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

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

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

Hello,

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

Thank you!
Lauren

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

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

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

Hi Team,

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

Best,

Laura

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

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

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

Cheers,
Thomas

Hi!

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

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

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

Thanks!

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

Thanks
Jerome

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

Hello,

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

Thanks

Hi Skyline Team,

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

Thanks!

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

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

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

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

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

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

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

Thanks as always,
Sam

Dear Skyline developers,

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

Giovanni

Hello,

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

Thank you
Anhdao

Hello,

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

Thanks
Anhdao

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

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

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

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

Best regards,
Jonatan Nygren

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

Thanks

Hello

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

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

"Please see the attachment."

Thanks,
Zhaoxuanxuan

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

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

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

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

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

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

Regards,
Per

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

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

But as the skyline is updated, some problems arise.

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

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

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

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

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

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

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

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

Hello,

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

Hi all :)

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

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

Thanks,
Lindsay

Hi,

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

Any help is much appreciated.
Thanks,
-Eric

Nick

Dear Skyline people,

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

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

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

Thank you very much for the help!

Hello,

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

For in silico digestion using various enzyme.

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

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

thank you.

Hi,

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

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

Hello,

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

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

Thanks a lot
best wishes

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

Thank you!
Seamus Kelley

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

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

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

Thank you for any help you can provide!

Sincerely,
Morgan Mann

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

How to fix that issue please?
Thanks
Nihel

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

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

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

Thank you in advance!

Alexander

Hello,

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

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

Hi team,

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

Cheers

Will

Dear Skyline team,

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

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

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

Thank you in advance!
Shimin

Dear Team,

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

Thanks in advance.

Kind regards
Vanya K N

Hi Skyline team.

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

Thank you

Stoyan

Dear Team,

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

Thanks in advance.

Kind regards
Vanya K N

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 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