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 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.

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

Hi,
We have calibration curve equations determined for all our molecules (Lipids) in an excel sheet, but we do not have all the raw files. Is there any way in Skyline that I can import the calibration curve equation and calculate the concentrations?

Dear Skyline Team,
This software helps me a lot in analyzing PRM and DIA data. Thank you.
And I am curious about how the software identify peaks in spectra. However I can't find the defination of that anywhere...
For example, this explanation (https://skyline.ms/wiki/home/software/Skyline/page.view?name=tip_peak_calc) makes me clear that how Skyline calculates peak areas, but why the time 14.4- 14.48 is defined as the start and end of the peak to calculate? I wonder if there is a document containing the algorithm about that, like treat the "10% intensity of climax" as the bottom.
Thanks again for your reply!

Best regards,
He Zhu

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

Hi Skyline Team,

I am analyzing DIA data from for cell line proteome generated on Sciex TripleTOF 6500.

Nick and I have previously tried to understand the Isolation Scheme issue and now I do it by manually inputting it (and have it saved) as we use variable window. But I have noted that when I use only DIA data to generate a spectral library and quantify, Skyline produces only 1800 proteins while from the same files DIA-NN is producing 3500 proteins. But if we use DDA data to build spectral library, then we are able to generate 6500 proteins and quantify. We have also used it to create a groupwise comparison. Now my queries:

1. We know that DIA produces more data then why are we able to capture much less protein than DDA. What can we check and improve ?
2. In the groupwise comparison, the proteins of interest (we have RNAseq for the samples and looking for those genes) are all non-significantly expressed across groups. How can that be possible even the proteins of highly expressed genes ? How can we check that and also what method is used to calculate the fold change (need it for writing the methods).

P.S. I have the skyline zip [9Gb] in Onedrive and can share as per your convinience.

Much gratitude for all the help and advice.

Best regards
Sangramjit

Dear Skyline,

Not sure where best to post this. Love the mirror-plot feature for prosit spectra but would really like to be able to compare mirror-plots of modified and unmodified peptides natively in skyline. Is this something you are working on or a feature you could add?

Cheers,
Luke Gamon

Hello,

I am importing and analyzing DIA-IM data using an Ion Mobility library and iRTs built from DDA-PASEF data. When clicking the apex of a peak, a purple IM extracted window is shown for the precursor but not for all fragments (with a label of IM = 0 above them). I'm a bit concerned that the fragments shown are not at the specific IM window but rather the entire IM range.

Is there a way to make sure the same IM extraction window is assigned to the fragments of a target?

Thanks,
Premy

Hi,
We are trying to build a library for DIANN search. When we export the library, it's only in .blib format and DIANN is not recognizing this format (it only takes .speclib). Is there are way to export the library that can be used in DIANN?
Thanks!

Lorrain

Good morning. I updated my skyline daily this morning. Opened my template file, saved as the new data set and then attempted to import data from my Sciex 6500+.
I'm getting an error when importing data that says "the file may be corrupted, missing or the correct libraries may not be installed. [WiffFile::create()] could not load file or assembly 'clearcore2.data.version= 3.0.0.0. Culture=neutral. Public key Token 2a79e0d8fd2e4eca or one of its dependencies. system cannot find the file specified.

I went to a different PC declined the upgrade and opened my template, saved with the new file name and imported the same data no problems. I suspect something is broken with the new version of daily.

Dear,

When importing timsTOF DIAPASEF runs in skyline daily 22.2.1.391, skyline closes down. Any idea what is going on?
List of peptides is not huge, only the promega isotopologues.....

Thank you for looking into this!

Kind regards,
An

Hi,
I use Skyline for metabolic flux analysis. I am tracing the conversion of isotopic labeled substrates into different metabolites. For this purpose I define the count of labeled ions in the precursor ion and let Skyline calculate the Ratio between the unlabeled and the different labeled metabolites in my samples.

Lately I want to trace the fate of nitrogen and on what position they will appear in amino acids (sidechain or headgroup). In this special case the precursor ions have the same mass but differ in only one product ion.

How can I define this ion to be the one being used as the quantifier for the calculation of the ratio?

I am using a prosit library and I would like to see how my spectrum compares to the one in the library (predicted by skyline) using mirror image display. However smth is clearly wrong:

1. in the mirror image the dotp 1, but in the target tab dotp is 0.89
2. the name "2022_08_24_BSA_ATG4 vs prosit", but the 2022_08_24_BSA_ATG4 is the name of the library generated using prosit
3. The mirror image includes the ions that I have removed from the analysis
4. all the errors are 0 ppm implying that skyline just plot the library vs library

(see the screenshot attached).

Why is skyline not showing the actual spectrum from the current analysis as part of the mirror?

Thanks again for all the support with Skyline!

I am developing a DIA-IM Lipidomics pipeline on Skyline and was wondering whether an isolation scheme would need to be defined for Bruker timsTOF data or if the isolation scheme can be read from my raw files using "Results Only"?

Thanks,

Premy

Hello,

I am performing DIA data analysis. I notice that only one replicate is showing up while the others are not visible. How do I resolve this issue?

Dear Skyline people,

I am sorry in advance to haunt you with ghosts from the pasts, but I have a problem with importing Thermo Fisher raw files in one our systems by using your last version of Skyline 32-bit. We have two systems which run Windows 7 32 bit.

System 1

• Xcalibur Foundation 3.0 SP2
• Xcalibur 3.0
• Q Exactive Plus Orbitrap MS 2.9

System 2

• Xcalibur Foundation 2.0 SP1
• Xcalibur 2.2 SP1
• TSQ Quantum 2.3.0

I want to import an SRM run into Skyline to check system performance. While it works flawlessly with system 1, I get the following error on system 2:

At 1:15 PM:
Failed importing results file 'C:\Xcalibur\data\Faelle\Test_TSQ\230210\Test_LXQ_Q1_SRM_pos_230210.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=2": [ThermoRawFile] The system cannot find the file specified.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Xcalibur\data\Faelle\Test_TSQ\230210\Test_LXQ_Q1_SRM_pos_230210.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=2": [ThermoRawFile] The system cannot find the file specified. ---> System.Exception: [SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=2": [ThermoRawFile] The system cannot find the file specified.
   at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
   at pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\skyline_20_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1064
   at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\skyline_20_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 538
   at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasCombinedIonMobilitySpectra() in C:\proj\skyline_20_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 599
   at pwiz.Skyline.Model.Results.FileBuildInfo..ctor(MsDataFileUri msDataFileUri, MsDataFileImpl file) in C:\proj\skyline_20_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1430
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 211
   --- End of inner exception stack trace ---

It doesn't matter where I try to import the results file from, it is never found. Do you have any idea why this might be the case? Is the Xcalibur version on system 2 too old?

I attached the file in question and the used skyline file for you.

Thank you very much for your help!

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

Hello,
I need help with processing hela-PRTC SRM data from tsq altis plus using skyline. Is there any tutorial available?

Thanks,
kmsd

Dear Skyline team,

I am performing SRM data analysis for small molecules on the beta version of skyline daily. I would like to display the skewness, kurtosis and shape correlation values in my table.

While I have integrated the columns in the report, the cells appear empty whatever the compound despite the presence of a chromatographic peak. I also searched the check box "Calculate skewness" in the advanced section in the settings, without success.

How can I solve this issue?

Thank you in advance for your feedbak.
Best regards.

Chloé C.

Hi all,

I want to use Skyline for MS1 filtering and quantification of collagen PTMs from crude samples, conceptually similar to what we did here:
https://doi.org/10.1016/j.mbplus.2019.04.002

Right now, I am trying to analyze crude cell ECM from cells where I knocked down a gene (n=2, four samples in total). This is basically a test run, to see whether we get enough coverage of collagens and other proteins of interest.

We use MyriMatch (2.2.19172) for peptide search of thermo .raw files and obtain results in .pepXML format and check them in ID picker (3.1). After the "fine PTM search" where we restrict the search on proteins present in the sample and include decoys in the fasta file, I loaded a merged session of my four files in IDpicker and exported the spectral library (.sptxt file) from the merged idpDB file.

I imported this .sptxt file into Skyline ("Use existing library") and then loaded the four thermo .raw files after conversion to .mzMXL via MSConvert (3.0). This seemed to work.

However, now, when I crosscheck the IDs and the retention times I get in Skyline, they are most of the time completely off compared to the scan times for the same peptides in ID picker.

Is there anything obvious I have been doing wrong? I would appreciate your help on this. Happy to upload files or give more information, of course, too, if needed.

Many thanks,
Claudia

Hi Skyline team,

I am looking back at a Skyline document that has 100+ runs in it. The Ctrl + Shift + Up/Down (arrow key) is very useful to go through the different runs based on the document order. However, I have noticed that it completely changes the arrangement of runs in the Replicates window. Any ideas why?

I noticed this a year ago, but tested it with the newest Skyline-daily available (22.2.1.391) and see that the problem persists.

As always, thanks for your time and help!
Sincerely,
Juan C.

Dear Support Team,
I had a doubt regarding the peak annotation is Skyline.
Under "Transform" option, there are four different options: None, Interpolated, Second derivate, Savitzky-Golay Smoothing.
In each view, the number of data points shows a different value (as in the attached picture)
While selecting the peak, which view should be considered for analysing the peak width or to know the number of data points in the peak? Can we consider the Smoothing view for this as that shows the maximum number of data points.

I look forward to this.

Best Regards
Mehar

Dear Skyline Team. Currently the max precision calculated for retention time prediction (reported in "Predicted Result Retention Time" field), is hundredths of a minute. For very fast runs with very narrow peaks, it seems like it would be useful to allow a more precise prediction. The current precision of 0.01 minutes is only 0.6 seconds, which for some separations is nearly the 4sigma peak width. Please see attached an example of a file where I think having a more precise result of the prediction algorithm would be useful for putting the prediction closer to the apex of the peak. Or please let me know if I've just not configured something correctly. Thanks for your help!

Cheers,

Will

Hellow World! I am looking at a SureQuant result and I found a parameter called Raw Number of Points, which is the number of ms2 spectrums acquired for a particular target. However, there is a separate parameter called Points Across Peak. When I look into the numbers there are far fewer Points Across a Peak than Raw Number of Points. This brings up a question: exactly what does the parameter Points Across Peak mean?

I'm attempting to do a DDA search with MSFragger. My overall workflow is as in the thread I started last week (FATAL: An exception occurred: 'c' is not a valid amino acid), and works fine with those old data. The same workflow also works fine with most of the new data I have collected. With some files, however, I will get 0 PSMs returned from the decoy search, which results in an error message when Percolator gets to that file (Error: no decoy PSMs were provided). I have attached the log from an attempt at running the search with one of the files in question (several files will trigger the same error message). This might, in part, reflect the small size of my database and relatively low sample complexity (289 peptides), but is there some way that I can adjust settings to encourage MSFragger / Percolator to find at least 1 PSM among the decoys so the search can complete with all files?

I'm trying to import DDA files and complete a search within Skyline. I can get the search to complete sometimes with MSAmanda, but when I use MSFragger, I usually get the error, "FATAL: An exception occurred: 'c' is not a valid amino acid." I'm not sure where this is coming from. There are no "c" in my fasta; I'm searching a peptide library that lacks cysteines, and I have checked / resaved-as / etc. the fasta without effect. I think it might be related to modifications because I could get MSFragger to complete when I set the maximum variable modifications to 0 (though there were few identified peptides when I tried that compared with when I have searched in MSAmanda or MaxQuant). My library has fixed C-terminal amidation, but otherwise should not have any modifications. I've tried making a custom C-terminal amidation modification instead of using the built-in modification without effect. I've attached the full MSFragger log from my latest attempt if that would be helpful. Does anyone know where this error is coming from an how to fix it?

Dear team,

Quite new with Skyline. I would like to run DIA analysis with quite large set of MS data. The idea:

• Prepare analysis configuration/libraries using Skyline and Skyline batch programs under a standard Windows machine.
• Execute this configuration on a powerful server using only the command line through Skyline.exe / SkylineBatch.exe, i.e., without any graphic UI that is not available on this machine (I checked I can execute SkylineCmd.exe --help on it).
• Go back with result files on the standard machine for Skyline visualization.

Is there any way to achieve this idea or alike and process the workflow with no more user intervention? Or suggestions ?

Thank you for your help.

It works for the interact-*.pep.xml and *_uncalibrated.mgf files from FragPipe 18.0 coupled with MSFragger 3.5. But not the files from the latest MSFragger. Could you please help to test, and tell me if Skyline or MSFragger need to be adjusted? All files have been uploaded to the attachments.

Thanks,

Fengchao

I am curious if Skyline could be used to generate data collected on a GC-QTOF. My instrument (Agilent 7200 GC-QTOF) is not capable of generating data using MRM\PRM acquisition modes - all data represents full product ion scans using EI ionization.

Would I be able to use Skyline to extract select ions from the full scan and compare their ion intensities relative to a spectral library entry to detect compounds and obtain quantitation?

Hello Skyline support team,

I have an issue when extracting MS2 fragments integration for quantification. The quantification results (showed in the "quantification" column from the "peptide ratio result" report) of my target peptides are correctly showed, but for the isotopically labelled (+8 for Lysine and +10 for Arginine) internal standards, the quantification showed "#N/A", even though the chromatogram was correctly extracted. I wonder if this is due to some settings about isotopic labelling that I've ignored.
I attached a snapshot from the "peptide ratio result" report preview. Any suggestions would be greatly appreciated!

Hi Skyline Team,

I have been getting myself familiar with skyline by doing the tutorials and when I was working on the Targeted Method Refinement Tutorial I got stuck. Whenever I need to import results I end up getting a Corrupt RAW file error. I have tried it with both with Method Refinement supplemental and if I skip that I get the same error when I try to import in the "Scheduling for Efficient Acquisition" step.

I am running skyline 64-bit 22.2 on Windows 11 using Parallels on a 2020 MacBook Air.

I have tried to access the file from a few different ways;
*direct by clicking on the tutorial inside the skyline app,
*downloading it from the website,
*using the hyperlink in the PDF that opens when you start the app (https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/MethodRefine-20_1.pdf)

It has also persisted through restarting skyline and using a completely fresh .sky file.

Error message:

At 3:13 PM:
Failed importing results file '\Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW'.
[RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file '\Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW'.
[RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW ---> System.Exception: [RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW
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 ---

Many Thanks,
Soren

Hello,

I am using Skyline Daily v 22.2.1.391 to integrate small molecules using a transition list. I noticed for a few metabolites, that I manually integrated, the exported Skyline report shows zero values even though there is a peak that has been integrated. In all cases these are low signal peaks. However, within the same dataset, Skyline allows integration of noise from blanks and samples that have even lower than the biological replicates that was "zeroed out".

In the retention time comparison graph, there is a retention time window showing the sample that has a zero value. The peak area graph doesn't have a line/bar for that sample because Skyline is recording it as a zero. Please refer to attached document.

Thank you for your assistance. Let me know if there are any other details you'd like from me.

Best,
Hanan

Hi,
I would like to include the number of spectrum IDs per peptide when exporting a document report. For example, if I have 8 samples and each chromatogram has 1 ID then I would be looking for a value of 8.

Hope you can help.

Thanks

Paul

Hi,
We are testing some standard peptides using SWATH DIA on SCIEX 7600 zenoTOF instrument. Our transition setting is attached. After importing the datafiles, for some peptides we were able to see MS2 fragments; for some peptides, there is no fragment data. I am wondering if this is due to our DIA method settings or skyline transitions settings. Would you please help?
Thanks!
Lorrain

Hi all,

I have a PRM experiment where I am trying to normalize each sample according to their respective TIC.
I have looked in the Skyline forum where the suggestion is to take modify the document settings and enable calculation of the Total Ion Current Area.
Subsequently, setting the "Explicit Global Standard Area" column to the TIC area

However, I have come across a couple of issues -> The "Explicit Global Standard Area" is under the "calculated" tab rather than "editable" in the document settings so I can't seem to copy and paste.

Ultimately, what I would like is when I view the peak areas of my peptides (with respect to the other replicates); I would like to normalize against the TIC -> Currently, the only options available are "Total", "Maximum" and "None"
And then to take these values through to MSstats

Is there any resource that provides step-by-step instructions in what I am attempting to do?

The version of Skyline I'm using is:
20.2.0.343

Hi team,

I am trying to fit some cross-linked peptides to an iRT scale (i.e. calculator) generated from endogenous peptides. Unfortunately the column had to be changed, resulting in different retention times. However, I am trying to compensate for this using the background proteome to define some iRTs.

Skyline allows me to add the peptides within the "Edit iRT Calculator" by clicking "Add Results..." but when I try to click OK I am getting a error message (attached).

You will know best, but to me it seems is that here Skyline is not recognizing cross-link modifications?

Any suggestions for a workaround or is it something that has to be patched?

As always, thanks for your time and help.
Sincerely,
Juan C.

Dear Support Team,
When I import the result files, while looking at the results, it seems as if the whole target list is not at all monitored in all the samples and it shows "Chromatogram information unavailable". However, I have used the same method to run all the samples.
When i checked the raw file, it is ok and have been monitored for all the precursor masses. I tried to import the results in a new Skyline document, some of the files which didn't show up before, they showed up and I could see the data for all the precursors in the target list. But the issue was still there if I remove those files or reimport.
As i have shown in this attachment, the peak is being shown for only sample 1 and not for sample 2-4. If I try starting the whole thing again in a new document It can happen the next time that I see the results for sample 3 or 2 or 4 and not for sample 1. So, it is just happening randomly. As this is not happening to a single or same set of raw files each time, I am not able to guess the issue.
Kindly help me fix this.

Best regards
Mehar

Dear Support team,
I am using Skyline-daily (64-bit) 22.2.1.391
I am planning for a PRM experiment on Thermo Scientific Orbitrap Eclipse mass spectrometer. How can I export isolation list for this instrument.?

Best regards
Mehar

Hi all,

I am currently analyzing some DDA- and DIA-PASEF data with the most recent Skyline-daily version and I am not being able to click on proposed peak boundaries as I used to.

Is someone else facing this same issue? Or is it my document and/or data the issue?

JC

Dear Skyline staff,

I’m trying to analyze a dataset by DDA MS1 filter to compare XIC of glycopeptides. The peptide search comes from MSFragger – glycol search –. I can create a library with different peptides from the proteins ID but I can see the glyco peptides identified by MSfragger, which is actually my interest in the analysis.
I have tried to add the glycosylation by peptide setting but Still, I only see unmodified peptides ( only Carbamidomethylation and met ox -.
Any suggestion to solve that?

thank you so much in advance,

Carlos

Hi,
I was wondering if it's possible to implement a slit retention time window for the explicit retention time.
In some cases we have shoulders or a bit of tailing. Of course this is not ideal but just the case sometimes.
adjusting the retention time window with different values would help in these cases.
Best, Dennis

Hello,
I have 15-20 peptides that I would like to confirm specificity against Human proteome. I took following steps:
Settings->Peptide setting->Digestion->background proteome
and have added human Fasta sequence.
Then I took following steps:
Edit-> Unique peptides
Under this option, I could see some peptides that have blue checks meaning they are not unique. However, if I keep scrolling I see a limited number of proteins in human. I am not sure if this option is covering all the human proteins. Can you please provide an insight to this?

Suppose I have a raw data, named N1, which generates 300 sky files in 300 spectral libraries. How should I merge these 300 sky files? I try import-document and set it to merge with existing results by duplicate name, but the new results after import have no peak. I want to ask how to merge the sky files of different spectral libraries.
best wishes.
Zhao

Hello,

We have user whose skyline app continues to crash. When She attempts to open it, she gets the attached error message telling her to check the log in Event View, also attached. I've already uninstalled and reinstalled the program and uninstalled windows updates.

Thanks so much for all the developments in Skyline. This tool has been an amazing support for my DDA and diaPASEF data!

I'm currently working with DDA-PASEF data (Bruker timsTOF) to build an ion mobility library for a set of lipid standards. I had a few questions regarding the 1/K0 and CCS values presented in the report.

1. Is there a way to get the raw CCS values (in case there are slight shifts in CCS between replicates)?
2. In the case that the IM extraction window needs to be corrected by overriding with "Explicit Ion Mobility Value", is there a way to also recalculate the CCS value in the report? It currently still reports the original CCS value and the newly corrected 1/K0 value.

I hope this makes sense and thank you in advance!

Premy

Hello,

Im trying to find out if the newest skyline upgrade is compatible with windows 10 22H2 and/or windows 11 22H2?

Thanks!

-Tatiana

Hello,

Can you please let me know to import FASTA file is this the only way:
File->Import->Fasta
Or is there any other way?

Hello Skyline team,

I would like to report an error issue that might not have been mentioned here yet.

I am working with Skyline Batch, where I shared a bcfg file and tried to import it back into Skyline Batch on a different computer. Both systems were using Skyline Batch 21.2.1.389. While trying to import the bcfg file I got an error complaining that the version of my file is newer than the version of the program, which was, of course, wrong (see screenshot).

Since the version number look weird with a comma in the error message, I checked the windows 10 region settings and realized that the decimal symbol on my German system was set as comma and the digit grouping symbol was set as dot. Reversing this in thw windows settings resolved the issue. I am not sure, whether something can or should be done here but I thought I better report this.

Thank you for this great software,
Roman

Hello Skyline team,

I accidentally clicked on Prosit in the Library Match window which lead to the window blanking out and becoming completely unresponsive, showing the message "Prosit is not properly configured".
Right clicking on the blank window resulted in an unexpected error report (submitted):

Message:
Object reference not set to an instance of an object.

Source code location:
System.NullReferenceException: Object reference not set to an instance of an object.
at pwiz.Skyline.Controls.Graphs.GraphSpectrum.get_SpectrumInfo() in C:\proj\pwiz\pwiz_tools\Skyline\Controls\Graphs\GraphSpectrum.cs:line 1466
at pwiz.Skyline.SkylineWindow.pwiz.Skyline.Controls.Graphs.GraphSpectrum.IStateProvider.BuildSpectrumMenu(Boolean isProteomic, ZedGraphControl zedGraphControl, ContextMenuStrip menuStrip) in C:\proj\pwiz\pwiz_tools\Skyline\SkylineGraphs.cs:line 1159
at ZedGraph.ZedGraphControl.contextMenuStrip1_Opening(Object sender, CancelEventArgs e)
at System.Windows.Forms.ToolStripDropDown.OnOpening(CancelEventArgs e)
at System.Windows.Forms.ToolStripDropDown.SetVisibleCore(Boolean visible)
at System.Windows.Forms.ToolStripDropDown.Show(Control control, Point position)
at System.Windows.Forms.ContextMenuStrip.ShowInternal(Control source, Point location, Boolean isKeyboardActivated)
at System.Windows.Forms.Control.WmContextMenu(Message& m, Control sourceControl)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.UserControl.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
at System.Windows.Forms.Control.WndProcException(Exception e)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
at System.Windows.Forms.NativeWindow.DefWndProc(Message& m)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.UserControl.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at pwiz.Skyline.Program.Main(String[] args) in C:\proj\pwiz\pwiz_tools\Skyline\Program.cs:line 312

Reloading the Skyline file did not fix the issue, I can see my spectra fine via View>Spectral_Libraries. I imported my library into the Targets list via the Spectral Library Explorer, but this also did not have any effect on the Library match window staying blank.
It seems like the window is stuck for now. Is there a workaround for this?

Best,
Tobi

Dear Skyline team,

I am trying to build spectral library from timsDIA PASEF data. I have raw files (.d folders) that were processed using FragPipe/MSFragger/Philosopher. Which, by the way, works great for these data.

I am not able to build a spectral library using the DDA-MS1 quant workflow and DIA workflow, with Import - Peptide results etc for building the list form the search results. Neither using MSFragger pepXML files nor using interact-.pep.xml files after PeptideProphet work. When using interact-.pep.xml files, I get an error that it cannot find spectral files. When using pepXML, it finds the files to get the spectra (from file_calibrated.mgf files produced by MSFragger) but gives an error at a later stage. Can some one help me to get the solution of it.

Hi,

I have found a previous post suggesting that Apex3D pepxml files can be uploaded in Skyline. I am planning to use skyline to identify two crosslinked peptides and I would like to use the output of UNIFI data search to build an ion library and gain confidence in peak picking. I understand from previous posts that it is possible to use PLGS csv output, however at this stage I would not know how to add additional modifications in PLGS. I can see that UNIFI allows to export results in mzml and mgf format. Is there any chance I could directly use these to built the ion library? UNIFI is doing a targeted analysis to match spectra to peptides from one or two proteins added to the processing method. Would this have an impact on dotp or confidence in peak picking? Many thanks, Floriana

I am trying to open bbCID (MS/MS) and MS from Bruker, it is a type of DIA. I applied this method for degradation of pharmaceutical compound (small molecule). What is the procedure, how I can insert the range not a list of masses?

Can I inspect the data manually? is the step seeing the precursor and products overlayed available? can I use the software to extract specific ions during by inspection?

I am working with v22.2.0.351 and trying to run a MSGF+ search.

However, when running the searches, I got multiple errors related to my digest and modification settings.

Digestion: I had made a customized trypsin with proline digestion, and that was not recognized. I had to revert back to the default Trypsin digest setting (no cut when Pro follows KR). Once I did that, that error went away. My customized digest setting was "-1", which was out of the expected range of 0-9.

Modifications: I got multiple errors for modifications that were defined by their chemical formula in my Peptide Settings. It did not like "H3N" that was defined for pyroGluQ. It did not like "H2O" defined for pyroGluE. It also did not like "O - HN" that was defined for deamidation. I tried clearing all modifications from my Peptide Settings, and re-entering all the default modifications that had these chemical formula definitions, but that did not help. I ended up deleting all the chemical formulas from the modifications, just leaving the numerical values in there, and now the searches run as expected.

Below is the error information I got for the deamidation and pyroGluQ chemical formula instances:

MS-GF+ Release (v2021.09.06) (06 September 2021)
Java 17.0.1 (Oracle Corporation)
Windows 10 (amd64, version 10.0)
java.lang.NumberFormatException: For input string: "O - HN"
at java.base/jdk.internal.math.FloatingDecimal.readJavaFormatString(FloatingDecimal.java:2054)
at java.base/jdk.internal.math.FloatingDecimal.parseDouble(FloatingDecimal.java:110)
at java.base/java.lang.Double.parseDouble(Double.java:651)
at edu.ucsd.msjava.msutil.AminoAcidSet.parseConfigEntry(AminoAcidSet.java:930)
at edu.ucsd.msjava.msutil.AminoAcidSet.getAminoAcidSetFromModFile(AminoAcidSet.java:797)
at edu.ucsd.msjava.msdbsearch.SearchParams.parse(SearchParams.java:337)
at edu.ucsd.msjava.ui.MSGFPlus.runMSGFPlus(MSGFPlus.java:77)
at edu.ucsd.msjava.ui.MSGFPlus.main(MSGFPlus.java:61)
Search canceled.

MS-GF+ Release (v2021.09.06) (06 September 2021)
Java 17.0.1 (Oracle Corporation)
Windows 10 (amd64, version 10.0)
java.lang.NumberFormatException: For input string: "-H3N"
at java.base/jdk.internal.math.FloatingDecimal.readJavaFormatString(FloatingDecimal.java:2054)
at java.base/jdk.internal.math.FloatingDecimal.parseDouble(FloatingDecimal.java:110)
at java.base/java.lang.Double.parseDouble(Double.java:651)
at edu.ucsd.msjava.msutil.AminoAcidSet.parseConfigEntry(AminoAcidSet.java:930)
at edu.ucsd.msjava.msutil.AminoAcidSet.getAminoAcidSetFromModFile(AminoAcidSet.java:797)
at edu.ucsd.msjava.msdbsearch.SearchParams.parse(SearchParams.java:337)
at edu.ucsd.msjava.ui.MSGFPlus.runMSGFPlus(MSGFPlus.java:77)
at edu.ucsd.msjava.ui.MSGFPlus.main(MSGFPlus.java:61)
Search canceled.

Dear Skyline Team,

I was just trying to set up a configuration for Skyline Batch. I added the path to the R script in "R script file path" then clicked "OK". However, I did not see the path appears afterwards. I have attached some screenshots to illustrate the problem.

Am I missing some steps? Is there anything I can do to fix this problem?

Thank you in advance.
Shimin

We analyze datasets with many samples (typically 96) and rely critically on the dotP values displayed above each Peak-Area bar. Depending on the monitor and resolution, sometimes the dotP text is so small that it's very difficult to read. It would be helpful if these values could optionally be colored based on the value -- something like >0.9 green, between 0.7 and 0.9 orange, below 0.7 red. Ideally these would be user-configurable.

Thanks

Hi,

As the titled states I am using skyline-daily in molecule mode. When I add all of my molecules from a spectral library from the Spectral library Explorer I find that if I navigate to View -> Document Grid -> Reports -> Molecules the columns with either Predicted Retention Time or Average Measured Retention Time contain #N/A.

I am wondering if one of the columns should have picked up the molecule retention time given that the spectral library contains scans at explicit retention times. Maybe there is a setting I am not correctly using...

Also I am wondering if this is the cause of some incorrectly selected peaks for precursor that fall outside of the observed retention time seen in my spectral library for some molecules.

Thank you!

Hi Skyline community,

I am using Skyline for small molecules quantification.
For the quantification of some metabolites, I don't have standard but I have two internal standards in the samples and relative response factors of my interest metabolites.
Is it possible to implement these data in Skyline workflow for the quantification of large-scale metabolites?
I have not found the documents in Skyline and please advise me.

Thanks and regards,
Nay Min

An error occurred trying to download 'https://skyline.gs.washington.edu/software/Skyline-release-64_22_2/Skyline.application'.
See the setup log file located at 'C:\Users\amahan\AppData\Local\Temp\VSDF115.tmp\install.log' for more information.

when I enter "https://skyline.gs.washington.edu/software/Skyline-release-64_22_2/Skyline.application" directly in browser I get xml code

<?xml version="1.0" encoding="utf-8"?>
<asmv1:assembly xsi:schemaLocation="urn:schemas-microsoft-com:asm.v1 assembly.adaptive.xsd" manifestVersion="1.0" xmlns:asmv1="urn:schemas-microsoft-com:asm.v1" xmlns="urn:schemas-microsoft-com:asm.v2" xmlns:asmv2="urn:schemas-microsoft-com:asm.v2" xmlns:xrml="urn:mpeg:mpeg21:2003:01-REL-R-NS" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:asmv3="urn:schemas-microsoft-com:asm.v3" xmlns:dsig="http://www.w3.org/2000/09/xmldsig#" xmlns:co.v1="urn:schemas-microsoft-com:clickonce.v1" xmlns:co.v2="urn:schemas-microsoft-com:clickonce.v2">
<assemblyIdentity name="Skyline.application" version="22.2.0.312" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" xmlns="urn:schemas-microsoft-com:asm.v1" />
<description asmv2:publisher="Skyline" asmv2:product="Skyline" asmv2:supportUrl="https://skyline.ms/support.url" xmlns="urn:schemas-microsoft-com:asm.v1" />
<deployment install="true">
<deploymentProvider codebase="https://skyline.gs.washington.edu/software/Skyline-release-64_22_2/Skyline.application" />
</deployment>
<dependency>
<dependentAssembly dependencyType="install" codebase="Application Files\Skyline_22_2_0_312\Skyline.exe.manifest" size="142364">
<assemblyIdentity name="Skyline.exe" version="22.2.0.312" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" type="win32" />
<hash>
dsig:Transforms
<dsig:Transform Algorithm="urn:schemas-microsoft-com:HashTransforms.Identity" />
</dsig:Transforms>
<dsig:DigestMethod Algorithm="http://www.w3.org/2000/09/xmldsig#sha1" />
dsig:DigestValuehj+8x2o0jbNyeVZb9efH6GInr0w=</dsig:DigestValue>
</hash>
</dependentAssembly>
</dependency>
<compatibleFrameworks xmlns="urn:schemas-microsoft-com:clickonce.v2">
<framework targetVersion="4.7.2" profile="Full" supportedRuntime="4.0.30319" />
</compatibleFrameworks>

<publisherIdentity name="CN=University of Washington, O=University of Washington, L=Seattle, S=Washington, C=US" issuerKeyHash="254581685026383d3b2d2cbecd6ad9b63db36663" /><Signature Id="StrongNameSignature" xmlns="http://www.w3.org/2000/09/xmldsig#"><SignedInfo><CanonicalizationMethod Algorithm="http://www.w3.org/2001/10/xml-exc-c14n#" /><SignatureMethod Algorithm="http://www.w3.org/2000/09/xmldsig#rsa-sha256" /><Reference URI=""><Transforms><Transform Algorithm="http://www.w3.org/2000/09/xmldsig#enveloped-signature" /><Transform Algorithm="http://www.w3.org/2001/10/xml-exc-c14n#" /></Transforms><DigestMethod Algorithm="http://www.w3.org/2000/09/xmldsig#sha256" /><DigestValue>AKRfQgZhA6beEuaNTdRclrAcMFKROjHT9gZcM/BHQlI=</DigestValue></Reference></SignedInfo><SignatureValue>gz9+hqU2fuNwJuwvS6F3Hu7KGrmCKFdG+kDl3M1fTyxFGty9n4EiUuFhckD0oYY5KfmQiMVdpCnWmHTt02KEfL0XZR+qiuMXHHK7YzZssuWTYGw4JvOuZAYqV0Ptio8V06agdP0+IJAV9lk48AqNuLkvQiaL+MhuRReImEZGuXYWeGKDvgNS9jacUm20HC+lVSKjlUwZM7HnWFPWKNq1Qwk3RL4o1hojDoWPxG9SGf+e7vPHMXwcPEMNpC6BGZFkN6msCA/cJ3VwyBgenTwegtVilZkgANr0Yrg6yqxQUTjR0jUUUQre2A8ckEDR+JempO+K6+RQ+1NqTAmtj7axNg==</SignatureValue><KeyInfo Id="StrongNameKeyInfo"><KeyValue><RSAKeyValue><Modulus>xtmaVGQm59JHOXiq6oJWkFTjjnuORjbC1asIEVfQa1xvRXXyk+jye7h3ezw6wBTJlNq3r0wgNIbAkk1wq59hndsBcRRSQJJdFScLQrD7Z5LDNrwFqZZFlCt0Yxl0ZfxPes/BNGgfnoGXA6cd7jFPbahElvmCJoXR96fcwx6mIOMtTuGr+YINFngeBEVHADYnt+/6i5c842rPjYQa1P2oCFZFwdsa8GUFIf4QLG3JVpyqTfNzn3sikKeFxFU8COnwv1MYQ7WEt/qkOujM9uF6MP7003tjtA6fuBwPTH8c0+laBTq+5MYIqvroPwnalSTJ/9EaBanhxxE0o0Ppb69Igw==</Modulus><Exponent>AQAB</Exponent></RSAKeyValue></KeyValue><msrel:RelData xmlns:msrel="http://schemas.microsoft.com/windows/rel/2005/reldata"><r:license xmlns:r="urn:mpeg:mpeg21:2003:01-REL-R-NS" xmlns:as="http://schemas.microsoft.com/windows/pki/2005/Authenticode"><r:grant><as:ManifestInformation Hash="524247f0335c06f6d3313a9152301cb0965cd44d8de612dea6036106425fa400" Description="" Url=""><as:assemblyIdentity name="Skyline.application" version="22.2.0.312" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" xmlns="urn:schemas-microsoft-com:asm.v1" /></as:ManifestInformation><as:SignedBy />as:AuthenticodePublisheras:X509SubjectNameCN=University of Washington, O=University of Washington, L=Seattle, S=Washington, C=US</as:X509SubjectName></as:AuthenticodePublisher></r:grant><r:issuer><Signature Id="AuthenticodeSignature" xmlns="http://www.w3.org/2000/09/xmldsig#"><SignedInfo><CanonicalizationMethod Algorithm="http://www.w3.org/2001/10/xml-exc-c14n#" /><SignatureMethod Algorithm="http://www.w3.org/2000/09/xmldsig#rsa-sha256" /><Reference URI=""><Transforms><Transform Algorithm="http://www.w3.org/2000/09/xmldsig#enveloped-signature" /><Transform Algorithm="http://www.w3.org/2001/10/xml-exc-c14n#" /></Transforms><DigestMethod Algorithm="http://www.w3.org/2000/09/xmldsig#sha256" /><DigestValue>DUzXbnrMpbSbDpg1/vOyeij67/Hf7/q3ROK732oBjQA=</DigestValue></Reference></SignedInfo><SignatureValue>GS3MBbhLTXUVxbwM8VyvpKjQq684tqVBMc0pomt6LJ9JzY945VGe3RTj5m+kaMCCoJ7H2wXrO9ycbkPpAV+b7I7T82o9sy3OJlT3RJJcRuc4vIxACWy45z5N8y0t7joEW+S/NG9FcEB9LfW1J+EkrqROW8EloLBj88vFwdcI8DH4xgHub5K8NFZcMRQxbgktFZ3BkUuP/GV2BpCJs6x/PD6nPtVsd5SYTwHnq+wheCauL6A4jrZKf2E5hQDvtjjB3foe3Jwn+Y6vaA7fUdD+TNeTdsG8pbDX1slmbTgHjajZb0fehL4ZQId4jtywvvZjAI2v/6eTvbeMwWYVrUfdxw==</SignatureValue><KeyInfo><KeyValue><RSAKeyValue><Modulus>xtmaVGQm59JHOXiq6oJWkFTjjnuORjbC1asIEVfQa1xvRXXyk+jye7h3ezw6wBTJlNq3r0wgNIbAkk1wq59hndsBcRRSQJJdFScLQrD7Z5LDNrwFqZZFlCt0Yxl0ZfxPes/BNGgfnoGXA6cd7jFPbahElvmCJoXR96fcwx6mIOMtTuGr+YINFngeBEVHADYnt+/6i5c842rPjYQa1P2oCFZFwdsa8GUFIf4QLG3JVpyqTfNzn3sikKeFxFU8COnwv1MYQ7WEt/qkOujM9uF6MP7003tjtA6fuBwPTH8c0+laBTq+5MYIqvroPwnalSTJ/9EaBanhxxE0o0Ppb69Igw==</Modulus><Exponent>AQAB</Exponent></RSAKeyValue></KeyValue><X509Data><X509Certificate>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</X509Certificate><X509Certificate>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</X509Certificate></X509Data></KeyInfo></Signature></r:issuer></r:license></msrel:RelData></KeyInfo></Signature></asmv1:assembly>

I tried to install Skyline 4.2.0.19009 on my computer running Windows7 64bit. The installation did not start giving the error message you can find in the attached screenshot together with the log file the message is referring to. Can you give me any advice to bypass the error and install the new Skyline version. Is there any possibility that the error is related to settings of the locales or regions because I run the English keybord and locales on the computer my IT installed a German version of Windows 7 on. If not I will keep the actual version 4.1 which is currently working without any problem.

I looked into the support issues list but I did not find any request with the same error description.

Thanks for your assistance.

Hey guys,

Is it possible to overlay the XIC of two samples/runs in Skyline of have to export the data?

Thanks.

Dear,
We have just installed AutoQC to monitor our System suitability runs, but although we were able to generate a file with 3 runs in Panorama, it does not update the new runs added to the folder. It does not move forward from "waiting for files". Could you help us troubleshoot this issue? I have attached the skyline file document (.zip) were using and the error log information. Please let us know if you need other files. Thank you very much. Best, Daniela

Hello Skyline team!

I have looked at the user instructions to view GC-MS data in Skyline, but I'm stuck without an initial transition list. The dream is to import my NIST .msp file and to be able to generate "transitions" from the 1710 small molecule spectra it contains to find them in my GC-MS results (using the strategy of selecting DIA in the MS2 transition settings and using a fake precursor mass). Unfortunately, the library explorer doesn't recognize the spectra in my .msp file, maybe because it's missing precursor mass and charge information. Is there a way to make this work? Do I have to learn how to code so I can pull the information from the .msp file and create the transition list I need? Any help you can offer will be greatly appreciated!

Cheers,
Brynn

Hi Skyline community,
I am new to Skyline and I found some issues with dual polarities. My .raw files are positive and negative polarities in one file. When I prepare the standards, QC and blank samples in positive mode, it worked well.
However, when running in negative mode, there is no chromatogram found.
Could you please advise how to work with these dual polarities .raw files? Or please check the transition lists I have prepared and let me know if anything is not correct.

Many thanks,
Nay Min

Hi,

I tried to import a large Spectronaut Mouse library - MouseRefSWATH_iRT.csv by Krasny, L. et al 2020 (https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/GetDIALibs). Skyline was able to read .csv and check the file for errors but the process ended up with an error message (please see the attachment). I have Skyline 22.2.0.312 running on 64-bit Win10 Pro 128 GB RAM. Would you have any suggestions?

Rita

Hi Skyline Team,

I am working on project in which inactive protein undergoes PTM (carboxylation) and leads to active protein (carboxylation at gamma position of glutamate (Glu) residues). I am not getting good response in positive ionization mode and I want to try in negative ionization mode.

Is it possible to have the negative transitions (and precursor) of a peptide in skyline? if I generate data in negative mode, Is skyline able to read that data?

Kindly suggest.

Regards
Dilip

Skyline (64 bit) 22.2.0.255 (c99206313)

When using the "Import DDA Peptide Search" workflow Skyline hangs on "Running percolator..." after the search has successfully finished. I have to manually end the "percolator.exe" task in order to make the software respond again.

The issue seems related to the FASTA file (attached; downloaded from NCBI; sequence is correct). If I manually remove line 11 and 12 everything works just fine. It is not the length, as everything works fine as well if I replace line 11 and 12 with A's.

Why does the percolator task hang when using a specific FASTA file? I don't see anything wrong with the file, and obviously want to be able to use the full sequence for my search.

Thanks,
-Remco

Hi,

As i cannot use the Prosit server on the Skyline software on Trust PC, i thought i could access it through my Laptop, however i am unable to do this on my laptop, is this due it being an Apple IOS and not on Windows? is there a way to overcome this?

Kind regards.

Hi,

I am using skyline-daily in molecule mode and using the built in feature to build a spectral library from a .ssl file and mgf files.

In mgf files you may now explicitly denote the product ion charge for measured peaks in individual scans. I am wondering why when I build the spectral library and add the molecules with transitions to the target space that the product ions are all assumed to have 1+ charge.

Thank you

Dear Skyline Team,

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

Regards,
Arttu

Hi,
I have a problem with iRT alignment with my gas-phase fractionation-SWATH-runs. As I am doing GPF for the SWATH-analyses some of the iRT peptides (Pierce C18) are fragmented only in fraction while others are fragmented in fraction 2 and so on, but none of the peptides are fragmented in the different gas-phase-fractions. I have full-MS-spectra for the entire precursor region in every run, though. Is there a possibility to align the runs according to the precursor signals or do I have to fragment each peptide in each run?
Many thanks!
Joerg

Hi All,

I am new to skyline. I am doing a research project in measuring a particular hormone. Is there a 101 document on how to use the Software, I have been through the tutorial (targeted method editing) but unable to replicate it for my project. We have the water Xevo. I wanted to do an in-silico for my peptide but dont know how to conduct it. Thank you for any support.

Kind regards,

Hello Skyline Team,

I ran 34 samples on Maxquant and tried to import the msms file on Skyline. However, Skyline only continued with 2 samples and 17 replicates of each. I attach a screenshort of the import and the msms file. What did I do wrong?

Thank you,
Yael

Dear Skyline Team,

Thank you for your continued development of an excellent software package.

I am having trouble importing scheduled PRM data from a timsTOF Pro instrument. When I look at the results in Compass DataAnalysis, it appears that the method worked as expected - MS1 signals for the expected isotope labeled peptides are observed and MS/MS spectra are acquired at the retention times of interest. However, when importing into Skyline Daily (20.1.1.32) no precursor and no product ions are observed.

I've successfully imported other PRM runs from the instrument. Only this scheduled method is causing problems. Any insight you could provide would be appreciated. Thanks.

Robert

Hello,
I have just updated Skyline to the most recent version and am getting the "Server Unavailable" error with Prosit (attached image), as are my colleagues so I don't think it's a problem my end with connection etc. Could you advise?

Best wishes,
Colleen