Hello Skyline team.
I am working on post-translational modifications and am using skyline to export PRM lists. I would like to generate a list of peptides containing only a specific modification based on the peptide search results, is there a way to do this?
Unfortunately, search results exported from PD cannot be filtered for specific PTMs and .pdResult files imported into Skyline cannot be filtered for specific PTMs.

Yours,
Zimeng

Hi Skyline team,
I have a use case where I'm comparing the performance of several spectral libraries for the same set of raw files. It would be great if I could compare different spectral libraries within the same Skyline document without reimporting the raw files. That is, I want to keep the settings in the doc exactly the same and just compare the results of loading spectral library A vs spectral library B.

I know that I can go to Peptide Settings > Library > Edit to import a new library, but I get the feeling that Skyline doesn't actually update the settings according to the new library. The only way that I've been able to successfully perform this task so far is by creating separate Skyline docs for each library, which is cumbersome and slow.

Is there anyway to do this within the same Skyline doc?

Thanks,
Lincoln

Dear Skyline team,

Two years ago a former colleague of mine posted a question titled: "Sciex TripleQuad7500 .Wiff2 support" (roosso 2022-07-05 22:41).
We still encounter this problem, also with the newest Skyline versions.
Some methods only generate ".wiff2" files. We think this happens when we apply experiment scheduling (3.5min of ESI- followed by 3.5 min ESI+)
On import of the .wiff2 file in Skyline we get the error below:

"Failed importing results file ...
The sample .... contains no usable data."

This also happens for methods where both the .wiff and .wiff2 files are generated and we try to import the wiff2 file.
I attached a Skyline file which we use for our 'System Suitability Test', including some raw data we would like to import. The .wiff file succeeds, the wiff2 file fails to import.

The last two years we used work arounds, so that SciexOS would generate .wiff files.
At moment we develop a method which requires experiment scheduling, and so will only generate .wiff2 files.
Could you please advise how to import wiff2 files?

Thanks a lot!

Regards,

Jeroen Kooistra

Hi,
Using the neat feature of the export (multiple) methods. Since Skyline is usually installed on personal computers and not on the MS-linked computer, it would be great to generate those methods anyway. Is this a thermo requirement, or is there a workaround to install the necessary software on the user PCs as well?

thank you!

Skyline-daily

An error occurred attempting to export.
ERROR: Registry key (Software\Thermo Instruments\TNG\Stellar) not found. Stellar is not installed on this machine.

Command-line: Method\Thermo\BuildThermoMethod -t -m "W:\User\yx.meth" "..transitions.txt"
Working directory: .....\

OK More Info

Skyline-daily (64-bit) 24.1.1.284 (bc93c2813)

System.IO.IOException: ERROR: Registry key (Software\Thermo Instruments\TNG\Stellar) not found. Stellar is not installed on this machine.

About 8 peptides were tested by waters QTof under MSMS mode. Peptide 1 resulted in "Chromatogram information unavailable" When I used skyline to analyze raw data, while others could work under same settings.

I am confused and want to know how to solve this.

I uploaded two peptieds information and results(peptide 1 and 2) in the file.

While you provide great tutorials and fantastic support, I was wondering if a readme is available with the full documentation of all features, settings, GUI usage, etc.? That would be fantastic for knowledge-based chatbots. Especially in the beginning, where finding some of the features sometimes can be a a little difficult.

Tanks you,
best
André

Getting error message when saving Skyline .sky files to our file server.

Error states "Failed writing to ... the process cannot access the file because it is being used by another process".

Please advise

Hello,

I was in the lipidomics webinar today and was curious if there's a way to do isotope corrections and isotope abundance calculations directly in Skyline?

Thanks!

Hi, I would like to use Skyline for untargeted lipidomics analysis. I have an Exploris240, and LipidSearch for lipids identification. I am doing AcquireX for high-throuput lipidomics. I am also using more than one internal standards for lipids class normalization.

I have never used Skyline, and I would like to know if you have a Tutorial for that.

Thank you very much
Nidia

Good evening,
lately I am unable to import raw files to the Skyline (I just uploaded the newest version). Everytime I try to upload my result files by the usual way, it starts importing and and after few seconds each of the files gives the "failed" which imideately turns into the "canceled" yellow saying. Sometimes first few injections upload, but the rest is always blocked from importing. Everytime the data are not open anywhere else. I tried rebuild the .skyd cache, reexporting the files in case there would be problem in them, saved the Skyline for those data in different folder in case there would be the clash there, checked the read/write permissions. But here I am done with what I had in my mind that could possibly be the problem. Could you help me solve this issue please? I am attaching the error statement.

So I´m facing some method development issues for Surequant with heavy labelled peptides. I followed the tutotrial for Surequant but when analysing my files, I saw there was no signal for my endogenous peptides which were spikedin at 250 ng. Could there be a specific reason for this?

I'm very new to Skyline and am trying to set up my project for some DIA data collected on a Bruker timsTOF HT. In doing so I have a few questions...

1. When configuring the full-scan settings, is it best to use the resolving power that is stated by Bruker for this instrument (60,000)? I ask as I read in another post that the resolution setting in Skyline is used to define the width of the XIC extraction, and if in MS1 it is set a little lower than the instruments stated resolution, it can account a bit better for potential mass accuracy deviation. If going a little lower is useful, do you have any suggestions on what this should be?
2. How do I determine what the ion mobility filtering resolving power should be? Is this something I should be able to ascertain from my data?
   Thank you for your help!

Hello,
I am wondering if we can import results from FRAGPIPE to create spectral libraries on Skyline. If yes, what formats are supported by Skyline.

Thanks

Hello,

I am trying to set up Skyline for the analysis of GC-MS data from an Agilent single quadrupole instrument. I followed Pawel Sadowski's protocol for Shimadzu and also tried adapting the guidance provided here: https://skyline.ms/announcements/home/support/thread.view?rowId=43600. However, no matter what changes I make to the transition list, the files do not process correctly. Skyline identifies only the precursor ion and cannot detect the fragments.

That said, when I manually click on the chromatogram, I can see that the MS/MS spectra have been recorded and are readable from the files.

I have attached the Skyline file, the GC-MS data file, and the transition list to make it easier to review the data.

Thank you very much for your help.

Best regards,
Daria

I am trying to create the library with msms.txt file from MaxQuant but I keep receiving a error. What do I need to do in order to proceed?
Thank you!

Similar to the existing routine for optimizing CE on a triple quad, is it possible to optimise % HCD and/or % CID on a Thermo Stellar MS?

Dear Skyline team,

I am currently working on generating calibration curves for several peptides. Despite observing good peak areas, I am unable to obtain a calibration curve for some peptides. I have attached a screenshot for your reference.

Additionally, I would appreciate guidance on selecting the appropriate quantification settings, as there are multiple options available (e.g., linear, bilinear). Could you please advise on which method is recommended and how to approach LOD calculation? Is it possible to choose different quantification options for individual peptides?

Thank you.
Akhila

Hello,

I'm running a DDA skyline file to look at certain peptides and it's giving me cut-off peaks near the middle. I'm using an orbitrap mass analyzer to run my samples. The peak and transition settings are attached below. The same samples were ran with PRM and the peaks looked completely fine.

Hi Skyline Team,

I am having an issue in my document regarding the "Enforce Peptide Uniqueness by: Proteins" function.

My target list contains a list of peptides that I thought were unique, but when I copied the peptides and pasted them back in the document, some were flagged as also being present in the background proteome. The proteins I am building a targeted method with are recombinantly expressed and are from another organism entirely.

It was my understanding that when you enforce peptide uniqueness by protein, any peptides in the background proteome should be removed from the target list of peptides, but this does not seem to be happening in my case.

When I select Edit>Unique Peptides, I can see that some of the peptides are shared with background proteins. So I am unsure as to why they are not being removed when I enforce peptide uniqueness.

Any help on this would be greatly appreciated.

-Peter

Hello Skyline team,

I am trying to build a chromatogram library using GPF runs by setting the parameter "run peptide search." When I finish, I get the "No matched passed score filter in." How do I solve this issue?

Thank you
Vanya

If data has been acquired using an acquisition method that uses both HCD (beam type fragmentation in a collision cell) and CID (Resonant fragmentation in an Ion Trap) on the same precursor, can those filters be separated in Skyline? Currently, when I try to process with Skyline, it treats the MS2 fragments the same, so the you get a noisy combined XIC.

Subject: Assistance with mProphet Peak Picking in Skyline

Dear Skyline Team,

I hope this message finds you well.

I have encountered some instances where Skyline appears to overlook clear candidate peaks, which results in the mProphet peak picking model selecting incorrect peaks. In the attached screenshot, you can see an example of a very straightforward peak that Skyline does not even recognize as a candidate.

Could you please advise on possible reasons for this behavior and suggest any steps I could take to resolve it?

Thank you for your assistance!

Best regards,
Anirudh

Hi, I am new to Skiline and beleive I must be missing something when importing my raw file. I can open the file in MassLynx with a problem, however, the file looks like it is importing up until the very end and then gives the error message.

At 6:07 PM:
Failed importing results file 'C:\Users\RobertRegister\Dropbox\Skyline\Tocilizumab 61 min scan.raw'.
error reading spectrum function=1 process=0 scan=24757
Under More Info it says

At 6:07 PM:
Failed importing results file 'C:\Users\RobertRegister\Dropbox\Skyline\Tocilizumab 61 min scan.raw'.
error reading spectrum function=1 process=0 scan=24757
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\RobertRegister\Dropbox\Skyline\Tocilizumab 61 min scan.raw'.
error reading spectrum function=1 process=0 scan=24757 ---> System.Reflection.TargetInvocationException: error reading spectrum function=1 process=0 scan=24757 ---> System.Exception: error reading spectrum function=1 process=0 scan=24757 ---> System.Exception: [pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: EOF Scan File Read Error
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_24_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1384
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetSpectrum(Int32 spectrumIndex) in C:\proj\skyline_24_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 995
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1109
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1160
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.Read() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 987
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.<RunAsync>b__33_0() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 845
--- End of inner exception stack trace ---
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Util\Util.cs:line 1926
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.NextSpectrum() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 950
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 273
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 253
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 617
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 388
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256
--- End of inner exception stack trace ---
Any idea what is going on? Also, it says Chomatogram information unavailable.

Any help would be appreciated !! Bruce

Hi,

I'm currently quantifying small molecules using Skyline. For some analytes, I have labeled internal standards, but for others, I don't. So, I'm considering using surrogate internal standards from different molecules. I'd like to assign these individually but haven't found instructions on how to do this.

Could you please guide me on where to find this information?

Thank you.

Dear Skyline,

Is there a way to set digestion enzyme as non-specific for the Background proteome? In the context of peptidome analysis having a non-specific background would be quite useful.

Thanks in advance,
Li

Dear Skyline,

I am now encounting an issue when I am importing the spectral library into the Skyline.

I used the .speclib format file and report.tsv files that are generated from the DIANN and renamed them as "NANE.speclib" and "NAME_report.tsv". I imported the spectral libraries through File-Import-Peptide Search-Add files...Then it came an error information which asked me if that report.tsv is the correct one for the .speclib file. Do you know what might be the potential cause for this issue and how I can slove it?

FYI I am using DIA-NN 1.8.2 beta 34 and Skyline daily versions. The error information is also attached.

Thanks in advance!

Best wishes,
Yuxing

Hi Nick,

I am doing ToF MRM for peptide quantitation in my sample with heavy-isotope labeled peptides.

1. Can you please help to calculate LOD and LOQ in skyline?
2. CV calculation?

Please share any tutorials having all these

Thanks
AD

Dear Skyline Team and Community,

I'm generating spectral libraries from ssl files via the command line tool with the following (simplified) code:

"D:\Software\Skyline\SkylineRunner.exe" ^
--in="tool_comparison.sky" ^
--import-search-file="results_tool1.ssl" ^
--out="tool_comparison.sky

How can I set the name of the generated spectral library? Currently, it is automatically named "tool_comparison.blib". Is there any option to assign a name or have it named after the ssl file (i.e. "results_tool1.blib")?

Additionally I receive the following warning: "Warning: Unable to locate results file 'results_tool1.mgf'". This does not seem to cause any major error for my analysis, nevertheless it would be nice to understand why this is happening. The mgf file is in the same folder as the ssl file and naming in the ssl file is correct.

Thanks in advance! Cheers
Jonas

Dear Sykline-Team and Community,

I'm looking for help in streamlining some skyline analysis using the SkylineRunner.exe / command line version.

I have a list of peptides in txt format (peptides.txt file attached). Is there any possibility to add these peptides to the Skyline document using SkylineRunner.exe? When analyzing the data using the GUI, I just copy them into the target list which creates a "peptides1" entry with all of the peptides - something similar would be needed.

I do have a spectral library for all the peptides in "peptides.txt" which I add using --add-library-path, peak boundaries from a search engine which I add using --import-peak-boundaries and a custom report which I can also create using --report-add. The only thing missing to have this completely in CMD is adding the peptides to the document.

Additionally, I add the corresponding raw files using --import-file and --import-replicate-name, i.e.

"SkylineRunner.exe" ^
--in="test.sky" ^
--import-file="condition_untreated1_20220522.raw" ^
--import-replicate-name="untreated" ^
--import-file="condition_treated1_20220522.raw" ^
--import-replicate-name="+treatment_1" ^
--out="id_all.sky"

However when doing this, the two rawfile end up both in the replicate "+treatment_1". How to have them in separate replicates as defined in the command? When I run the imports after each other, it works fine.

Thanks in advance for you help!
Jonas

Hello,

I am working on a project with tmt labeled small molecules. I would like to assign colors for the MS1 peaks I have assigned for each of the small molecules and their tag. I noticed there is a tutorial for this for peptides, but I can not seem to make it work for my small molecules.

Dear Skyline support,

I am looking for peptide quantitation in my sample using heavy standards of my peptides. After making the calibration curve based on serial dilutions of heavy standards, if selected normalization method as heavy in the quantification tab, I am not getting R2 of 0.9. While keeping quantification method as none, I am getting R2=0.9.

1. Am I doing something wrong in making calibration curve?
2. I am not able to see the peptide quantitation in (nmol) normalized to heavy.
3. In the calibration curve, peak area is there on y axis but not light to heavy peak area ratio.

Please guide me, which settings I need to follow to get my results.

Thank you
Arpita

Since diann 1.9 version, diann results could be directly export to skyline. Thanks to this very useful option.
To the slice-PASEF or Diagonal-PASEF acquisition, skyline is able to extract MS1 scan of the peptide but fail to extract MS2 ions.
Something was wrong in my parameters or skyline is not compatible to these acquisition methods?

Thanks
Jerome

Dear Skyline team,

We were trying to build a spectral library using DIA raw files in Skyline v24.1 through import->peptide search. In the build option, while we were trying to add the raw files, it seems the file format was not supported. In the previous versions of the Skyline, we have found an option to "start from -> DIA raw files". Kindly guide us to generate a spectral library using the DIA raw files in the recent version.

SCIEX TOF

I have a set of precursor masses and/or formulas (say n different one), and a set of collision energies (CE) (say m different one). I would like to create n x m individual method files, all with different precursors and CEs. The name should somehow represent the formula/precursor mass and CE, say sample_precursormass_CE.dam

I see that maybe it might be done in a batch using
BuildAnalystFullScanMethod.exe
but I cannot figure out in what format I need to provide the precursor/CE list, and what parameters I have to use with the EXE. I know that I need a file with the precursor and CE values, and a template method file. I was able to do one inside the software, but even if the CSV file has multiple lines, it just created new method for the first line. If you have a tutorial on that or a video, I would appreciate that, too, I could not find it.

If you could send me an example command line , that would be appreciated.

Thank you very much in advance, Vilmos

PS: Bonus: if I could also do the same, PRM methods with given inclusion lists for a Thermo QExactive, that would be phenomenal.

Hi,

I'm trying to run an EncyclopeDIA search through Skyline with an experiment-specific FASTA file. This has worked for me in the past, however, when I run the search now I get a "GO AWAY" error from Koina before it's finished processing the FASTA and the search fails. The text of error is attached. When I check the Koina configuration in Skyline (Skyline (64-bit) 24.1.0.199 (6a0775ef83)) it says the connection is active.

Any help is much appreciated.
Thanks,
-Eric

We were curious if it would be possible to add a different option for Skyline's Molecule Quantification LOQ . The Max LOQ CV is useful, but typically for our PFAS quantitation it is more common to see the LOQ calculated like the LOD, which would calculate LOQ based on Blank plus 10 * SD.

• Kara Joseph, Baker Lab at UNC Chapel Hill.

Hello,
I imported an MSP format spectral library on Skyline as the Human Phosphopeptide Label Free Library. However, it does not show any spectra for the phosphopeptide target list. I am wondering if Skyline supports MSP format files

Hi,
I've been using Skyline to process data from our Shimadzu 8060NX for a while now and everything works well.
Recently we acquired a new Shimadzu 8060RX but for some reason when I import the results from this machine an error occurs: "The sample contains no usable data".
When I use labsolutions from Shimadzu there is clearly data in the file.

When I use SeeMS from ProteoWizard there is also no data to be seen.

Do you have any idea what is going on here?

I have included two files, one from 8060NX that works and one from the 8060RX that doesn't work. These are similar analysis.

I am very grateful for your help.
Wesley.

Hi all,

Where can I find the list of exact masses and ratios assigned to each of the isotopes used for Skyline?

I am seeing some inconsistencies with the exact mass determined up to the 6th decimal for some large glycan PTMs and I am trying to find the source of the error.

Sincerely,
Juan C.

Hi Team,

I am looking at diaPASEF data in Skyline and want to include IM values in the export report. However, the IM values all say "NA" and when I want to show IM on the chromatogram (by right clicking) it doesn't show anything. But when I double click the peak I can see the mobilogram so the ion mobility data is definitely read in correctly. How can I fix this?

Best,

Laura

Hello,
We are using Skyline for quantification, but we're unable to find the intensity values; we are only getting the retention time values. Please see the attached pictures for reference. Kindly assist me to get actual intensity value for quantification.
Thanks
VC

Hi
I'm facing an issue with the detection of the peptide "R.ALLNAIGNLELTGAFAEALK.N [146, 165]" across different replicates in my analysis (photos attached). This peptide is successfully identified in replicate 1_RA1_01_2682, but none of the other peptides in this sample are detected, and this same peptide isn't identified in the other replicates.
Interestingly, this peptide wasn't initially identified either, and I'm unsure what I did that led to it being detected. I would really appreciate any guidance on what steps I might have accidentally taken to identify this peptide, as this could help me replicate the process for the other replicates. This is quite important for my analysis.

Thanks so much for your assistance!
Amir

Hi Skyline Team,

Is it possible to construct a standard curve from 5 isotopoluges of a peptide within Skyline? Currently Skyline treats these as 5 different peptides and shows the peak areas individually so it seems only way is to export the total peak area for each of the heavy labelled versions and further process externally from Skyline.

Hi,
I am wondering if we can use the “DIA-NN” predicted spectral libraries with Skyline.

Hi skyline team!

I just imported my results (.raw files) after loading my fasta, but found out that my fasta was missing a pepitde... is there a way i can add this peptide now and integrate it for the result files? The loading of the files took about 4 hours, so i would prefer not having to start from scratch...

Best Shelley

Hello, my name is Amie and I taught the Ion Mobility filtering for lipids tutorial during the last online Skyline course (last week). During the tutorial, we discussed how it would be helpful to be able to change the integration bounds within the drift time dimension, similar to how we are able to with chromatograms. Is this possible? Normally, we get around this by changing the resolving power window in the ion mobility transition settings, but sometimes there are peaks that fall outside the horizontal violet box that we would like to be able to include in our integrations (i.e., conformers), but only for certain targets, so I wouldn't want to change the filtering for the whole document. That is why I think being able to integrate within the drift time window just like we do for LC would also be super beneficial.

I've included an example data file where this would be helpful. The retention time of interest is 9.6 mins, but at this retention time and m/z there are multiple peaks in the drift window. I would like to be able to integrate the first two peaks at drift times 23.56 and 25.36 ms (summed), but not the peak at 33.96 ms. Can you make this possible in Skyline?

Thanks in advance for taking a look at this!

Hi Skyline team,

I tried following the advice on the post below but it didn't fix my problem.

https://skyline.ms/announcements/home/support/thread.view?rowId=42832

My orginal search against my raw data was done w/out a percolator but with fixed value PSM Validator since I was only searching against one protein. I received the same error message as that given in the post above but unlike them, I couldn't get the warning to go away even after setting my score threshhold to 0. Am I filling out the wrong part of the form? Thanks!

Cheers,
Martín

Dear skyline supporter, I am doing PRM to quantify my protein A. My heavy protein B is not the same as light protein A. B and A has many similar peptides, so I want to use one peptide of heavy protein B to quantify one peptide of protein A. How can I show them together with chromatograph?

For small molecules containing two heavy tracers with similar heavy-added mass (e.g., C13 and N15, or C13 and H2), it seems that Skyline does not recognize as distinct any adducts that contain combinations of these tracers that have highly similar m/z. For example, for glutamine as the small molecule, the following transition list only gives a single M2C13-H transition instead of a separate one for each row. For high-resolution instruments we know it is possible to discriminate these. Is there a way to import a transition list into Skyline so that these appear as separate precursors?

Transition List (of 3 distinct precursors with very slightly different m/z):
Molecule Name Molecular Formula Precursor Adduct Precursor Charge
Glutamine C5H10N2O3 M2C13-H -1
Glutamine C5H10N2O3 M1C131N15-H -1
Glutamine C5H10N2O3 M1C131H2-H -1

Resulting Targets List (just 1 precursor)
Glutamine: precursor: 147.0686[M2C13-H]

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,

When setting the Resolving Power under Transition Settings > Ion Mobility, does this always reference the RP in milliseconds?
I know when searching for a targeted value, Skyline prefers CCS over arrival time when available. I've tried creating a library with only CCS and another with only arrival times to test this but the resulting bounds of the filtered mobility bands appear to be the same.

Thank you!
Lauren

Hello, back to Skyline after some time. Now testing metabolites precursor only HR workflow -with glycans, but question applies to metbolites in general I believe. Is there a way to filter so that skyline would choose only the nearest by RT in transition list as a hit, and discard other isobaric hits in transition list, and results -when there is only one retention time present?
With metbolites, and glycans in particular, isobaric structures are separated by retention time. Skyline picks all isobaric close-in-retention time compounds as hits despite they share the same RT/EIC. Restiricting retention time too tight dosen't work because retention times in transition list are predictions and because they can wobble a bit in hilic. In the attached screenshot Skyline marks all isobaric glycans to be there, G0, A2 and A1B, showing same EIC three times. The glycan nearest to predicted RT, A1B, is the only one actually there.

I generated a DDA library on FragPipe (.tsv), and now I want to use this library on Skyline to analyze some DIA data.
I had no success. Is there any way to do that?

Hi there,
I'd like to use tools Protter and SRM collider, but links to both tools are 'dead'.

Clicking on Protter in Skyline leads you to this
https://wlab.ethz.ch/wollscheidlab/#file=5fa311d8-5fea-4770-9f45-d80a65716917.csv

The link to SRM collider returns a blank page, is SRM collider still active?

Cheers,
Thomas

Hi!

I was asked to download the human, mouse, and bovine proteome (reviewed) to load into Skyline. I've used programs with spectral libraries before, like Bruker's XRD spectral libraries, Thermo's FT-IR databases, etc... Usually they're pre-loaded or my lab purchased them for a few years, but I'm still new to Skyline and haven't really done this before.

My instrument is a Thermo Scientific TSQ Altis (QQQ), and I just needed some help on trying to understand where I go to find these databases/libraries so that I could download it and upload to Skyline and presumably use the library to extract/detect as many proteins as I can in a sample.

The best I've done is using the PRIDE Peptidome website and trying to download their spectrum libraries, but I'm not able to open or import the .msp.gz files into any program. I'd be grateful for any help!

Thanks!

Is a spectral library also required for dia-PASEF as is for DIA data acquisition?

Hello,

I observed the red X in the target panel What does it mean and how can I fix it? Please see the attachment t for more information.

Thanks

Hi Skyline Team,

I want to export the normalized peak areas of heavy and light versions of my target peptides in my Report. I know I can customize my report and include "RatioLightToHeavy" but I was wondering if there is a selection for the normalized area specifically for the heavy peptide?

Thanks!

Compound 1 uses the same parameters and at the same concentration shows:
Analyte area in MassLynx: 38511.961
Analyte area in SKYLINE: 2356949

Compound 2 uses the same parameters and at the same concentration shows:
Analyte area in MassLynx: 30827.227
Analyte area in SKYLINE: 1851899

I am experiencing the above discrepancy. Would you suggest any possible explanation as to why the data analysis through SKYLINE is 100 folds more?

Hi team,
Thanks so much for all these new command line additions!

Is there a way to set number of sig figs in exported reports? My goal is to get higher precision peak boundaries via the command line. I can right click on the column in Document Grid and set to Full Precision, but this isn't sticky. Precision resets to 2 decimal places after I close Document Grid.

Right now, peak boundaries are truncated after two decimal places and this is enough (.06 seconds) to be a noticeable wiggle.

Judging from line 3358 in CommandLine.cs, it looks like number of sig figures might be persisted in viewInfo? I'm happy to submit a patch if you can give me a rough outline of what to do, or maybe I'm just missing something obvious in the GUI.

Thanks as always,
Sam

Dear Skyline developers,

I am encountering an error while running Koina for EncyclopeDIA library generation.
The error is attached, thank you very much for support.

Giovanni

Hello,

I have just downloaded a new version of Skyline. However, in the "peptide setting" section of the new version, the normalization method shows no option of "Ratio to Heavy." Do you know why? Please see the attachment for more information.

Thank you
Anhdao

Hello,

Please let me know when the next online courses for beginners will be.

Thanks
Anhdao

Hello Skyline team,
I'm new to this software and I'm trying to figure out if it can help me with what I currently need to do.
After going through some tutorials, webinars and questions in the forum, I thought it better to ask if this is possible.

I am analyzing polychlorinated alkanes (PCAs) using direct injection and HRMS data.
A common way of doing this with HRMS data is to, for each congener, extract the two (or three) most abundant m/z of the characteristic isotope clusters of highly chlorinated compounds.
For example for C14H20Cl10, that would be [M+4], [M+6] and [M+2], see attached image.
The relative intensity between the most abundant m/z and the second (and third) most abundant m/z are used to identify the congener.
The most abundant m/z is then corrected by the injection standard and used for quantification.

What I hope that Skyline can help me with is to match the theoretic isotopic pattern to the experimental isotopic pattern, and integrate the potential EIC on matches.
So far, I've learned how to get matches based on masses but not on isotopic patterns of chlorine. Is that possible and if yes, are there some functions/tools that I am missing?

For a deeper explanation, see the materials and methods section of Bogdal et al 2015: https://pubs-acs-org.vu-nl.idm.oclc.org/doi/10.1021/ac504444d#_i2

Best regards,
Jonatan Nygren

Hello

"I want to optimize CE using 'Automation' as shown in the figure."

But why doesn't my operation panel have 'Automation'?

"Please see the attachment."

Thanks,
Zhaoxuanxuan

Hello, dear Skyline team!
I want to optimize collision energy using Skyline now. I saw a tutorial that said it could be used with Masshunter, but why can't I use it? Here's a screenshot of my problem.
Zhouyanan

Dear Skyline Team,
I'm having some issues with Skyline to optimize MRM parameters, and I don't know how to automatically optimize collision energy and declustering voltage. Since my previous protein and peptide identification was done by sending samples, using Thermo Fisher Fisher Scientific's fluid, the raw data was imported into Skyline and the ion pairing information was exported. Later, I used my own Agilent instrument to verify that the peak was too low, and I wanted to optimize the MRM parameters, but I haven't found how to use Skyline optimization, do you have any good suggestions? Or am I using a 2023 version that needs to be updated?
Zhou Yanan

Version:Skyline-daily (64-bit) 24.1.1.254 (ba32f7f50)
I have had problems to export MRMs to .exp file for waters TQAbsolute. I was able to find the cause. I used a template file with an MRM created in Masslynx.

Skyline writes the "CollisionEnergy(V)_10.000," (for a collision energy of 10eV) this results in an error in Masslynx since collision energy is not regognized.

In masslynx files the collision energy is written as "CollisionEnergy(eV)_1,10.000" the missing e results in that the CEs are not read by masslynx. This can be fixed in word by replacing the wrong term with correct but it would be better if skyline developers fixed this.

Also please make it possible to export retention times (start and stop) and polarity from skyline to masslynx

Regards,
Per

I have untargeted DDA data for metabolite samples and have used public .msp libraries from RIKEN for MS-DIAL data analysis.
I have access to .raw files of the samples & .msp libraries.
I identified over 100 differentially abundant metabolites that I want to further investigate.
How can I develop an MRM method for these metabolites?
Does anyone have a latest tutorial or guidance on how to approach this? I’ve heard that Skyline might be useful, but I'm unsure where to start. Any advice would be appreciated!

We are trying to build a library using raw files of mass spectrum data contributed in other existing studies.

But as the skyline is updated, some problems arise.

During the initial file upload process, RAW format files cannot be uploaded. (picture 1)

At this stage, only ".dat" file formats are read, and ".mzid" and ".mzxml" files are not readable.

At this stage, may I know if there are any file formats that can be uploaded?

If only a specific file format is possible to uploaded, I would like to know if the library cannot be built if the contributed mass spectrum data does not have that file format.

We built the library by uploading the ".dat" file in the first step and registering the ".raw" file in the next step.

In the next step, enter “search condition”, and under “modification” I want to select carbamidomethyl(M).

However, carbamidomethyl(M) is said to already exist, but cannot be found in the list. (picture 2)

In this case, carbamidomethyl(M) is not in the list because it is a modification that is applied by default, and I would like to know if it is applied automatically or if another error occurred.

Hello,

I have generated an unscheduled PRM method using Skyline for 200 precursor ions. Could you please provide a guide on how to analyze the resulting PRM data in Skyline? Moreover, Is it necessary to import spectral libraries for this analysis?

Hi all :)

I seem to have lost my little red scan number demarkations with the small red number that indicated how many scans were across my peak when I was on the Transition Targets level to view my chromatograms. I attached a minimal doc that I think captures the issue, I'm sure I'm just missing some Magical Checkbox in the settings!

If you need another other information or details, please let me know

Thanks,
Lindsay

Nick

Dear Skyline people,

I am trying to extract pressure traces from our Thermo Fisher raw files recorded using a Q Exactive couples to a Dionex Ultimate 3000. However, I am not seeing any pressure traces, although they are stored in as "Pump Pressure" within the file (verifyable using Freestyle). It works for files recorded using a Vanquish HPLC.

Can you help me find out what's going wrong?

I attached you example raw files as well as an example document.

Thank you very much for the help!

Hello,

I collected MRM transitions on an Agilent QqQ. I had 2 scan segments. One segment was from 0-1.1mins, in which solvent was diverted to waste. The second segment was from 1.1mins to the end in which solvent was diverted to MS for data collection. When I import the results into skyline it only shows that first segment from 0-1.1 mins. I want to see the data from 1.1mins to end which is when analytes in the sample will have abundant MRM transitions. How do I get Skyline to be compatible with this data?

For in silico digestion using various enzyme.

What do the following mean (X= some amino acid):

[X | P]
[X | - ]
[- | X]

thank you.

Hi,

Is there a way to construct a spectral library such that Skyline will just "trust" the user that parameters are correct? I am trying to build some empirical (e.g. constructed from DDA) spectral libraries for DIA runs where the peptides have exotic/unlocalized modifications - and I am really struggling with how to get Skyline to accept a spectral library.

Ideally, I can just pass in a list of arbitrary ID, precursor m/z, precursor charge, product m/z, library intensity and have Skyline accept it without any "error" checking (e.g. do not try to match the product m/z to the peptide sequence). Is there a way I can fool Skyline into accepting such a list? I don't want to have to manually add the modifications to Skyline, since I have 1000s of them. I am happy to manually construct a library in whatever input format is needed.

Hello,

I wanted to know if Skyline is able to process top down MS data? If so, is there a tutorial for it?

Dear Skyline team,
I won't be able to attend the upcoming online course and was wondering if you will record it? In case yes, would I still need to sign up for the course to watch it?

Thanks a lot
best wishes

Hello, I'm interested in adding Skyline to my group's proteomic workflows. We are working with the proprietary sequences. Does your software protect the proprietary nature of these sequences?

Thank you!
Seamus Kelley

Hi, I'm experimenting with small molecule work in Skyline and have a few questions that I have not seen addressed in the documentation or other questions.

1. When building spectral libraries, right now I am converting a zenoTOF wiff2 file into mzml, and selecting scans manually for entry into an ssl file. This works, but has been very tedious. In the documentation, there is a list of supported search engines, but they all seem to be for proteomics. Is there a supported search engine for small molecules to aid in library building/annotation? If not, are there any faster ways that would be useful for me to be aware of?

2. With the above method of creating library entries, I often have high-abundance precursor peaks in the library spectrum. Ideally, I'd like to filter these out from quantitation/selected transitions automatically. The 'Precursor m/z exclusion window' under Transition Settings > Filter seems like it should do this, but in practice it has not worked to filter out these peaks. Can you advise?

Thank you for any help you can provide!

Sincerely,
Morgan Mann

I have under my skyline file one same metabolite showing in two lines, and I would like to add the second transition into one same SRM
I tried adding transition, copy paste from another skyline file where they show up together, but the second transition is gray with no data when I do this.

How to fix that issue please?
Thanks
Nihel

Hey! For our proteomics project we are using the automation tool to connect Skyline with Masshunter from Agilent. Therefore we have two questions.

Recently, we have compared the export methods that we made by going through the steps manually and doing it with the automation tool. Luckily, the results were completely the same!
However, Skyline comes up with the starting point for CE optimization, using step size and step count. We would like to further optimize with the results of the first experiment as an starting point. How could we approach this?

Secondly, when processing our results in Skyline in the replicate comparison, the different CE are always displayed as step (-2, -1, 1, 2 etc.). See figure in the attachments.
We would like to know whether it is possible to have the CE settings shown in de legend as there are many differences between peptides.

Thank you in advance!

Alexander

Hello,

I am analyzing my PRM results using Skyline and encountering an issue with quantifying certain targets. Specifically, in the attached file, there is a peak at 69.x minutes in the bottom panel, which corresponds to one of my targets. The MS/MS data from Thermo Fisher software confirms this match. However, when I import the same RAW file into Skyline, this target is not being quantified or integrated. Could you please help me understand why this might be happening and how I can resolve this issue?

I´m missing the heavy fragment ions for some of my peptides from a SureQuant run. I can see that these were triggered ( precursor traces)but no fragment ions, unfortunately. These fragment ions were there in the method used for the run. So I´m a bit confused.

Hi team,

I have a working document for small molecules (attached) and I'd like to use the document grid to add information into the KEGG/CAS/HMDB fields. However, these fields are grey in the document grid and not editable. Please advise on how to batch-enter values into these fields in a manner such as copy/paste from excel, or importing an annotations file?

Cheers

Will

Dear Skyline team,

I encountered an error while trying to import peptide search result (MaxQuant msms.txt file) into Skyline (see attached error message). I have tested this in Skyline Daily 23.1.1.520 & 24.1.1.254 as well as Skyline 24.1.0.199 and got the same errors from all.

I uploaded both mqpar.xml and msms.txt file to https://skyline.ms/files.url. Because the msms.txt file is quite large (50 GB), I uploaded a smaller version (only the top 10000 line in the original msms.txt). Since I encounter the same issue with the smaller version, hopefully you will also be able to reproduce the error on your end. I also had to change the mqpar.xml file name because there is already a file named after that in https://skyline.ms/files.url.

Please let me know if I can provide more information about the issue.

Thank you in advance!
Shimin

Dear Team,

We are failing to train mProphet model in skyline version 22.2.0.527 (841287d47) and 24.1.0.199 (6a0775ef83). But unable to reintegrate. It's showing pop-up "failed attempting to reintegrate peaks. The index 6 must be between 0 and 5". As per the suggestion obtained from previous queries we tried to perform the rescoring but failed to reintegrate. Kindly advice to resolve the issue.

Thanks in advance.

Kind regards
Vanya K N

Hi Skyline team.

We are setting command line based data extraction where a custom report is meant to be exported at the end of the analysis. When trying to export a standard report template everything runs fine but we get an error when we try to export a custom report. I have attached some sreenshots. Please advise further,

Thank you

Stoyan

Dear Team,

We are failing to train mProphet model in skyline version 22.2.0.527 (841287d47) and 24.1.0.199 (6a0775ef83). But unable to reintegrate. It's showing pop-up "failed attempting to reintegrate peaks. The index 6 must be between 0 and 5". As per the suggestion obtained from previous queries we tried to perform the rescoring but failed to reintegrate. Kindly advice to resolve the issue.

Thanks in advance.

Kind regards
Vanya K N