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

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

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

Thank you for any help you can provide!

Sincerely,
Morgan Mann

Hello,

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

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

How to fix that issue please?
Thanks
Nihel

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

Hi team,

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

Cheers

Will

Dear Skyline team,

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

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

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

Thank you in advance!
Shimin

Dear Team,

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

Thanks in advance.

Kind regards
Vanya K N

Hi Skyline team.

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

Thank you

Stoyan

Dear Team,

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

Thanks in advance.

Kind regards
Vanya K N

Dear Team,

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

Thanks in advance.

Kind regards
Vanya K N

Dear Sir,

,sky format is propriety of MacCoss lab ? If yes, how do we convert raw file to this format ? Is .sky format can be used for AI/ML ?

Sachin

When using iRT for the replicate comparison plot, I get different a mixture of RT and iRT plots in the chromatogram window for different samples. I would like both the RT comparison plot and the chromatogram to show the same thing for all samples, in this case iRT instead of RT on the x-axis of the chromatogram.

Hi,

I see a signal for my selected fragment ions for peptides but somehow its transparent. I don´t quite understand the reason why and can it be used for atleast saying that this peptide was trigerred with atleast fragment ions

Dear Sykline-Team and Community,

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

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

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

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

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

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

Thanks in advance for you help!
Jonas

Hi,

How can I define my data as standards, blank, and unknowns?

Also, how can I calculate LOD-LOQ using 10 blank data?

Sincerely

Hello, how to use skyline to obtain linear curve for peptide (PRM data)? is there any SOP? Thanks~

Hello,

Our group had previously been using a custom transition list for glycomics data analysis that included both protonated and sodiated adducts. However, recently when trying to use the same list it does not seem to properly extract the sodiated XICs, even on old data files where it had worked fine before. Based on the timeline of this problem, it's possible that this began with Skyline 24. Is there a way I can install Skyline 23 to troubleshoot? I can't seem to find any archive of older releases.

I have attached the transition list in .csv format here. I have only tested this on Agilent .d files.

Best,
Trevor

There are many transitions listed for one precursor. I just want to keep the highest 3 transitions when I export to be a SRM method. How can I do that?

I tried to predict mz for certain compounds using publically available isotope exact masses and my calculations agree with the raw data much worse than skyline calculations (I interpret this as skyline using more accurate exact element massess). Is there a way I could get the element masses out of skyline and also understand where the developers took them from?

Hello,

I have built a spectra library in Skyline and now would like to use this in DIA-NN. Since the BiblioSpec output is an SQLite3 file and DIA-NN needs a .tsv as specified here: https://github.com/vdemichev/DiaNN?tab=readme-ov-file#spectral-library-formats.

I have found this program which might be able to do the trick, but wondering if I can have any help with this? https://gist.github.com/icedraco/78d9879f2171ecebcbfb

Thank you very much for your time,
Ronnie

Hello,

I am trying to build a spectral library using timsTOF data that was searched using Peaks Studio. I exported the db.pep.xml file from Peaks.

I am receiving an error message that reads ERROR index 10095 out of range in mzXML file ".\081023 C1 SDS 1 - 45 min_Slot2-1_1_1119.d.mzxml. I know this type of error was posted on the support forum previously and mentioned "this type of error was due to pep.xml file claims to have a spectrum match #, but there aren't that many spectra in the mzXML file."

I'm not quite sure how to fix this issue, but I would appreciate any feedback.

I have uploaded a folder with the error message, raw, mzxml, and pep.xml file to the file sharing folder for skyline support titled "Skyline Issue timsTOF" .

Thanks,
Ashley

Hi Team!

I updated to Skyline daily and when I use the new bilinear turning point LOD calculation, almost everything has LOD of the highest point on the curve. I have heard from a few others that they are seeing the same issue. Do we need to somehow update the Skyline document for these calculations to work?

Thanks,

Lilian

I am trying to run Skyline from the command line inside of an apptainer container as part of a Nextflow workflow.

There are a couple of steps in the workflow which are failing with the error:

Error: Failed saving to the user configuration file.
Failed to save settings: A configuration file cannot be created for the requested Configuration object.

Specifically, the steps that are causing the error are when I try to run protein parsimony in Skyline and when a new report template is added to a document.

If I set the --verbose-errors flag, this is the stack trace:

Error: Failed saving to the user configuration file.
System.Configuration.ConfigurationErrorsException: Failed to save settings: A configuration file cannot be created for the requested Configuration object. ---> System.Configuration.ConfigurationErrorsException: A configuration file cannot be created for the requested Configuration object.
   at System.Configuration.MgmtConfigurationRecord.SaveAs(String filename, ConfigurationSaveMode saveMode, Boolean forceUpdateAll)
   at System.Configuration.ClientSettingsStore.WriteSettings(String sectionName, Boolean isRoaming, IDictionary newSettings)
   --- End of inner exception stack trace ---
   at System.Configuration.ClientSettingsStore.WriteSettings(String sectionName, Boolean isRoaming, IDictionary newSettings)
   at System.Configuration.LocalFileSettingsProvider.SetPropertyValues(SettingsContext context, SettingsPropertyValueCollection values)
   at System.Configuration.SettingsBase.SaveCore()
   at System.Configuration.SettingsBase.Save()
   at System.Configuration.ApplicationSettingsBase.Save()
   at pwiz.Skyline.CommandLine.<>c__DisplayClass136_0.<HandleExceptions>b__0() in Z:\pwiz\pwiz_tools\Skyline\CommandLine.cs:line 4438
   at pwiz.Skyline.CommandLine.HandleExceptions[T](CommandArgs commandArgs, Func`1 func, Action`1 outputFunc) in Z:\pwiz\pwiz_tools\Skyline\CommandLine.cs:line 4451

It looks like Skyline is trying to save settings to a config file which does not exist or can not be written to because of the user's permissions in apptainer. I understand why saving previously used settings makes sense in the GUI, but I am not sure why this would be useful or desirable when running Skyline from the command line. It is especially pointless in a container where the user settings do not persist.

I have attached a minimal set of files to reproduce the error I am getting with the commands:

unzip minimal_example.zip && cd minimal_example
apptainer exec --no-home -B "$(pwd)" 'docker://quay.io/protio/pwiz-skyline-i-agree-to-the-vendor-licenses:3.0.24172-63d00b1' wine SkylineCmd --in=final_minimized_annotated.sky --report-add=replicate_quality.skyr

Is it possible to modify Skyline so the user settings are not saved when running the command line version of Skyline, or at least print a warning instead of raising an error when the file can not be written to?

Thanks,

Aaron

Hi!

I am trying to write a tool that plots cal curves. So I need a report that has information on intensity for each replicate, as well as cal curve information (slope, LOD, LOQ, etc). I can write this all to one report, but it takes forever. If I remember correctly, it recalculates LOD and LOQ for every line in the table. I really only need that once per peptide, but I may have 12+ rows per peptide. Normally I just write two reports: one that has info on cal curve (one row per peptide), and one that has a row for each point on the cal curve. Is there a way to get an external tool to use these two reports? Or would you suggest another way to do this? It can take a very long time to write the report as is.

Another small thing, I am unable to get Skyline to recognize the path to R.exe. The only reason I can think it's doing this is because there is a space in the path (C:\Program Files\R\R-4.3.1\bin\x64\R.exe). When I run in command line I normally have to add quotation marks around the path name, but Skyline still can't find path when I manually specify pathname, and the macro also has space in name and it gives the same error. Is there a way around this besides moving R installation directory?

Thanks,

Lilian

Hi,

I tried to run the EncyclopeDIA workflow through Skyline using the latest Skyline daily version. I got an error in the step of building spectral library using koina. Attached please find a screenshot of the error I got.

Thanks,
Bo

Hi!

I am trying to optimize my PRM method on Exploris 120 coupled to Vanquish Neo.

I am getting pointed transition peaks on skyline for few peptides for some LC-MS methods. I have attached the pics. Could you explain the reason for pointed peaks and can it be considered as good identification?

I have attached 5 pics. However, I know Pic4 right side seems good. But I am unsure what causing pointed peaks in remaining pics.

VM

Hi there,

I am trying to prepare some EIC plots with multiple peptides selected at the same time. I noticed that when this is done, the summed peptide EIC is disregarding if a transition was labelled as non-quantitative and shows the EIC considering all.

Is it possible to exclude the non-quantitative transitions for the peptide EIC or should I delete them for this purpose?

Thanks in advance!
Sincerely,
Juan C.

Dear Skyline team,

We have found a response regarding query on Precursor exclusion m/z window. However, we were not able to understand importance of "Precursor exclusion m/z window" instead the option to use "DIA precursor exclusion window".

Thanks in advance

Vanya

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

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

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

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

Thanks as always,
Sam

Hi Skyline users, a new user here.

I was able to follow the tutorial on "Absolute Quantification.' Now, I'm interested in learning about TMT-based quantitative proteomics. My questions are the following: 1) Is there a skyline tutorial on TMT quantification? 2) If the TMT-labeling is applied only to the samples but not the heavy standard target peptide (used to generate calibration curve), what is the correct way to quantify the labeled samples?

Thank you so much!

Hi Skyline team,

I am trying to run an EncyclopeDIA search through Skyline (v.Skyline (64-bit) 24.1.0.199 (6a0775ef83)) and it fails almost immediately when trying to submit my FASTA file to Koina to generate a Prosit library. A picture of the Skyline error is attached as a PNG. Any advice you have is much appreciated!

-Eric

Dear Skyline Team,

I recently tested the script MALDISkyLink from C. Ashwood for MALDI support and found that it worked quite well. It essentially duplicates the spectrum and brackets it with background values to add a "fake" time value, allowing Skyline to calculate areas. Could you consider implementing something similar directly into Skyline for the import of MALDI data?

The limitation of this script (in my hands) is that it can only convert MALDI spectra one by one, whereas MALDI users usually have thousands of spots to analyze.

It would be a great addition, as Skyline is a fantastic tool for processing and curating large sample batches. MALDI-MS still has numerous applications in proteomics, glycomics, and the world of small molecules.

Thanks

Best regards

Hello Team Skyline,

I am currently trying to export CE optimizing methods for 2x Sciex Qtrap systems (6500+). Both systems got Skyline installed on their own individual PCs. When I try to export CE optimizing method using Skyline, I am getting two different errors from each Skyline (please find attached). I have fed in the same transition list, both have the same transition settings, and I selected the same export method settings. Any idea on how to fix this? FYI, I do not have any problem exporting them as transition lists.

Thanks a lot for your time.

Kind regards,
Alex

Hi there,

We are fairly new to Skyline and targeted MS methods in general. Our goal is to target certain peptides in a complex sample using a Brukers TimsTOF mass spectrometer.

We have compiled a bunch of DDA data which should be used for the creation of the spectral library. Whenever we look through the data in Skyline, the MS1 spectra of the identified peptides look fine. The corresponding MS2 spectra however, usually have very weird, angled shapes.

In the attached picture SADPNFLRF_MS2, you can see how the spectra for the fragments are triangular. Compared to this one, many other spectra are very messy, some of them making little sense at all (the chromatogram being just a straight horizontal line...)

Does anyone know what could be causing this and/or could share some tips with this kind of method development?

Thank you very much,

Julian

Hi Skyline Team,

We recently moved a TSQ Quantis to our lab from another lab in the company, and so I'm starting to work with it and noticed there are no native method export settings that seem to work for this instrument. We are able to work around it by using TSQ Ultra CE settings and using the File/Export Transition List for TSQ Altis, then importing the transition lists into the method editor. Obviously the native method export is an additional convenience. Are their settings I am missing or is there no plan to support this instrument in native method export?

Cheers

Will

Dear Skyline Team,

Could you please inform me on how to re-pin the target panel to the left side of my main skyline window? I accidentally unpinned it, causing it to merge with the other tabs displaying the peaks. Additionally, I would appreciate guidance on pinning other viewing options such as RT replicate comparison. Typically, I have them displayed on tabs or separate windows. Thank you!

Dear Skyline Team,

I hope you've had a great ASMS. I've been working on my reports, and noticed that when I import a transition list, only one form of note is available, "Note". However, when I try to export the same note from the document, there are four different note types (transition, precursor, molecule, molecule list). Skyline seems to default that "Note" in the transition list import is a "Transition Note", and this cannot be changed in the transition list import stage. This becomes problematic when exporting MS1 data with isotopes because only the monoisotopic line in the export features the imported note.

Could the transition list import feature please be updated to include all existing note forms? I would like to say that a note is a precursor note, ensuring the note is specified across different isotopes coming from the same precursor.

Cheers,
Chris

I am attempting to perform label-free quantitation on two proteins in human macrophage cells. I prepared the samples using the EasyPep™ MS Sample Prep Kits from Thermofisher (Pierce), from the required amount of cells and passed over the cleanup columns.
I then injected the sample onto an Agilent 6545XT QToF running DDA MSMS over a 50 minute reversed-phase gradient on a peptide mapping column (fairly standard proteomic methods). The TIC itself looks quite dense, so there should not be a lack of data for the algorithm to mine.
I am attempting to build a spectral library for the samples, but keep encountering the following error: ERROR: No spectra were found for the new library.
I was first trying to build the library using the FASTA sequences for only my proteins of interest, but just to double check I also ran the search against the human proteome and encountered the same issue. I tried lowering score cut-offs and loosening mass tolerances, but nothing has prevented this error from showing up.
I have attached the error message, are there other pieces of information I should upload to help figure out the problem?

Hi there,

I am processing my first set of data since I upgraded to 22.2.

I use the MPPreport external tool to export my DDA results, and the tool no longer works. I have attached a screenshot of the error string from the "immediate window".

Can this be repaired? I am trying to find a different way to export my results, and I tried to export report from the file menu, and it sort of works, only my protein groups are not rolled up together, and I am not sure if there is a way to fix that? Or just to fix the MPP report external tool?

Thanks.

Denina

Is there an available version of Skyline for MAC OS?

Hi,
I am trying to visualize my proteomics samples after running on Sciex 7500. The samples do get imported in the Skyline but I don't see any chromatogram of corresponding peptide transitions.

I reached out 08/13/2024 about difficulty filtering in msconvert to filter thermo ascend data by scan event. I beleive the issue is related to syntax, as the GUI MSconvert operates when scan filter is not used.

Hi,
is there a tab that we can add to the results grid so that we can manually assign a normalization factor for each replicate?
i.e. we know for each replicate/data file, different amount of total peptides were loaded, and we hope to assign a normalization factor for each of the data file such that the final quantification results will be normalized to total peptides.

Thanks!

Lorrain

I'm trying to use Skyline to replace Tracefinder in my workflow. I'm collecting HRMS on a Thermo Orbitrap in MS1 full scan mode. I've got a list of targeted analytes, and while Skyline in general works well, all the documentation I can find for small molecules recommends making manual adjustments to peak integrations, which is not feasible for the number of samples I need to process per day.

I haven't exhaustively read every document, so I'm hoping there is a resource I've just missed!

Hi,

I have a list of peptides that I am trying to quantify. I spiked in heavy standard and collected the data with FAIMS. if I don't use compensation voltage filter, everything looks fine. But if I select compensation voltage filter, the peak looks nice but in the report all ratios become N/A. Any idea what might have caused it?

Thanks,
Qing

Dear,

We are having some issues in submiting the skyline file to Panorama. We are receiving the error below. Could you please help us to troubleshoot this issue? Thank you very much. Best, Daniela

08 Aug 2024 05:17:48,369 INFO : Starting to run task 'org.labkey.targetedms.pipeline.TargetedMSImportTask' at location 'webserver-high-priority'
08 Aug 2024 05:17:48,370 INFO : Starting to import Skyline document from Corridas_PRM_Amostras_Injeçao1e2_184pepsquantificados_2024-08-08_09-14-59.sky.zip
08 Aug 2024 05:17:48,370 INFO : Expanding Peps_Mix_Saliva_PRM_Unscheduled_364peps_17fev23.blib
08 Aug 2024 05:17:50,589 INFO : Expanding PRM_Final_21_07_23.blib
08 Aug 2024 05:17:55,067 INFO : Expanding PRM_Final_21_07_23.redundant.blib
08 Aug 2024 05:18:16,718 ERROR: Import failed
java.lang.IllegalArgumentException: malformed input off : 26, length : 1
at java.base/java.lang.String.throwMalformed(String.java:1242)
at java.base/java.lang.String.decodeUTF8_UTF16(String.java:1198)
at java.base/java.lang.String.newStringUTF8NoRepl(String.java:732)
at java.base/java.lang.System$2.newStringUTF8NoRepl(System.java:2398)
at java.base/java.util.zip.ZipCoder$UTF8ZipCoder.toString(ZipCoder.java:199)
at java.base/java.util.zip.ZipCoder.toString(ZipCoder.java:66)
at java.base/java.util.zip.ZipInputStream.readLOC(ZipInputStream.java:302)
at java.base/java.util.zip.ZipInputStream.getNextEntry(ZipInputStream.java:124)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:106)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:82)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:69)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:64)
at org.labkey.targetedms.SkylineDocImporter.extractIfZip(SkylineDocImporter.java:1005)
at org.labkey.targetedms.SkylineDocImporter.importSkylineDoc(SkylineDocImporter.java:317)
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:249)
at org.labkey.targetedms.pipeline.TargetedMSImportTask.run(TargetedMSImportTask.java:63)
at org.labkey.api.pipeline.PipelineJob.runActiveTask(PipelineJob.java:833)
at org.labkey.api.pipeline.PipelineJob.run(PipelineJob.java:1075)
at org.labkey.pipeline.mule.PipelineJobRunner.run(PipelineJobRunner.java:40)
at jdk.internal.reflect.GeneratedMethodAccessor1156.invoke(Unknown Source)
at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
at java.base/java.lang.reflect.Method.invoke(Method.java:568)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invokeMethod(DynamicEntryPoint.java:312)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invoke(DynamicEntryPoint.java:259)
at org.mule.impl.DefaultLifecycleAdapter.intercept(DefaultLifecycleAdapter.java:193)
at org.mule.impl.InterceptorsInvoker.execute(InterceptorsInvoker.java:47)
at org.mule.impl.model.DefaultMuleProxy.run(DefaultMuleProxy.java:470)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$CallerRunsPolicy.rejectedExecution(ThreadPoolExecutor.java:1486)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.reject(ThreadPoolExecutor.java:391)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.execute(ThreadPoolExecutor.java:865)
at org.mule.impl.work.ScheduleWorkExecutor.doExecute(ScheduleWorkExecutor.java:39)
at org.mule.impl.work.MuleWorkManager.executeWork(MuleWorkManager.java:277)
at org.mule.impl.work.MuleWorkManager.scheduleWork(MuleWorkManager.java:244)
at org.mule.impl.model.seda.SedaComponent.run(SedaComponent.java:483)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:650)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:675)
at java.base/java.lang.Thread.run(Thread.java:833)
Caused by: java.nio.charset.MalformedInputException: Input length = 1
... 39 more
08 Aug 2024 05:18:16,745 INFO : Failed to complete task 'org.labkey.targetedms.pipeline.TargetedMSImportTask'
08 Aug 2024 05:18:16,747 ERROR: malformed input off : 26, length : 1
org.labkey.api.pipeline.PipelineJobException: malformed input off : 26, length : 1
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:276)
at org.labkey.targetedms.pipeline.TargetedMSImportTask.run(TargetedMSImportTask.java:63)
at org.labkey.api.pipeline.PipelineJob.runActiveTask(PipelineJob.java:833)
at org.labkey.api.pipeline.PipelineJob.run(PipelineJob.java:1075)
at org.labkey.pipeline.mule.PipelineJobRunner.run(PipelineJobRunner.java:40)
at jdk.internal.reflect.GeneratedMethodAccessor1156.invoke(Unknown Source)
at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
at java.base/java.lang.reflect.Method.invoke(Method.java:568)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invokeMethod(DynamicEntryPoint.java:312)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invoke(DynamicEntryPoint.java:259)
at org.mule.impl.DefaultLifecycleAdapter.intercept(DefaultLifecycleAdapter.java:193)
at org.mule.impl.InterceptorsInvoker.execute(InterceptorsInvoker.java:47)
at org.mule.impl.model.DefaultMuleProxy.run(DefaultMuleProxy.java:470)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$CallerRunsPolicy.rejectedExecution(ThreadPoolExecutor.java:1486)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.reject(ThreadPoolExecutor.java:391)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.execute(ThreadPoolExecutor.java:865)
at org.mule.impl.work.ScheduleWorkExecutor.doExecute(ScheduleWorkExecutor.java:39)
at org.mule.impl.work.MuleWorkManager.executeWork(MuleWorkManager.java:277)
at org.mule.impl.work.MuleWorkManager.scheduleWork(MuleWorkManager.java:244)
at org.mule.impl.model.seda.SedaComponent.run(SedaComponent.java:483)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:650)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:675)
at java.base/java.lang.Thread.run(Thread.java:833)
Caused by: java.lang.IllegalArgumentException: malformed input off : 26, length : 1
at java.base/java.lang.String.throwMalformed(String.java:1242)
at java.base/java.lang.String.decodeUTF8_UTF16(String.java:1198)
at java.base/java.lang.String.newStringUTF8NoRepl(String.java:732)
at java.base/java.lang.System$2.newStringUTF8NoRepl(System.java:2398)
at java.base/java.util.zip.ZipCoder$UTF8ZipCoder.toString(ZipCoder.java:199)
at java.base/java.util.zip.ZipCoder.toString(ZipCoder.java:66)
at java.base/java.util.zip.ZipInputStream.readLOC(ZipInputStream.java:302)
at java.base/java.util.zip.ZipInputStream.getNextEntry(ZipInputStream.java:124)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:106)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:82)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:69)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:64)
at org.labkey.targetedms.SkylineDocImporter.extractIfZip(SkylineDocImporter.java:1005)
at org.labkey.targetedms.SkylineDocImporter.importSkylineDoc(SkylineDocImporter.java:317)
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:249)
... 24 more
Caused by: java.nio.charset.MalformedInputException: Input length = 1
... 39 more

Hi,

I was wondering if anyone has encountered a similar issue. I'm trying to access results files to import from a mapped network drive. However, I can only see the local drive (C:). I've tried making a shortcut of the network drive to "This PC" and restarted as well but still can't see any other option other than C:.

Thanks!

Hi,
We're evaluating some Astral data with Skyline doc previously optimized for PRM on Lumos Fusion (MS2 filtering matched Fusion Orbi res 60K, At 400 m/z and selected PRM and Orbitrap as instrument in the MS2 filtering). I would like to know if you've observed any issues or have experience with Astral run in targeted mode. We use the latest Skyline Daily build Skyline-daily (64-bit) 24.1.1.202 (c511d22bf). We noticed the Astral data seems very noisy with very choppy peak shape so curious if alternate instrument selection (under full scan filter settings), resolution, etc. would improve this (we already have Savitzky-Golay smoothing active). The Astral experiment acquires targeted MS2 scans for ~60 peptide precursors. The Astral method has Experiment 1 and 2 in parallel: Exp 1 set as Orbi for full scan MS and resolution of 240K, while Exp 2 Astral MS2 scans were set for 1.6Da isolation and 20ms injection. Should Skyline MS2 filter be PRM & Orbitrap or should it be TOF (PRM?)? What would be optimum resolution for Astral MS2...80K? Are there any other settings we should be concerned with, especially since this instrument is scanning so much faster than our older instrument?
Thanks!
Monica

Hello,

I have the following error message : "No product ion chromatograms found" and indeed I don't see any fragments for any ion in any of the proteins monitored. Sometimes the error message don't appear but the signal associated to the fragments is extremely low (a line on the 0)

What I tested :
• crosslinked peptides / non-crosslinked peptides
• changing transition settings / peptide settings (and re-importing the results each time that I was doing significant changes) for the transition/peptide settings modified I tried to stick with what you said in this similar request : https://skyline.ms/announcements/home/support/thread.view?rowId=43123
• using different raw files with different protein concentrations to verify if it was a sensitivity problem (I can send the data if needed)

Thank you in advance for your help,

Best regards,

David

Dear Skyline Team,

I'm working with DIA-NN 1.9.1 and Skyline (64 bit Administrator Install), but DIA-NN doesn't generate "report_skyline.sky/skyd".
Could you tell me how I could get over it?
I'm using the Administrator Install Skyline, because DIA-NN 1.9.1 hasn't recognized the conventional one.
Thank you very much.

Kazuki Sasaki

Hi

When I was trying to set up Koina to generate a spectral library in Skyline, it says server unavailable. Do you know what could be causing this?

Thanks,
Qingling

Dear Skyline Team,

could you please consider implementing support for DIA-NNs .speclib spectral libraries? Its a highly convenient tool for predicted libraries and much faster than Prosit.

https://github.com/vdemichev/DiaNN

With best regards,
tobi

Hello Skyline Team,

Is there a way to change the isotope envelope of multiple peptide precursors?
I am working on some long glycopeptides with many precursors (see attached .png). So I wonder if the M+3, M+4 can be included by editing the .sky using a text editor.

Thanks in advance.
Ho-Tak

Dear Skyline Team,

I am hoping you can help me with a problem that is causing me great frustration.

Recently when loading LysC digested PRM data into skyline, there is no longer a predicted retention time associated with the data. This seems to be a relatively recent (we started noticing it around June) problem for us. I've attached two files to this message. The first is a file entitled HA_May2024. This data was acquired in may of this year and as you can see when you open the data, there is a predicted RT time associated with the data. The second file is entitled HA_V2d. This data was acquired on July 27th and as you will see, there is no predicted RT time associated with this data. Interestingly, if you remove the data from the May2024 file and import the data from the V2d file into the May2024 file, there is again no predicted RT time associated with the data. Perhaps even more perplexing is that if I take the data acquired in May and import it into the recently made V2d skyline file, the data has predicted RT times! To me this is suggesting that the HA_V2d skyline document (and associated blibs and iRT databases) are not the problem but perhaps there is a problem with the way the data is being read??? One thing to note is that we are only seeing this with LysC data, our Trypsin digested data does not have this problem.

If possible, could someone on your team take a look at these files and see if they can identify why this is happening.

Thank you for your time,
Christie

NOTE: the data for this request has been uploaded to your file sharing folder. The title of the file is problems_w_RT_predictor_20240801

Hello,

I was able to successfully install Skyline 24.1 after previously uninstalling the older version. However, my current issue is that now I am unable to open previously saved skyline files by clicking on them and the icon has also gone missing. I can still view them by opening the Skyline software from the start menu and manually loading the file, however this is a huge inconvenience. Attached is a screen clip of the skyline file.

Thank you!

Hi Skyline team,

I have found that when I was working on a Full scan spectrum from Sciex QTOF 6600 system, I was not able to extract the correct peaks after entering the molecule's formulas. For example, I input the molecular formula of Molecule 1 as C16H20N6O3, and after adding an H, the software calculates the monoisotopic m/z to be 345.166965, but I didn't get the peak. Only after I manually changed the monoisotopic m/z to 345.1193 which was read from the instrument, I could get the correct peak.

I tried to change the "Method match tolerance m/z" in the Transition settings to 0.6 m/z, but it didn't help. So I wonder what I can do to increase the tolerance so that I don't have to change the m/z manually every time. Thank you!

Best wishes,
Mengchun

Hi,

I would like to set up DDA method for linear peptide, which got 1unusual Amino acid.

The amino acid is 2 aminoisobutyric acid.

I tried to include in modification but option is either N or C terminal. This modification is at C next to N terminal. Do you have any suggestion, how i can incorporate that.

Hi Skyline team,
Thank you for your contributions to the proteomics community.
There is a growing interest using Mass Spectrometry for mRNA analysis in academic and pharm industry. I wonder if functions for mRNA analysis could be added to Skyline. Similar to protein analysis, mRNA sequence is digested into oligonucleotides with enzyme (such as T1), and the oligonucleotides of interest are quantified with MRM.

Thanks,
Zhijun

Hello,

I cannot connect to the website of MSstatsShiny (link below). There is no problem with my internet and I tried to connect in different browsers. Is there a general problem?

https://msstatsshiny.com/app/MSstatsShiny

Thank you
Oyku

Hello everyone,

First of all, I'd like to thank you for all your hard work. Skyline is a very powerful tool (even if I haven't mastered it perfectly yet) and it allows us to do so many things.

I have a challenge that I think is quite unique. I'm working in diaPASEF on a timsTOF Pro and I'm looking to do absolute quantization in label free mode.
I'd like to know if it's possible to make a calibration/spreading curve on diaPASEF acquisitions from a DIA-NN library?
If so, can you tell me how?

Thanks in advance!

I got my IT people to try and install 24.1 using the setup.exe from here, which is labelled as version 24.1.0.199. But after installation, the About says that it is 23.1.0.268.

In addition, I am confused by the labelling of the Unplugged and Administrator versions as they are both labelled as 23.1. Is 24.1 not available as these versions?

Hi Skyline Team,
I want to run a Quality check on peaks that have been identified by my mprophet model trained on second best peaks. Could you guide me on what Z score and Q value I need to keep? Thanks!

I had the v22 installed on my PC (Win10, 64-bit, 32GB memory), but failed to installv23, now failed again on V24. The message said "Application validation did not succeed. Unable to continue." I have attached the log file.
Could you please advise? Thank you very much.

Hi,

I am working on MSstats in Skyline, and was following the steps of the attached tutorial. However, the version of skyline is old in the tutorial. Is there any updated tutorial about MSstats?

Best regards
Oyku

Dear Skyline team,

We really enjoy using your software to analyse our PRM measurements. However, there is one thing which caused us some trouble when dealing with larger data-sets. You are probably already familiar with the phenomenon - you create a method based on an initial measurement, validate it and start a big sample set.

However, in our case (and I can imagine that we won't be the only ones affected), we sometimes have slight shifts in our chromatograms when measuring several hundreds of samples. No problem - the PRM windows are large enough. Unfortunately, however, it can happen that the integration of the peaks does not always work automatically in these cases. Therefore, in the past we have often had to reintegrate ‘by hand’...it would therefore be an absolute quality of life improvement for us if we could ‘jump’ between the ‘tabs’ (i.e. between the individual measurements) in Skyline with our left hand, while we could integrate or reintegrate with the right hand (using the mouse). We know the "ALT + arrow key"-way, but this can only be done left-handed by real piano players ;)

Thank you very much in advance!

Hi Team,
I had tried many times to install the skyline 24.1 - 64 bit and the skyline daily(beta), but I failed.
Sometimes, it says I'm running out of disk space. In fact, the disk space is enough. In other cases, it suggests that it is a network issue. The attachment is one of the log files.
I really need your help. Thank you very much!

Hi Skyline Team,

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

Thanks!

Hello Skyline team,
I upgraded Skyline to version 24.1.0.199 (6a0775ef83) this week. After that, I am not able to export transition list using multiple methods anymore. Normally I select 50 max transitions per sample injection.
In this new version, when I enter with 50 (max transition) it doesn't display the number of methods anymore.
But, If I try to export, I get an error message " The precursor XXX requires 600 transitions to optimize, which exceeds the current maximum 50".

Instrument Sciex 7500 Qtrap, proteomic interface, optimizing methods for quantification of surrogate peptides
Could you please help/advise?

thanks!

Hello,

I am trying to copy and paste rules into the rule set editor in the document settings for result files. I have a number of different conditions, and right now I cannot paste in or import a table to set up my result file name rules. Happy to discuss more if needed, thank you so much!

Hi,

I have MRM data obtained by SCIEX ZenoTOF7600.
Product ion chromatograms look jaggy.
What kind of parameter do I need to change or use?

Best regards,
Satoshi

Hi there,

I am wondering if there is any default filter being applied for Peptide Spectrum Match Count in the document grid? I see that for some peptides this has 0 despite having a library match. Example in the attached figure.

If data is required I can provide some through Panorama, but this is more of a general inquiry on how this is counted in Skyline and what would happen if there are multiple spectral libraries active in a Skyline document?

I had not seen this PSM counting before, but looks very interesting for generating UpSet plots from a Skyline report.

Sincerely,
Juan C.

Dear Brendan and Team

Just tried to use the Import DDA Search wizard for the first time since updating to 24.1. It appears that the wizard in the current version has deleted the ability to start from raw files, and instead goes straight to the screen where you point to the search results. To reproduce:

File New/Start
Select Import DDA peptide search

Result: There is no option to choose start from raw files
Desired Result: There previously was an option to start from raw files, search results, etc.

Has this functionality changed in the current version, or is this a bug?

Cheers

Will

Dear Skyline Support Team,

I hope this message finds you well. I am writing to seek assistance regarding an issue I am experiencing with the Skyline program. Specifically, I am encountering difficulties importing a peptide with a PTM (Post-Translational Modification) due to a configuration mismatch with my current settings.

Here’s a brief overview of the problem: Skyline indicates that it cannot import a specific peptide, even though there is a similar peptide in the library with the same modification mass that Skyline does recognize. This discrepancy has left me unsure about which settings I need to adjust in order for Skyline to correctly recognize and import the peptides from my library.

Could you please provide guidance on what configuration settings I should review or modify to ensure that Skyline recognizes the peptides with PTMs from my library? Any advice or steps you can provide would be greatly appreciated.

Thank you very much for your assistance. I look forward to your prompt response.

Best regards,

Dear developers,

are you planning to implement workflows which will allow processing timsTOF data acquired by the newest flavours of PASEF, like synchro or midia-PASEF (Vista-scan option in timsControl)? I guess some preprocessing of the data has to be done before this can be achieved?

Best

Robert

Dear Skyline Support,

I have acquired MRM using the skyline "isolation list" export method. Now, I want to acquire data with different sets of Collision energy to identify the best transition for my peptide.
So, while exporting an isolation list for acquisition, CE optimization is not showing up in the dialogue box (attached). As mentioned in one of your manuals, I have checked Use optimization values.

Can you please tell me, how I can export the CE-optimised acquisition method for MRM in Waters Xevo-qTOF?

Regards,
Arpita

Hi, I'm having an issue getting samples to import, I have in the attached example 4 injections in the same file where only the first sample imports, the error is attached. This only occurs when I'm trying to import scheduled results.

Hello,
I'm trying to download a new library but I get this error message "ERROR: no such table: MassSpectrumItems.
I don't know what to do because it usually works.
Thank you for your help

Dear Skyline Support Team,

I hope this message finds you well. I'm reaching out to seek guidance on two specific features within the Skyline command-line interface:

1. Removing Empty Proteins: Is there a command-line equivalent for the "Refine -> Remove Empty Proteins" function available in the GUI?
2. Missed Cleavages Not Appearing: Despite setting --pep-max-missed-cleavages=0, I am noticing that some peptides which do not associate with the provided FASTA file but have a spectral library match still appear in my results. Here is the command I've been using:

%SKYLINE_CMD% --in=%INPUT_SKY_FILE% --import-search-file=%LIBRARY_PATH% --import-pep-list=%PEPTIDE_LIST% --pep-max-missed-cleavages=0 --associate-proteins-fasta=%FASTA_FILE% --refine-auto-select-transitions --refine-missing-library --refine-auto-select-peptides --save --out=%OUTPUT_FILE%

Attaching an image for better reference

Hi,
I am using EncyclopeDIA wizard to create chromatogram library and analyse my wide window datasets, however when I attempt to do it I am getting a Percolator error. Please see the attached log for details. I would appreciate if someone could let me know if there is anything on my side I can do to fix this.

Thank you.

Kind regards,

Michal

I am encountering an error where single scan frames will produce peak areas that measure much higher than they should. In the attached file "skyline error" the three samples are replicates, you can see that scan 3 has a much higher measured area. When you shift the scan over a single frame in either direction like in file "skyline error corrected" the peak area changes by ~100% and the replicates will have similar areas as expected.

I saw this thread and it seemed similar to my issue: https://skyline.ms/announcements/home/support/thread.view?rowId=49486

I tried to follow the steps (changing filters, turning off filters, etc.), but my peaks still look the same (nonexistent lol). I do have one spectrum that does show data, and it's a 60-minute runtime whereas the others are 30-min. It doesn't seem to be an issue with the intensity. I feel it's some filtering issue, but I'm not experienced enough with Skyline.

Thanks for any help anyone can give me!

Hi Skyline Team,
Thanks for the beautiful software.

I had one question,

View -> RetentionTime -> Alignment, doesnt seem to work. I'm not sure if I'm not loading the data correctly? Is there documentation available to learn how to use this. That would be very helpful.

I would like to synchonyse zooming for all the plots in my skyline analysis, but synchronisation works for only a subset of plots, while other chromatogrammes remain unsynchronysed (full RT range is shown). You can see the example in the attached presentation

Hi,
I am working with skyline (64-bit) version 23.1.0.455 using the small molecule settings. Since I cannot find the original transition list of a very important Skyline file, I was wondering if there is a way to export the precursor formula with chemical symbols and not the resulting mass. I tried exporting different parameters, but all of them just show masses and not the chemical symbols.

Is there a way to solve this? Many thanks for your time.

Bests,
Elia

I attempted to run an encyclopeDIA search using the new release, however, I get an error when Skyline is about halfway through requesting predictions from Koina. The full error message attached as textfile, but the first two lines are:

System.Reflection.TargetInvocationException: Status(StatusCode=Unavailable, Detail="GOAWAY received") ---> pwiz.Skyline.Model.Koina.KoinaException: Status(StatusCode=Unavailable, Detail="GOAWAY received") ---> Grpc.Core.RpcException: Status(StatusCode=Unavailable, Detail="GOAWAY received")

Thanks for any help you can provide.

Hi,
I have recently used Skyline for peptide absolute quantitation. Do you have the tutorial for getting started in Skyline with all the parameters (peptide settings, translation, modifications, instrumentation, etc.)?

Thank you
Anhdao

Hi team,

I had a few basic doubts regarding developing the PRM method as I am working on it for the first time. What are the points/parameters mandatory during a method development? (Ex: pooled QC, intraday, interday variations, calibration curve, reverse calibration curve). Should I run in triplicates when I have to check the sensitivity and specificity of the assay using serum samples?

Hoping for a positive response.

Thank you.
Akhila

I have two questions

1. Understanding Default Peak Picking Models in Skyline: When you import data into Skyline, does it utilize an mProphet or another default peak picking model? Furthermore, is it recommended to train an mProphet model enhance peak identification accuracy in Skyline?

2. Exporting mProphet Features via Skyline Runner: Is it possible to export all mProphet features (for all the skyline files) using Skyline Runner? While exporting a report file is straightforward, incorporating mProphet features into this report seems challenging. Are there any methods or workarounds to include these features in the export?

Hi All,

I have acquired my PRM data and using Skyline for computing my Fold change using MS stats.

I have 2 peptides corresponding to one protein and would like to know the Fold change at the protein level.
I am getting a foldchange at peptide level. I would like to know how can i get it right at protein level. Do i have to do any offline calculation for protein level?

I followed the Tutorial 20_1, however, my data is displayed in Fold change of each peptide with Adjusted P-Values.

I am ready to share my Skyline document too. Could you please provide me with some ideas how to do so. Than would be very helpful

Thank you.
Varsha

Dear Skyline team,
Thanks for an amazing tool and support.

I'm trying to set up a library for DDA with MS1 filtering, which works perfectly fine for many of my samples. However, one specific sample consistently gives an error (attached) while importing the pepxml with the Skyline wizard. I have both pepxml and .raw files in the same folder. I've also tried to use .msf file instead of pepxml: I can build a library if Score threshold=1, which is rather useless, but not when Score threshold=0.05 (same error message as attached).

I've used Proteome Discoverer 1.4 for my pepxml and .msf files.

Can you please help?

Many thanks
Adnan

Dear Skyline Team,
I would like to use Skyline to analyze immunopeptidomics DDA, DIA and directDIA (DIA-Umpire) data. I'm using the wizard to do it but I'm stuck at the creation and importation of fastA file. We usually use "unspecific enzyme digestion" for normal Discovery approach and I don't know how to do it especially for directDIA (DIA-Umpire).
Your help to overcome this would be great.

Thanks in advance for your help

Best

Hui Song

Hello,

I have acquired Full Scan MS2 data from Orbitrap Exploris 240. I wanted to know if Skyline is able to identify product ions based. I see green dots next to the precursors ion indicating that the precursors are present however all b,y,a ions are red. Is Skyline able to detect data only in MRM format or all MS/MS data?

Dear Nick, Brendan and/or colleagues from Skyline support,

To satisfy a prerequisite of a journal, reviewer and to attempt to comply with FAIR data principles, we are trying to upload MRM3 data to Panorama. I have previously asked for help to upload the data making a method, we have found a work-around and have succesfully uploaded data to Skyline in a method.

Unfortunately, a substantial part of the data-files failed to upload, while others do. Uploading the 'faulty' data provides this error message:

At 10:05:
Failed importing results file 'C:\Users\Hyung\Surfdrive\Projects\2020\P20-0001_COVID-19\Data\20240425_Exp134_RepositoryUpload\220503_Exp111_batch02\renamed_files\220503_019_MRM3_30089A1_10uL.wiff2'.
[WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\Hyung\Surfdrive\Projects\2020\P20-0001_COVID-19\Data\20240425_Exp134_RepositoryUpload\220503_Exp111_batch02\renamed_files\220503_019_MRM3_30089A1_10uL.wiff2'.
[WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object. ---> System.Exception: [WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object.
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\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 200
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 295
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 191
--- End of inner exception stack trace ---

It seems to me that the built-in msconvert module is throwing an error, pertaining the serial number of the MS. I checked the original Sciex data file that fails in Sciex OS (version 2.1.6.59781), and it does not seem to be corrupted, at least it opens normally. The MS data and metadata (serial number in the 'sample info') are also retrievable using Sciex OS (explorer module).

I also tried to convert the data in MS convert (version: 3.0.24094-d2966db), but I am not able to execute it properly. It also returns an error ([WiffFile2Impl::setSample()] Value cannot be null.). I think my colleague has previously reported this. I also tried to look in previous reported Skyline issues if I could find an answer to my question, but I was unable to find a solution for this problem.

Back to the Skyline upload: Is there a solution to upload all of our files?

Best regards,

Hyung Elfrink
Leiden University, Metabolomics and Analytics Centre.

P.S. I have attached my method file and I can share the data files per FTP.
P.P.S This is a link to the data that succeeded to upload, to Skyline and subsequently Panorama: https://panoramaweb.org/Leiden University - MAC/COVID-19 Mass Spectrometric MRM3 repository/targetedms-showPrecursorList.view?id=204275