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

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?

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

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 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

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.

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?

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 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

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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

Hello,

I had an idea for an enhancement request. There is a workaround involving highlighting potentially 100's of lines and deleting them, it just isn't as nice. I created a targeted assay in a two-proteome mixture, and before performing a dilution curve that would take a few days, wanted to confirm with a quick experiment that the peptides chosen were from one proteome and not the other. Two replicates were injected and imported at each of two concentrations, and a Group comparison performed between the concentrations. The sorted Bar plot does a good job to show which peptides do not exhibit the expected fold change. Using Refine / Advanced / Group Comparison and entering a Fold change value appears to be useful in the case where one is interested in peptides that change by more than some ratio, up or down. But in this case, I am interested in accepting peptides with a range of acceptable fold change values, for example between 0.5 and 1.5, and rejecting the others. Does that make sense? Something you could add to your queue of new features?

A related feature request for these types of two-proteome experiments is the ability to filter out peptides that are homologous between organisms. This comes up also when using a carrier proteome: one wants to make sure not to pick peptides that could possibly exist in the carrier proteome. One could imagine a filter tool that allows to enter a path to another fasta file, and to filter out any peptides in common between the current document fasta and that one.

Thanks
Philip

Hi,

I would like to verify an option of cross linking between two peptides:

ATAQDNPKSATEQSGTGI (with BS3 linked on K) and SYCR (with BS3 linked on S which is the amino-term)

Is it possible in the panel modify, clicking on the sequence of one of the two peptide, insert two different site of modification for the two peptides?

or is it possible to insert my modification with K as a position and also C term, in the same modification? I know that is possible but then the program search for K only in the position C term and in the peptide ATAQDNPKSATEQSGTGI it's not on the c term

many thanks

Hello,

I am trying to exclude all redundant transitions in a DIA assay library from quantitation. I was able to export all transitions and manually determine which ones were identical with others based on their molecule list name, precursor m/z, precursor charge, product m/z, and fragment ion.

I tried manually deleting all transitions in my DIA assay library and reimporting the "mixed transition list" containing only the non-redundant transitions, but this didn't work. Is there a better way to exclude the redundant transitions from quantitation?

Thank you so much!

Hello,

I am working with skyline-daily in molecule mode and trying to understand an error in my spectral library build.

I have used Molecule settings -> Library -> Build... to attempt a spectral library build from empirical data from an .ssl file. I have constructed multiple ms2 data files from multiple DDA experiments and referenced each in the .ssl file it by the "file" and "scan" columns.

After inspection in the Spectral Library Explorer, for each molecule only the first spectra from the first file for each will display in the viewer. When I change file to another scan I am met with the error "Failure loading spectrum. Library may be corrupted." When I explore Library Details many of the Files have 0 matching Spectra even though I have found the molecules' scans in the .ms2 file.

Is there a setting I am not using correctly?

The docker image of Skyline is a bit behind - can anyone help me find previous releases for windows? See below.

Thanks! Nathaniel

"
The document format version 22.2 is newer than the version 22.13 supported by Skyline-daily (64-bit : automated build) 22.1.9.244 (804ca65).
Exiting...
"

Hello,

I'm trying to build a library of Yeast peptides labeled with 11-Plex TMT, using Eclipse DDA MS2. I've tried doing this in several ways:

1. Search the data in Proteome Discoverer, import the .pdResult files. There are 17k peptides with q-value < 0.01, however I'm not able to successfully import the results. We get the old, "Importing the FASTA did not create any protein targets".

2. Search the data in Skyline. I tried using MSAmanda, and MS-GF+. There appears to be an error parsing the TMT modification string. I'm pretty sure that this is the string that Skyline supplied for that modification.

Based on this thread I made sure that there were a number of good targets in PD. I've had no trouble importing standard, unlabeled HeLa results from PD or searching them in Skyline, so it seems like the TMT modifications must be the issue. https://skyline.ms/announcements/home/support/thread.view?entityId=5a28316e-8430-1035-b2a7-e465a393b02f&_docid=thread%3A5a28316e-8430-1035-b2a7-e465a393b02f

I've uploaded the pdResult files, raw files, and Skyline files to the file sharing server with the name ForSkyline.zip. Could you have a look and see if the problem is on my end, or the Skyline end?

Thanks
Philip

Good Morning, I hope this email finds you well.

I am using Skyline to interpret ThermoFisher Exploris data. My method performs SIM analysis followed with MS2 analysis if about a certain ion threshold. What I have noticed is that Skyline extrapolates data for the entire LC run time. Therefore, if the ddMS2 data was only collected for 30 seconds and the run is 5 minutes, it will extrapolate the baseline from 0 - 5 minutes. See attached picture. This causes a problem when attempting integration. Is there any way to prevent skyline from doing this?

I've also attached what ThermoFisher's qualitative analysis software shows for the same MS2 experiments.

Thanks again!

Johnny

Hi Skyline team,

I'd like to ask, whether the notation for heavy atoms has changed recently?
I used to enter deuterium into transition lists as H' (one deuterium being H'1), which worked fine for Skyline (64-bit) 21.1.0.278.
I can still use this older Skyline version without problems, but the newest one shows an error when trying to load such transition lists.
The error message I get is:

Failed reading the file C:... .csv. The given key was not present in the dictionary.

The same transition list without any H' is loading fine in the latest Skyline version.
Is there a way I can generate transition lists with deuterium containing precursors and products that will work in all versions of Skyline?

Thanks,

Phil

Hello Skyline Team,

I have a set of TimsTOF pro2 files (.d) that Skyline sees them as normal folders rather than spectra files. These data are recorded as MS1 or MRM mode without TIMS enabled. Is there a way to force Skyline to read them?

MSConvert also see them as normal folder, so I cannot convert them manually.

I can upload a few of them if that will help.

Thanks
Ho-Tak

We have an issue with Skyline 22.2.0.312 which looks like a potential bug. Maybe you could have a look at it, here is the description:

• We set up a scheduled targeted method for our lipidomics measurements with (among others) a retention time window from 13.9 to 15.9 min.
• An example is shown in the attached Image Analyst-correct-retention-time.png. You can see that both chromatograms are aligned.
• When importing the data (minimal example in: Test.zip), we get a shift in retention times as visible in Image Skyline-incorrect-retention-time.png.
• However, this shift should not be there, it should be aligned.

Thank you for your support.

Dear Skyline team,

I am trying to setup a PRM method on our new Exploris 240 and to analyze the data using Skyline. There is no "PRM" mode per se on the Exploris and the engineers have told me to use "product ion scan" mode which should result in equivalent data. The resulting raw file, opened in FreeStyle, contains no ms1 spectra, but ms2 spectra continuously acquired for the precursor mass added in the inclusion list during the time window specified. I am new to PRM so I have no comparison but it's exactly what I expected. I imported the file into my Skyline project and it says "Chromatogram information unavailable". I figured my Skyline document might have wrong settings, so I manually added a BSA peptide and fragment masses from the Skyline PRM tutorial and imported one of the associated Agilent QTOF data files (3-BSA-1fmol). The file imports without problem and the precursor and fragment ions are displayed.

Another user had submitted a PRM&Exploris question in 2020 so I assumed it should be possible to analyze data from that instrument with Skyline, generally. However, not sure if relevant, I noticed that user used a different "MS/MS filtering" - "Acquisition method" --> "Targeted (obsolete)" which is not available in my Skyline (see his post).

Some info that might be relevant:
I am working with small molecules, in particular I am targeting derivatized fatty acids. I am transferring info from a MRM method which is why I only have 2 product ions at this point. Data was acquired in positive mode. I'm using Skyline 64-bit 22.2.0.255, Windows 10. I will upload the skyline document with the name "2022-09-29_NPH-OnlyButyricAcid.sky".

+Is there an issue with the way I acquired the data?
+Is the data ok but Skyline is confused by the derivatization adduct?
+Is there a mistake in the way I setup the transitions?

Any help is appreciated! Thanks!

Hi skyline team,
I used skyline to extract the interested peptide precursors from the FAIMS-DIA files. The interested peptide precursors were almost extracted from files acquired from Q Exactive HF instruments or TripleTOF instruments. But there are some weird things about those peptide precursors extraction from FAIMS-DIA files. 1. Some of the peptide precursors show good MS1 extraction but without fragment ions. 2. Some of the peptide precursors could extract fragment ions from A files but could not extract from B files. The A and B files are technical replicates.
I do not know whether there are some issues with skyline settings, so I upload the skyline documents if you can check it would be better. Many thanks.

I have uploaded the file to the link. http://skyline.ms/files.url
Best regards,
Winnie

Hello,

I am trying to build a library from my spectra files that contain peptides digested with Pepsin, grown in SILAC media, and that have phosphorylation on S/T/Y (PTM search).
I get mzID and mzML files from MetaMorpheus but I get an error that No spectra were found for the new library.
I tried changing files to lowercase letters mzid and mzml both or each and it does not work.

I did read another request that said PTM searches can cause library build issues and wanted to reach out to you if this might be another issues with PTM or SILAC.

Thank You,
Gabriela

Hi, I want to use Protter tool to show the unique peptide selected in skyline in the protein structure (Skyline--Tools--Protter)

But I am getting error, attached herewith.

My operation system is windows 10 enterprise with 11th Gen Intel ® Core (TM) i7-1165G7 @2.80GHz 1.69 GHz; RAM 16 GB, 64-bit operating system
Skyline (64-bit) 22.2.0.255 (c99206313)

Could you take a look at this problem? Thank you very much!

Regards
Dilip

Dear Developers and all researchers,

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

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

I would be very appreciated for your help!

Best Regards,
Peter

Dear Skyline time,

I am trying to download encyclopedia-1.12.31 but I get an error "SSL_ERROR_RX_RECORD_TOO_LONG". could you please check your ssl record?
Thank you
Ebru

Dear Skyline Team,

I am trying to use Protter from tool store. To do so, I need to add "Protter" in external tool option.

I am getting error, which is attached for your reference.

Kindly suggest solution.

Regards
Dilip

Hello, I am new to skyline so I am still learning new things. I have followed MS1filtering tutorial to import my short listed protein target into skyline along with 8 orbitrap pdresult files. After cleaning them now I need to export out the protein-peptide list from skyline to hard drive for future use. Is there any supporting tutorial available?

thanks,
kmsd

Hi All, does anyone have the latest list of CIRT peptides skyline looks for? Preferably in a bilb library with predicted ion mobilities ? Just curios , I looked and couldn't find it

Cheers

Brett

Hi,

I am having difficulty exporting a scheduled method for collision energy optimisation.

My colleagues ran a panel of peptides and I have the RT for these peptides, however I don't have the .raw files. I have pasted the results into the iRT calculator.

I am trying to run these same peptides using the same chromatography on a different platform, so I need to optimise the CE and as I am sample limited would like to do it in as few injections as possible. When I try to export a scheduled method optimising CE I get the error "Retention time predictor is unable to autocalculate a regression". Is it possible to export a method for peptides that you've used to train the iRT calculator?

Many thanks
Matt

Hi Support team,

Could I export dot p value of MS2 spectra comparison between MIDAS vs library?

thanks,
Heyi

Dear Skyline team,

The samples run from Our Q Eaxctive had a hard time recording the UV data, so we could not retrieve the UV data after the sequence run. So when I am trying to import the raw file into skyline, this error message pops out "Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type". (The full error message can be found below)

Do you have any suggestions to recover the data from my raw file, or can skyline ignores this UV signal but import my MS data?

Full error message:
Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type ---> System.Exception: [ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type
at pwiz.XX.msdata.ChromatogramList.chromatogram(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetQcTraces() in C:\proj\skyline_22_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 846
at pwiz.Skyline.Model.Results.GlobalChromatogramExtractor..ctor(MsDataFileImpl dataFile) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 129
at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 87
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1306
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 244

Hi, skyline,

I extracted chromotogram of targeted peptide precursors from FAIMS-DIA files by skyline, the xic show rugged profiling. The screenshot is attached. I was wondering there are issue on ion moblity library because i did not set ion mobility sepctral library. The sepctral library was imported by the following strategy. File>import>assay library. I found the column named precursor ion mobility in this spectral library is NA. The spectral library was build by Fragpipe with files acquired by FAIMS-DDA. I first communicated with Fragpipe team, they told me it is normal the column named precursor ion mobility is NA, because they this the FAIMS is low resolution device for ion moblity. So they set NA of precursor ion mobility for the library genetated by FAIMS-DDA.

So, did you have suggestion on the rugged xic and how to spectral library generated by FAIMS-DDA

Everytime I try to conncet to the prosit server, it says "server unavailable".
I have updated my software to Skyline (64-bit) 22.2.0.255 (c99206313) but the same things happens.

Any way to fix this?

Dear Skyline support team:

When launching Skyline-Daily or even trying to run the program, we are getting the same error that we have attached to this trouble ticket. We first thought that RAM was problematic as another product had taken so much, but even when the other program is not running and the server is rebooted to clear resident memory and caches, the error occurs. It states that the system did not have enough resources to complete the operation. The server is pretty beefy with 128GB of RAM, plus 2 Xeon 3.2GHz CPUs in an enterprise level Dell server running Windows 2016. The drives all have plenty of disk space (500GB or more) available, plus the NAS drives not only have plenty of free space, but the Network load does not reach above 10% utilization on the 1TB network interface. So not sure of the resources needed, other than we thought that the browser level was still using the old deprecated IE 11 ( we just installed Edge, but IE 11 is still on the system) and maybe the .NET needs to be updated (it is v4.8). So rather than guess at the issue, we are asking if you could read the error and determine the possible issue at hand.

Thank you for any help or assistance on this matter, in advance.

Sincerely,

Henry Chandler

Hello,

I hope you're well.

Is it possible to install and use an earlier version of MSstats in Skyline? I currently have the most recent version installed, but would like to use an earlier version to compare my data to an analysis I had done a couple of years ago

Kind regards,
Susannah

Hi, good afternoon

Is there a way of getting the peak intensity and background signal amplitude to determine the LOD and LOQ (being LOD 3x and LOQ 10x)? And is this the best way to determine these parameters using DIA MRM-like data?

Many thanks,

Ricardo Gomes

Hi Skyline support team,

I have some data acquired from Agilent 6560 IM-QTOF. I noticed that for some analytes, the IM drift times of the fragments are lower than their precursors, leading to unsuccessful alignment. In a literature, I found a function called "HighEnergyDTOffset", which could set in skyline transition list. This function indicates how much the fragment drift times could be lower than their precursor. I added the "HighEnergyDTOffset" column to my transition lists of different batches of data. Something interesting is that this function only works for certain transition lists. In other transition lists I loaded, the IM drift time window applied in MS/MS spectra was the same as the precursor, not having any drift time offset at all. I wonder if there is a guidance on how to set "HighEnergyDTOffset" correctly, or if there is any restriction on how the data is acquired by MS (AIF, PRM, etc.) in order to enable this "HighEnergyDTOffset" function.

Thanks,
Anne

Dear Skyline Team:

We have 5 replicates of predigested BSA injected with fixed window SWATH on our Sciex a 7600 ZenoTOF. The BSA Fasta was imported and the 5 .wiff data files were imported. I have tried a several different settings in the Transitions form but still do not see the fragments.

I have confirmed by viewing the data in OS Explorer that there is response for at least some of the fragments.

Thanks,
John

Data files available to upload if helpful.

I am using MacBook. I can’t able to use the software from my MacBook. Is there any solution?

Hello, I have a list of peptides that I want to monitor against lumos LFQ (DDA) raw data file. I know there are lot of good articles and Ques/Answer available in skyline but I am new in this platform and need some guidance. Can someone direct me how to do this using skyline weblinks/tutorials etc?

Thanks
kmsd

Hi all,

I have a problem with my transitions. I work with GC-(EI+)-HRMS and LC-(ESI+/-)-HRMS data acquired in full-scan mode and I use the small molecule part of Skyline.

I created my excel sheat with 1 quantifier ion and 1 or 2 confirmation ions, the quantifier in the upper line of my sheat. When I imported this sheat with copy/paste, I selected all ions as precursor (it didn't work with precursor and product ions, no data in the chromatograms). Next, in the left side, for each molecule, my precursors were rearranged from the smaller mass to the highter.
My problem is that for all the molecules, the quantifier ion is automatically the first precursor ion, the smaller one, even if it's not the best choice. I tried to select the "Quantitative" choice only for my quantifier ion but, when it's not the smaller ion, the message "Error: All of the external standards are missing one or more peaks" appears in the calibration curve window. I can only quantify with the smaller ion...

I tried to change the monoisotopic and average masses of my molecules, which are always the mass of the smallest ion, to my quantifier ion mass but Skyline automatically adds the difference between the former masses...

How can I choose my quantification ion and confirmation ion in another way?

Thanks!

I've been running samples on a Bruker Scimax MRMS. When I import the data I get this error message:

At 3:47 PM:
Failed importing results file 'D:\Users\Data\2020-11-24-Aminoacid mix\Amino acid_1_2_1_123.d'.
unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'D:\Users\Data\2020-11-24-Aminoacid mix\Amino acid_1_2_1_123.d'.
unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7 ---> System.Exception: unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7
at pwiz.CLI.msdata.ReaderList.read(String filename, MSData result, Int32 runIndex, ReaderConfig config)
at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 194
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188
--- End of inner exception stack trace ---

Do you have an idea what the issue could be here?

Cheers,
Dennis

Good morning

I have a question...How to I know each peptide and proteins are considered identified and present in a sample using DIA SWATH data?

I am doing an untargeted global analysis of all possible proteins in the library. Is the library match peptide score?

Many Thanks,

Ricardo Gomes

Hi,

I was at the training on October 10 and 11 this week.Now I am trying to use a Skyline for the lab's needs. I was wondering if you could, please, give me some directions on a couple of problems. I was able to create a transition list for the peptide we are studying. However I would like to export a method for the Q Exactive mass spectrometer. When I go to file-export-method, I do not see Q Exactive in the list "instrument type". Also, when I tried to export one of them, the program is asking for a template. I would appreciate if you could direct me how the template should be for the Q Ex method and how to find the Q Ex in the drop down list. I tried to export the transition list method from a Fusion mass spectrometer since it is showing in the list of instrument just to practice, but I get an error message that I am attaching to this request.

Thank you very much
Rita

I am working on developing PRM-PASEF methods. I have encountered some issues with importing data and have some questions about the mobility filtering/method exporting.

Importing: Importing the same raw file with the same peptide/transition settings into separate skyline files produces A) peak area, RT, and spectral data and confident identifications or B) no peak areas or RT data displayed, unconfident identifications. I cannot figure out why this is happening.

Mobility filtering: The filtering window is frequently offset from observed maximum - is there a way to mediate this in skyline? When exporting a bruker method, how does skyline incorporate the mobility filtering? Is it necessary to refilter the data with the same library used in the method/data acquisition or should I use "Use Data" and refilter for a given dataset?

Attached is a powerpoint with screen grabs demonstrating the above challenges, as well as descriptions. The slides are segmented into "Issue 1" and "Issue 2".

Any help will be greatly appreciated - thank you!!

Hi

I was trying to look for a custom fragment ion from ECD and ETDhcd spectra for the isoAspartate residue of the peptide that gives off either c+57 or z-57 ions. I am able to add c and z ions in transition settings but unable to add any other loss/ gain to it. One workaround I had to this problem was to use a peptide modification of 57 gain/ loss and then select the c ions with a 57 loss or z ions with 57 gain. So is there any other simpler way to do this custom fragment ion search?

Thanks
Ankit

Hi,

I was wondering if you could, please, give me some directions on a couple of problems. I was able to create a transition list for the peptide we are studying. However I would like to export a method for the Q Executive mass spectrometer. When I go to file-export-method, I do not see Q executive in the list "instrument type". Also, when I tried to export one of them, the program is asking for a template. I would appreciate if you could direct me where to get a template for the Q Ex method and how to find the Q Ex in the drop down list.

Thank you
Rita

I noticed the collision energy calculated by Skyline Daily for Thermo Altis were dramatically changed in recent updates. They are much higher than the previous versions. Would it be possible to have the conversion of CE between current version and previous version? The collision energy of previous versions work better for me on my Altis. Thank you!

Hi,

I have attended the introductory training on October 10 and 11 but I am not sure how to apply the training to my immediate problem. My boss wants me to establish MRM conditions for a QExecutive mass spectrometer to check on a synthetic peptide. I will attach the slides I have. The peptide has modifications as oxidation, acetylation and has an amide attached to the n-terminal. It can be analyzed as heavy versus light. I was wondering which tutorial I should follow to create the assay. Any advice would be greatly appreciated.

Thank you
Rita

Dear Skyline team, I am working on the automation for Skyline. I am wondering is there any command lines or ways to set one peptide as the global standard?

Regards,

Jianqiao Shen

Hi all,

I could have sworn that a recent version of Skyline-daily was displaying at least one of the PSMs for one of the charge states if the selection was focused on the peptide. But on Skyline-daily 22.2.1.278 this useful feature seems to be gone (attached pic).

Would it be possible to get it back? :)
Sincerely,
Juan C.

Hello Skyline team,
I have been using Skyline to do the quantification for small molecule and recently we found a ghost peak integration issue. In Skyline, you can see that the software integrate a peak at m/z 1055.5057, but when I plot m/z 1055 in Freestyle (Thermo software), no peak was found. I really want to know if there is a way to fix this as we use Skyline heavily for quantification. If the peak integration was not good, then the data validation will be very difficult. I have attached the skyline file and two graphs demonstrating the peak
Thank you

Dear Skyline team,

I am trying to import 2 Waters Xevo G2 raw files into skyline. The skyline document has only 1 protein and the raw files being imported were collected using MSe method. I began importing the data and didn't see any chromatograms exported. After a few mins, the program skyline closed down by itself.

I am using Sklyine (64 bit) 22.0.255 (C99206313) on Windows 11 Pro. The processor is an 12th Gen Intel(R) Core(TM) i7-12700H 2.30 GHz and 40 Gb of Ram.

Many thanks,

Juan

I am doing quantification on amino acids so I am using the Molecules layout for my analysis. I read previous posts about using Concentration Multiplier in the Peptides layout. Is this function available in Molecules and, if not, will it be added in the near future?

Hi,

I learned how to use Skyline on the job for a peptide absolute quantification project. I read a lot of the tutorials, some of the posts I found here from different google searches, and looked at the methods sections of diverse papers. I think what I did makes sense I just want to make sure I didn't make a mistake because of some blindspots, so any help or advice is welcome.

We want to quantify some peptides from a hydrolysate (not for protein quantification but just for peptide quantification so we didn't have the choice of which peptides would be best). We have heavy variants of each of them. We built an internal calibration curve for each peptide using varying concentrations of heavy peptides. The first series of runs (PRM on a QE) uses a wide scale of concentrations to find the approximate concentration of light peptide, then another series of runs to adjust the calibration curve for each peptide to quantify and then replicated that again.

I used Skyline to analyze these data. I used the retention time of these peptides in previous DDA runs using the same LC parameters and the presence of peaks for the heavy and light products to select the correct peaks. Removed the product ions interfering with the peak. I then extracted the ratio of heavy/light for each analytical replicate and for each concentration. I used these ratios to make a calibration curve (linear regression) and I looked for the ratio heavy / light == 1 to find out the concentration of light. I had to go outside Skyline for this step as all my samples are marked as Standard, no sample being just Unknown, but being both at once, and as far as I know I cannot ask Skyline to look for a ratio of 1.

I hope someone can tell me if I am completely off the tracks or not.

Thank you

Jean

Dear Skyline team,

I hope you're doing well. I'm loving the new Skyline-daily update featuring the pressure traces, but currently the chromatogram visual alone makes automatic QC difficult. Could you please add a parameter in the report section that provides the maximum height observed in the pressure trace? I imagine it would be similar to the "Total Ion Current Area" parameter except only returning the maximum pressure. It would also need to ignore the automatic peak picking of the pressure trace since the maximum height would be the maximum y-axis value for the entire retention time range.

I've attached a mock-up of what I think it should look like.

Cheers,
Chris

Dear Brandon and Skyline Team,

I would have a question regarding S/N, is it somehow possible to access it in Skyline?
We currently run an instrument benchmarking experiment (small molecules, neg and pos mode, high resolution) and would like to compare also the signal-to-noise values for several metabolites in a fast and easy way (if possible). Would you have any recommendations for us?

Thank you for your help!

All the best,
Kathi

Hi all,

I've seen this requested a few times, but is there any update on the ability to export the Prosit dotp scores for each peptide in a mirrored database match-based spectral library?

Best,

chris

Hi all,

Simple question, but is there a way to highlight which peptides are matching to multiple proteins? I have a proteogenomics type of effort going on and there is utiility in me finding protein evidence for multiple isoforms that might all individually have the same tryptic peptide, but that tryptic peptide may only map to that novel isoform group and nothing else, which would be helpful in and of itself for me as I want to keep all of the isoforms in there.

Best,

chris