to log10(PEP) at the spectrum level)

I have uploaded an example results file to https://skyline.ms/_webdav/home/support/file sharing/%40files/results.sage.tsv

The raw data corresponds to (b1906_293T_proteinID_01A_QE3_122212.mzXML) from the paper An Ultra-tolerant Database Search Identifies more than 100,000 Modified Peptides (PXD001468). I can upload the mzML to S3 if needed.

Please let me know if there is more information, files, etc I can provide!
Best,
Mike

Hi,

I am trying to build the spectral library using a mouse and one the files don't have any issues but it's still not showing the finish button after the files are processed,
I am sending you the screenshot. Please help.

Hi,
I am new to skyline and i am trying to build a spectral library using peptide raw files but i am facing this error like:-
Specified argument was out of the range of valid values.

I am also sharing the screenshot of the error.

Please help.

Hi,

I am new to skyline, I am trying build a spectral library and after all the files are processed i cant see the finish button popping up build a redundant spectral blib file.

Please help.

Hello! As the title states I'm having trouble doing PRM on small molecule targets when the data was collected with an inclusion list. When collecting data with an inclusion list the precursor targets get "shuffled" into the available MS2 events on the fly. On my Sciex qTOF they don't always get put into the same experiment (sciex terminology for ms events per duty cycle), where the 1st experiment is a TOFMS scan, followed by several experiments for the MS2 events.

This is an issue when trying to integrate with the vendor software, since I have to choose one of the numbered ms2 events to quantitate from across the entire chromatogram and samples, yet it will not necessarily fall into the same experiment.

When attempting to integrate with skyline, I could not tell if it was operating in the same way.

Can skyline pull out any ms2 events that are from a particular precusor, since I know those well, and look for the transitions I tell it? Or does it also use the same ordered ms2 event across the entire chromatogram?

Hello, I am new to the skyline.
I am trying to build a spectral library using mouse peptide raw files. But Skyline is shutting down automatically whenever i am trying to run the file.
please help.

Hi,

I am new to the skyline and I am trying to build a spectral library using mouse plasma raw files, but after the raw files are completely processed and i am getting this error where it shows importing fasta did not create any target proteins.

Please help.
Thanks.

Hi!

I am wondering if I can use the skyline logo in a publication figure where I show a workflow where I used Skyline. The editor asked me whether the image is protected or it can be used for this purpose?

Thanks in advance for your help.

Best,

Ana

Hi Skyline team,

I am trying to import TSQ altis file and suddenly I started getting this error

At 15:30:
Failed importing results file 'F:\GluC_02.raw'.
[ScanInfoImpl::initialize(corrupt scan filter for scan 1)] Specified argument was out of the range of valid values.
Parameter name: scan
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'F:\GluC_02.raw'.
[ScanInfoImpl::initialize(corrupt scan filter for scan 1)] Specified argument was out of the range of valid values.
Parameter name: scan ---> System.Exception: [ScanInfoImpl::initialize(corrupt scan filter for scan 1)] Specified argument was out of the range of valid values.
Parameter name: scan
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_22_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_22_2\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 199
--- End of inner exception stack trace ---

Hi Skyline team,

Can you point me to any documentation that outlines how Skyline calculates FWHM and FWB?

Thank you

Hello,
I have made several lists which contain protein/peptide/ms1/ms2/rentention time using skyline from my discovery untargeted proteomics data. Now I want to transfer them to TSQ altis plus for monitoring them. Can you show me tutorials for this task?

Thanks,
kmsd

Dear Skyline teams,
I’m analyzing some small molecules in SIM mode acquisition with an Agilent QQQ 6490, with polarity switching. I have this error when I try to process the data:
Failed importing results file 'C:\Users\strassel\Desktop\RKF\DATA_TestColumns\20230510_Biphenyl_OS_MIX1SIM.d'.
[translateAsPolarityType] Error parsing polarity type. (see screenshot attached)
I’ve tried to convert it with MSConvert using the commandline : msconvert -z --ignoreUnknownInstrumentError
But due to accessibility issues I wasn’t able to change the file.
Is there another possiblity to process this type of data? I didn't have any trouble processing fullscan acquisition with polarity switching.
Thank you and have a nice day !
Oriane Strassel

I want to build a spectral library through metabolome raw file, how can I build it?

I'm trying to next follow

1. enter molecule interface
2. menu tab -> settings -> molecule settings
3. library tab -> build button
4. input Name & output path -> Next
5. Add files -> input raw data

but i'm met the "The following files are not valid library input files ..." error.

input the raw file is downloaded from metabolight database, study name is MTBLS3916.

also, not only those files but also the other raw files failed to build the spectral library.

there are various file extensions such as ".raw", ".dat", and ".wiff" ...

please help me..

Dear Skyline team,

I hope you're doing well. I've been showing a colleague Skyline and she has been following the tutorials, but had been using the tutorial tab on the Skyline Start Page instead of the skyline.ms website. She hit a few snags with the tutorial steps because the tutorial links are to the older versions of the tutorials, specifically related to importing transitions as that interface has changed since the tutorial was made. She is now using the web page for the tutorials but I could see other Skyline users possibly using the Tutorial tab instead of the website and getting confused.

I've only checked the small molecule tutorials, but it might help other users if the tutorial links are updated in the app because they have no way of knowing what version tutorial they should be following along with (the skyline.ms tutorial page is up to date as far as I can tell):
Small molecule quantitation (Skyline links to the 20_1 version but it should be 21_2)
Small molecule targets (Skyline links to the 20_1 version but it should be 21_2)
Hi-res metabolomics (Skyline links to the 20_1 version but it should be 21_2)

Cheers,
Chris

Hi there Team,

Is there a way to lock a Skyline file from accidental changes or further modifications in the setting and peak integrations of files etc...?
This becomes an issue when multiple people have access to a file that is supposed to stay untouched and I personally have burned a few times from accidental changes to a Skyline file that was marked as FIANL!

Cheers,
-Vahid

Hi,

I am using Skyline-Daily 22.2.1.488, trying to import peptide search of DDA-PASEF data from MSFragger, using uncalibrated mgf files.
Although the files are in the same folder with .d files and interact-filename.mod.pep.xml files, I get the error saying the software can't find the files.
I have tried Skyline as well, the same error.
Using calibrated.mzML I can load the files but no chromatogram will be extracted eventually.
I appreciate it if you can help me on loading uncalibrated mgf files to the skyline.

Thanks,
Mohammad

Hi Skyline Team,

I hope you're doing well. While plotting the CE regression with my results, I'm noticing that the equation is stating iRT and measured RT instead of collision energy and precursor m/z. The actual slopes/intercept and equations work fine, so I think it is just a labeling issue. I've attached a screenshot of the regression plot.

This is Windows 10 and Skyline-daily 22.2.1.488.

Cheers,
Chris

Hi Skyline team,
I have a specialized analysis for in vivo protein turnover kinetics data analysis. In a nutshell this is for D3-Leu in vivo enrichment in people, where I run the PRM with precursor selection on average of the H3-Leu/D3-Leu precursor mass (and wide selection window) and very-high res MS/MS (240k). I want to be picking up D3-Leu fragments selectively from A+3 of the H3-Leu fragment. I tried to upload this data into Skyline, but it does not recognize the precursor correctly (because it is average of the H3-Leu and D3-Leu version of the peptide - e.g. for peptide IGVELTGR 422.748 and 424.2574 the precursor in mass spec was set to 423.5080). Is there a way to trick Skyline to upload this data? I believe that the resolution for the MSMS should be easy to set.
Thanks,
Tomas

Hi Skyline team,
Just updated to 22.2.1.488 this morning and noticed about half of my Export Report options disappeared. They appear as greyed-out in the Manage Reports window, but I can't seem to get them active. Could someone let me know if this is a setting vs. bug?
Thank you!
Sara

Dear Skyline support,

I noticed two small bugs in the small molecule interface. One of them I found back in a post from Chris Ashwood in 2019: https://skyline.ms/announcements/home/support/thread.view?entityId=712b51a4-fe69-1037-86c0-e465a393641d&_docid=thread%3A712b51a4-fe69-1037-86c0-e465a393641d

I have the same problem: when I open a new Skyline file, and load my small molecule data, the initially calculated IDOTPs are very low and not correct. Saving, closing and restarting the file solves the problem. This is an easy workaround, but confusing for people that are not familiar with the problem.

The other thing I encountered is also in the small molecule interface:
My settings are: Transition Settings>Full scan>MS1 filtering>Isotope peaks included, count 3 (also happens when using percentage)
When I then load the transition list, still only 1 isotope is included.
The problem is solved by changing the isotope settings to something else and back.

Thank you and best wishes,
Noortje

Hello,

I'd like to put columns into a transition report with the first and last raw time. I go to Settings/Document Settings and try to Define Annotation. Select Calculated, and for Applies to set Transitions, then set Precursor/Peptide/Transitions/Transition Results/Chromatogram/Raw Data/Raw Times. For Aggregate Operation, select Min. Now I go to a report, find this new annotation "Min Raw Time" and add to the report. It has all the Raw Times in each cell, in their comma delimited form, but I wanted to find just the minimum. If I go back to try to Edit this modification, the Aggregate operation has always switched to Count. Am I misconstruing how this operation should work? Perhaps the list of Raw times is an unparsed string and Skyline doesn't know what the minimum value would be?

Thanks
Philip

Unusual request but is there a way we could get access to an old release of skyline? -Release 21.1.0.146(9b577b3b8) or a release close to this.

Hello,

Is there a way to compare/export the intensities of precursors when importing raw data from a direct infusion experiment. I have been trying to do this in
the molecules interface mode. I can see and export the TIC for all my raw files but cannot figure out how to look at the intensities of individual precursor masses. When I click through my transition list, I only see the TIC. If I check "precursor" under transitions, the error says no precursor ion chromatograms found.

I am trying to compare signal intensity of specific precursor ions as I am tuning different mass spec parameters (not CE optimization).

Best,
Hanan

I was just wondering if it is possible to use files from a Maxquant DDA analysis (having gone through the Maxquant’s andromeda search engine) to builds a spectral library for a DIA analysis in Skyline. We generated our DDA and DIA data on a Thermo Q-Exactive instrument.

Many thanks,
John

Dear Skyline team,

Suppose I have 120 replicates, I can use View/Arrange graphs/Groups... to split them across e.g. 6 windows with each 20 tabs. Now I can browse through the replicates by clicking ctrl+up or ctrl+down, but I still need 120 clicks to browse through all replicates. Is there a way to simultaneosly switch to the next tab in all 6 windows? This would allow me to browse through all 120 replicates in just 20 clicks.

Thanks,
Philipp

P.S. I am using Skyline v21 on Windows 10

I have many questions about the DIA analysis. Does the Skyline team provide consulting services?
If so, which email contact should I forward a request to?

Thank you!

Litiele

Hello,

I'm getting an error when installing he current version (351) with either the standard install or the unplugged, see attached log.

Installing the previous unplugged version (312) works fine but check for updates does not prompt to update to 351.

Thanks,
Simon

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 ?

Hi,

Problem: I have data for endogenous peptides and want to export an isolation list that includes both heavy and light peptides. However, Skyline does not let me export the isolation list, saying that I need to either select a retention time predictor or else import results for all the peptides. I think that actually Skyline has all the information it needs, since the light and heavy peptides have the same retention times. Do you agree, and if so, could this be added as a feature?

More Details:
I have a Skyline document where a Proteome Discoverer result was imported. Offline I compared the results to a list of heavy labeled peptides, and then filtered the results with Refine/Accept Peptides. Then I added the R/K heavy modification option from Settings/Peptide Settings/Modifications. I select Export Isolation List/Thermo Fusion Tune +3 with Scheduled Times.

Thanks
Philip

Hello Skyline team,

I am stuck with an error message after trying to build my peptide library : I am trying to load the file msms.txt , then I receive this message.
Error parsing mqpar file / / mqpar.xml (line 408) parameter group index was 0 was outside the range of parameter group (0).
I did my search on Maxquant Version 2.3.1.0 and I'm trying to use the msms.txt to build a library on Skyline-daily version 2.0.9.4899. I need to use the daily version because of some modifications to add.

I don't know if the problem is in the versions that I use.

Hope you can help me.

Thank you!

Liti

Hi,
Is it possible to import/insert a custome protein list?
The default protein list accepts only name, description, sequence, accession, preferred name, gene and species. How do I add extra column to this list or change some of the column headings?

Thanks

Hi,
We would like to use Skyline to analyse our GCMS data recorded on an Agilent instrument.
When importing the data I get the following error:

At 3:22 PM:
Failed importing results file 'D:\User\djakob\GCMS\30-Test110423\S0.D'.
[ReaderFail] don't know how to read D:\User\djakob\GCMS\30-Test110423\S0.D
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'D:\User\djakob\GCMS\30-Test110423\S0.D'.
[ReaderFail] don't know how to read D:\User\djakob\GCMS\30-Test110423\S0.D ---> System.Exception: [ReaderFail] don't know how to read D:\User\djakob\GCMS\30-Test110423\S0.D
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_22_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_22_2\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 199
--- End of inner exception stack trace ---

Do you have an idea on how to solve this? For some reason I managed to import one file from the whole batch which is also attached.

Thank you very much,
Dennis

Hello,

I am processing DIA search on a large batch using a high end server. All went fine but at a certain point during result import of pepXML/mzML files, the system memory went full and a crash occured.

Is there a way in Skyline to recover a session so that it continues the import process after the last imported result file ?

Thank you.

Hi, I'm a grateful user of Skyline.

I'm reaching out to you with a few questions.

First, is it possible to convert the color of the bars for retention time or peak area on skyline to a different color? It's set to blue by default, which makes it hard to see the error bars, so I was wondering if a user could change it to a lighter color or other colors.

Also, we would like to introduce how to use skyline for doping control, and I was wondering if you could allow us to use a screenshot of skyline as a figure in a paper to create educational materials on doping analysis using skyline.

Hi there - I am having an issue where the TIC area for some of my replicates is calculating as "NaN". Interestingly, when I view the TIC chromatograms, they are showing up as expected for all of the replicates. I tried re-importing results and that didn't fix the issue. I'm on Skyline-daily (Windows 10, 64-bit) version 22.2.1.425, using the small molecule interface. Will upload the .zip file to the file sharing as well, including results from one replicate with TIC NaN (AK_10) and one replicate that looks good (AK_11). Any help would be appreciated!

The program was running normally in the morning and then suddenly refused to open. When I launch the program I do not even get a loading screen, nothing happens at all. I ran task manager simultaneously to see if it was running as a background process or something and I notice that the program would pop up as "Suspended" and then close within around three seconds every time I tried to launch it. Have deleted and redownloaded with the same result. Help much appreciated.

Edit from previous support request:
I have uploaded one DAT file to the file sharing folder named F001441.dat.

I am doing proteomic work and wanting to build a peptide spectral library using .dat files from Mascot. My software and system specifications are:

Skyline (64-bit) 22.2.0.351 (28f9b9301)
Windows 10 Pro
Intel(R) Xeon(R) CPU E5-2660 v3 @ 2.60GHz

I generated mgf files from MZML files using MSConvertGUI (64-bit). My Mascot peptide searches have thousands of results. When I go into Skyline to build a spectral library, I go Settings>Peptide Settings>Library>Build. I choose my output path, and add my .dat files. Skyline then returns the error message "ERROR: no spectra were found for the new library".

I have attached 3 screenshots for 3 different instances where I received the error messages, described below:

1. First error message after uploading my .dat files to build a peptide spectral library.

2. After looking at different support threads on here, I tried converting my MZML files to mgfs again, this time making sure TPP compatibility was selected. I did a test search on Mascot with 1 mgf file, with over 1000 peptides being identified. To get my .dat file, I clicked "save link as" and changed the file name to end in .dat. This error message came after I uploaded that singular dat file.

3. I thought there was an error in my dat file based on how I was exporting. Using the same TPP compatible mgf from above, I went into the search result report on Mascot and created a dat file by specifying "Export format: Mascot DAT file" and clicking Export Search Results. I did not change any file names and still got a Skyline error.

Can anyone tell me how to fix this issue? Is it a problem with my DAT file, or a problem with how I am generating my spectral library in Skyline?

Hello,

I have a question regarding a combined spectral library.

When generating a spectral library containing multiple result files, is there an order in which the final spectral library entry to be matched to is picked?
Meaning if I have identified the same peptide in multiple result files that are used to generate the spectral library, how is Skyline deciding which of the spectra to use to match the PRM targets to?

I attached an example of a peptide that is identified in two result files that were used to generate one spectral library. The first result file is from a MQ search containing multiple fractions and the second result file is from a PD file. As you can see the peptide is identified multiple times and the spectra quality differs. Skyline decided to use the first entry in the spectral library for the result matching. This decision does not seem to be based on intensity, spectral quality or on alphabetical order, nor on the oder of loading the result files to generate the spectral library.

Could you provide me with some information on this decision process or if it's a random decision?

Thanks,
Nadine

Hello,
I'm trying to import a fairly large (~10 gigabytes with slightly more than 20 million precursor peptides) spectral library generated in silico and formatted as a .blib file just like the kind Skyline's built-in Prosit API generates. Anyhow, when I go to load this library, Skyline successfully builds the .slc binary file only to throw the error shown in the screenshot I've attached. For context, Skyline works flawlessly when on a simple toy library containing a few thousand peptides, which, coupled with the error message, leads me to believe this is some sort of memory issue; is there anyway to overcome this, whether it be changing Skyline's settings or by somehow (further) compressing the .blib file?

P.S.
The spectra are already compressed using zlib as per the .blib file format specifications:

https://raw.githubusercontent.com/ProteoWizard/pwiz/master/pwiz_tools/BiblioSpec/tests/reference/tables.check

Hi! I have a TTOF experiment with one TOF scan and several Product Ion Scan. I wonder when I select the peak from one of product ion scan by specifing precursor and product ion. Is there any way I can collect all the MSMS tranistion from this segment of time from the product ion scan. Basically I want a set of data with m/z versus intensity for selected time slot. And is there way i can batch export this type of data

Hi Skyline team,

I am just trying to save an "imported DDA results" experiment but I get hit with this message upon trying to "save" or "save as",
"Failed writing to C:\my_folder\experiment.sku"

Additional text is in the attached screenshot but here is the first line of the "more info"
System.Reflection.TargetInvocationException ---> pwiz.Skyline.Util.AssumptionException
at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Util.cs:line 2080

Thanks,
Nick Hartel

Hello! We're working with small molecules and while our chromatography methods are fairly stable, we will sometimes have small shifts in RT across batches that require us to manually integrate each replicate as the linked integration doesn't adequately capture the peaks. We aren't looking to predict RTs such as with iRT, but rather to align each peak within a batch. We tried view > Retention Times > alignment but because we don't have a reference library built with our own compounds we don't have a library to enter. Is there another feature or method we're missing? Our core is making the switch from vendor software to skyline, but are missing the alignment option. We've only encountered errors when following the wiki to download proteome wizard to use BiblioSpec (windows) and can't figure out how else to make a custom library. Any input is appreciated - thanks!

When creating a method for bruker timsTOF for PRM PASEF I get this message see attached.

The spectral library viewer shows the ionmobility values and I have it selected in transition settings, 'use spectral library ion mobility value when present'..

Is there some where else I need to specify this in skyline so it can creat the method?

bw and many thanks!

rob

I work within a commercial sector for a large biopharmaceutical CDMO company and I finally have a project where I could utilise Skyline software. I would like to set up a PRM method within my lab for a project I am working on.

I would like to check with Skyline if there are any legal restrictions on the use of the software from a commercial or industry perspective? Are you aware if the software is being used in the pharmaceutical industry at all?

Additionally our IT and legal departments have the following questions:
• How long does it take Skyline to fix a bug?
• What training do Skyline provide?
• Do Skyline have an applicable enterprise level Software License and Services Agreement that includes key topics such as Confidentiality, Data / Information Security (e.g. there is no known illicit Code in the software), Intellectual Property, Compliance with Anti-Corruption Laws, applicable Warranties / Indemnification etc.

Many thanks for your help.
Oktawia

Dear Skyline Team,

I am working on Sklyine v22.2.0.351 to quantify single peptides in an enzymatic digest.
These peptides are stemming from a single protein/polypeptide, but are fragmented and re-ligated, and thus non-contiguous. I am not interested in protein association, but would like to quantify individual peptides.
However, when I am importing my fasta file the majority of peptides are dropped as "Unmapped" (see screenshot attached). Is there a way to turn this off?
I would like to avoid to downgrade to the previous version 21.2, since the 22.2 version has a clearly improved functionality in other aspects.

Best wishes,
Hanna

I don't have an heavy internal standard for all molecules I want to quantify. It depends on the mix of internal standards use and the biological sample analyzed (matrix effect can prevent detection of internal standards less abundant).
How can I choose if I apply and internal or external calibration?
thanks a lot,
Maud

Hi, I started using Skyline (version 22.2.0.351) recently and have been trying to set up a peptide peak quantification protocol but I have trouble effectively defining retention time windows for peak detection.
I already have individual retention times for peptides of interest and would like to narrow down the window in which skyline searches for them. Very similar to this support case.

I managed to define the Explicit RT and Explicit RT Window in the Document Grid, but the issue I keep having is that the right peak is picked only if Explicit retention time falls exactly within a peak. More precisely, it has to fall within a peak integration boundaries that Skyline does defines every time, but are presented as either black or grey dashed lines, depending on the picking success. If the defined explicit retention time is outside of these boundaries, random noise is picked near the defined retention time, even though the actual peak might be eluting just a few seconds before or after the explicit RT and is well within the RT window. Re-scoring and re-importing doesn't help.
Is this behaviour expected and and is there a way to make skyline pick the actual best peak within the defined window? It could be that the problem is related to ppm which is sometimes lower for the picked noise than the actual peak. I have attached a screenshot of the chromatogram where the issue is visible.

Thank you!

I am doing proteomic work and wanting to build a peptide spectral library using .dat files from Mascot. My software and system specifications are:

Skyline (64-bit) 22.2.0.351 (28f9b9301)
Windows 10 Pro
Intel(R) Xeon(R) CPU E5-2660 v3 @ 2.60GHz

I generated mgf files from MZML files using MSConvertGUI (64-bit). My Mascot peptide searches have thousands of results. When I go into Skyline to build a spectral library, I go Settings>Peptide Settings>Library>Build. I choose my output path, and add my .dat files. Skyline then returns the error message "ERROR: no spectra were found for the new library".

I have attached 3 screenshots for 3 different instances where I received the error messages, described below:

1. First error message after uploading my .dat files to build a peptide spectral library.

2. After looking at different support threads on here, I tried converting my MZML files to mgfs again, this time making sure TPP compatibility was selected. I did a test search on Mascot with 1 mgf file, with over 1000 peptides being identified. To get my .dat file, I clicked "save link as" and changed the file name to end in .dat. This error message came after I uploaded that singular dat file.

3. I thought there was an error in my dat file based on how I was exporting. Using the same TPP compatible mgf from above, I went into the search result report on Mascot and created a dat file by specifying "Export format: Mascot DAT file" and clicking Export Search Results. I did not change any file names and still got a Skyline error.

Can anyone tell me how to fix this issue? Is it a problem with my DAT file, or a problem with how I am generating my spectral library in Skyline?

We are doing a Surequant analysis run with Heavy peptides spiked into biological samples. Basically what is happening is the Mass Spec is collecting 15-20 MS2 of the Heavy peptide but when we try to import the results into Skyline only maybe 6-8 MS2 are actually being imported. We thought it might be because of the mass shift but when we calculated the mass error it was only around 5ppm which was well within the mass error we select in the Skyline software. And we know the MS2 of the Heavy peptides are good because they are triggering an MS2 for the light peptide in the Surequant runs on the mass spec.

Windows 10 pro 64-bit PC
Skyline(64-bit) 22.2.0.351(28f9b9301

I have a 4 proteins XL sample which I searched using PD (Xlink + Sequest + Xcorr CSM scoring). Now I want to build a library from the .pd result file. I am having issues because there is no qvalue (i.e no percolator qvalue) and even setting it to 0 raises an error.

I have seen https://skyline.ms/wiki/home/software/Skyline/download.view?entityId=ac4c6cc4-97c6-1038-8aae-e465a393ee52&name=CrosslinkingInSkyline.pdf and I wonder what would be the easiest way to get all the XL peptides imported into skyline.

Ideally I would prefer to not research my raw files with one of the proXL supported search engines. Is there any way to directly import XL peptides from PD?

Thanks!

Andrea

Hello,

I am very new to proteomics and am trying to decide if using Skyline is appropriate for the purposes of my experiments, and if so, how I would be able to effectively use it. My project currently revolves around detecting electrophilic small molecule modifications of cysteine residues in a purified protein.

I am currently using a bottom-up approach, digesting the protein and analyzing it in a data-dependent acquisition mode on a QTOF 5600. So far I have only been attempting to obtain good sequence coverage and to ensure that I see all of the cysteine-containing peptides appropriately labeled with iodoacetamide.

Moving forward, I will be using less common small molecule electrophiles that are hypothesized to favorably react with specific cysteine residues in the protein. The idea is that as I treat my protein with decreasing concentrations of these small molecules, fewer and fewer cysteines will be modified
until eventually our (hypothesized) most reactive cysteine is the only one modified. Previously, I was doing my data analysis with Protein Pilot, but it seems to me that Skyline is more amenable to inputting and detecting these novel small molecule modifications.

In my first experiment, I treated my protein with the small molecule sulforaphane (monoisotopic mass of 177.0282). I followed the DDA search for MS1 filtering tutorial and the working with modifications webinar, but I'm still unsure if I'm using Skyline to its fullest capabilities, given that all of this is so new to me.

So, does Skyline seem appropriate to use for my experiments? If so, are there any recommendations as to how I could use it effectively?

If this kind of question is beyond the scope of this support board or if there is a better place to ask it, please let me know.

Thanks for your time!

I am trying to set up a Skyline that will allow us to quantitate the sum of two forms of a peptide, a version with an additional amino acid at the c-terminus and a version lacking this terminal amino acid. I can set up the target list fine by entering an adduct for the peptide version that has one less amino acid. However, the integration for the two peptides is linked and due to the structural difference the retention time is not identical. Is there anyway to independently integrate each peptide but have the calibration curve be a sum of the two peptides?

Best,
Jay

Hi,

So I'm working on developing a big assay for targeting ~300 proteins. During the peptide selection process , the libraries were created with PRM raw files using Proteome discoverer. Now, when I'm merging this big assay with all the individual libraries. Some peptides are missing the dotp values eventhough they had it earlier. So basically I wanna know how can my peptides be assigned a library spectrum,

Hello,
I need to quantify ~100 metabolites with a regression curve but they don't have the same standard concentration.
How can I change the concentration of the standard used in the calibration curve depending on the metabolite of interest?
thanks a lot,
Maud

I hope I can explain clearly what I would like to do. We work with small protein domains called EGFs, each of which could potentially be modified with as many as four EGF specific O-glycans. There is also non-stoichiometric hydroxylation of aspartates and asparagines. Additionally, mucin type O-glycans are possible in some cases, and we also have the potential for N-glycans. As you can imagine, if one is very unlucky, their peptide of interest could have a dozen or more potential glycoforms, in addition to the unmodified form. These modifications are often not stoichiometric, so we could potentially see all glycoforms in a given sample. When I make an extracted ion chromatogram (EIC) in Qualbrowser, I have masses for my peptide for all glycoforms. We use the observed values +/- a chosen ppm error for those we get hits for, and a calculated range for those we didn't get hits for. This allows us to plot an EIC that will show, from the raw data, that indeed, there is no peptide present for glycoforms that the software didn't identify. I am struggling to replicate this procedure in skyline. Firstly, it would be very advantageous to us if we had the ability to import a file of set modifications during each analysis, rather than having to imput them manually, or query a long list each time to make sure everything of consequence is selected. Secondly, I don't know how to get a curve in skyline for non-hits. We would very much like to query what came off the column for the non-observed glycoforms to show that they are not present in our data. I would much appreciate if anyone could give me some direction on how, or if this is possible in skyline. Thank you for your time.

Hi,

I used Skyline quite a lot in the past for target peptide quantification, and loved the software (so a heartfelt thanks to the team!)

I am starting a new project, which is a small molecule project, and I want to use Skyline if possible. However, our lab setup for small molecule quant is based on three LC channels (MS1, DAD/UV and a Chemiluminescent Nitrogen Detector).

Is it possible to use Skyline for this type of multi-channel analysis?

When I last used Skyline, the Small Molecule features were gaining traction, but where still early dev. and I can see how much has happened since then! I have searched the documentation but so far without result.

thanks,
Daniel

Dear Team,

I have a problem in importing the DIA PASEF spectral library that is generated in Spectranaut version 17. While importing the .csv file in Skyline as assay library, it's giving some errors (attached below).I have attached the library as well for your reference.

Looking froward to your answer.

Hi,

I followed the Webinar yesterday about how to use DIA results/libraries for SRM method setup/optimization.
I was now trying to do it myself using a Spectronaut output instead of EncyclopeDIA. I already asked yesterday during the webinar if this is possible and the link you provided says that I can use a csv SN output file. However I tried both building a library using the SN report file in .tsv format and using an existing library created with SN.
When trying to build the library I get the Error: Error getting score type for this file. The csv format as stated on the website https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild is not even supported, so I cannot use that one.
When trying to use an existing library I am having the issue that the library format (.kit) from SN is not supported.
I feel like I am missing something here.

Do you have any information how I can Import DIA peptide search results from SN?

Cheers,
Nadine

Hi
I am trying to create spectral libraries from PD results, using Skyline's 'Import Peptide Search' functionality. When I import from the .pdResult file, the scores are not recognized and all set to 0. When I use the corresponding .msf file, the scores are imported, even though - obviously- they are exactly the same and have the same column name (PercolatorqValue). Is that intended behavior or is there something wrong?
A second question is about the SpecIDinFile parameter. This ends up being a strange looking float like -1602.53037. In all cases the number before the decimal point (here 1602) corresponds to the WorkflowID from PD, but the rest has no obvious relation to file ID or scan number as one would maybe expect. Do you have any info on how this parameter is calculated and/or if and how you could retrieve the actual scan number from it?
Thanks a lot for providing these great tools to the community!
Hans Voshol

Hi,
I want to analyze timsTOF data and want to check the ion mobility. After updating skyline daily to 22.2.1.425 the ion mobility view is now empty.
To set up appropriate target windows it is necessary to check the ion mobility in addition to e.g., RT.
The view did work in the previous version.
Hope this bug is fixed soon.

Best
Kristina

Hello,

I have a collection of pepXML and mzML files previously obtained using File/Import/Peptide Search menu, including Oxidation and Carbamidomethyl modifications.

My goal is to import these files in Skyline for full visualization. The pipeline cited in title seems to be a tool created for that purpose.

I am following the indications Skyline is asking but there is only one modification displayed in the "Add Modifications" window (Oxidation). I also need Carbamidomethyl. When I try to add it in the list, I get "The modification Carbamidomethyl already exists".

Could you confirm that this popup window is asking NEW modifications I may need without discarding Oxidation and Carbamidomethyl I used to generate pepXML and mzML files ? This point is confusing me because all I can do on this step is to check or uncheck Oxidation modification, nothing else.

Thank you for your help.

Hi Skyline team,

I am in the process of building SS protocols across our LC systems using Skyline and Panorama AutoQC function. In this request I am investigating DDA MS2 data acquired on a bruker maXis 2 for a single compound, resperine, ran on with a c18, 4 min uplc method with bruker autoMSMS detection.

I have followed the tutorials for small molecule quantitation and generated two skyline documents, one from scratch and one using the "Auto MSMS_MS and MSMS filtering" template. In the scratch generated document, I am unable to see MS2 information. In the modified template, I can see my MS2 transition of interest, but the chromatogram appears wonky with a straight line. It seems selecting "DIA" or "none" I see in another post this may just be a bug. Is there something I can do within my transition settings or transition list to get the transition ion (which appears in the MS2 scan of m/z 609) to appear and be useful for SS? We may ultimately just quantify on the MS1 TIC, but for further use (including using GCMS data) I'd like to find a way to use a fragment ions in quantitation, without a MRM method.

Attached are screenshots of the skyline documents. For the auto MSMS template file there are two shots, one with DIA and one with the targeted(obsolete) method (which strangely disappears once you modify the template)

Hi all,
I'm looking to generate an external calibration curve but the column called $BioReplicate I used in the past to specify the replicates of a given point no longer exists. This means that the replicate data points are simply aggregated and the individual points do not show up on my curve. Which column should I use instead?

I'm using Skyline version 22.2.0.351

I'm attaching a screenshot of what the document grid looked like in the past.

Thanks,
Franziska

OS/Skyline version: Windows 10, Skyline-daily 22.2.1.425

Mode: Small molecule data

I hope you're all doing well. I am performing QQQ CE optimization for a single compound per run, with 14 product ions and 10 collision energies. As it currently stands, I have to modify the transition list after importing into Thermo software (Xcalibur 4.6 on an Altis Plus) as the export results in more than 10 rows with the same compound name and precursor m/z which causes an error. My understanding of the cause of the problem is below.

As Thermo method editor is limited to only a maximum of 10 rows with the same compound name and precursor m/z, Skyline appends a .2 to the end of transitions when there are more than ten. E.g. When I have 14 product ions and am performing steps in collision energy, there are ten product ions listed as "moleculeName(-charge).4", and four listed as "moleculeName(-charge).4.2". This behavior is expected and works fine.

The middle CE for a given molecule and product ion is just "moleculeName(-charge)" and, as I have 14 product ions for that name, there are ten product ions listed as "moleculeName(-charge)", and four listed as "moleculeName(-charge).2". This naming convention causes a problem as the second step in collision energy optimization uses the same suffix. E.g ten product ions listed as "moleculeName(-charge).2" and 4 product ions listed as "moleculeName(-charge).2.2". As a result, I then have a total of 16 compounds named "moleculeName(-charge).2", which is 6 more than allowed by the vendor software. In addition, it's confusing as I have to work out which of the "moleculeName(-charge).2" are from step 0 or step 2 in the CE optimization.

I currently append a B to the end of 4 of the compound names, but I'm not sure if Skyline can then read those files correctly. One possible solution could be changing the naming convention for CE optimization to add a 0 for the middle step in CE optimization, giving 10x "moleculeName(-charge).2" and 4x "moleculeName(-charge).0.2" in the example I'm encountering above instead of 16x "moleculeName(-charge).2". This should prevent any collisions in the naming as the transitions will then uniquely named.

Please find attached an example of the transition list that gets exported from Skyline, note the middle step for the last four product ions have the .2 suffix.

Cheers,
Chris

Hi Skyline team,

I am trying to build the spectrum library based on the searching results of the heavy labelled peptides. When I uploaded the mzid file, I got the error message shown in the attachment. I am not able to understand the issue based on the error message. Can you help take a look and advice what is the issue?

Thanks,
Chen

Hi Skyline Team,

I am trying to analyze some small molecule data acquired on TimTOF pro2 in MRM mode (Skyline-daily 22.2.1.391 (1e3a4e917)). However, Skyline is not able to integrate the peaks. DataAnalysis from Bruker showed that the peaks are there. What change can I make to have Skyline to integrate the peaks?

Thank you in advance.

Ho-Tak

Hi Skyline Team,

I found a bug parsing Fragpipe 19.1 result using Skyline 22.2.0.351. When I load the interact-filename.pep.xml with filename_uncalibrated.mgf in the same folder. Skyline was unable to find the .mgf. I have to rename the file to filename_uncalibrated_uncalibrated.mgf for Skyline to find it.

Ho-Tak

Hi all,

I am having an issue where Skyline is integrating peaks incorrectly for one out of 6 replicates, but for thousands of precursors in the file. This is data from the same support ticket as a previous issue with importing the DIA-NN spectral library (https://tinyurl.com/2x326w3a) - thanks Nick for helping on that.

I've attached screenshots of the issue. I have tried refining the peak picking several times and I think I have a really good peak scoring model, but it still is mis-integrating peaks. Sometimes it does pick the correct peak (slide 4), but this is uncommon. I obviously would prefer not to have to manually go through roughly 10,000 precursors and correct integration on what appears to be 80-90% of them. One way I tried to do this more quickly was by looking at run-to-tun regression and finding outliers, but this didn't work. As you can see in slides 5-8, as highlighted with the red box on the regression plot, there isn't much deviation for the misintegrated peaks, and so no good way to target the worst cases first. It's so many anyway that it wouldn't end up being a "targeted" fix.

One thing I did notice is that the replicate that has a lot of integration issues does seem to be unusual in that it has more ID matches for than other replicates, and they tend to skew towards the leading tail of the peak. See slides 8-10. Could that be the issue? Not sure why that would have happened, other than that was the first sample in the file list run through DIA-NN. The spectral library is based on all 6 runs, but the DIA-NN log does say:
"DIA-NN will optimise the mass accuracy automatically using the first run in the experiment. This is useful primarily for quick initial analyses, when it is not yet known which mass accuracy setting works best for a particular acquisition."
I know that this optimization can be turned off by specifying a mass accuracy setting for the run, but I don't know if that would fix the issue I'm seeing.

I have compressed my entire skyline document and can share that if needed, although it's 4 Gb zipped.
Thanks,
Josh

Hi Skyline team,

I am trying to develop a standard library with DDA data but I cannot see the MS/MS data in Skyline. I can check only MS1.
Could you please check and advise if somewhere is wrong?

Thanks and regards,
Nay Min

Hi Skyline team,

I know this issue has been in the forums before (https://tinyurl.com/446rjst8), but I tried the suggestions in that post and I still seem to be having issues.

Basically, I created a spectral library of a basic yeast search with DIA-NN and am trying to import into Skyline. I have moved the speclib and accompanying tsv file to their own folder, so that they are in the same directory and there isn't any confusion on which tsv to import. However, I am getting an error stating that Skyline cannot find the accompanying tsv file. See attached ppt for screenshots. I can upload the speclib and tsv if you'd like.

I'm running Skyline 22.2.0.351. Any advice?

Thanks,
Josh

Hi Team,

I have ended up with two separate accounts in your system. One is through my gmail address and the other one, which is more focused on PanoramaWeb is through my work email address. I'd like to consolidate them into one account with my gmail address. Is that possible?

Best,

chris