When a peak has very low intensity (close to background noise), I observe incorrect quantification in Skyline, but only for compounds present at very low concentrations. In these cases, the QC samples fall outside the acceptable range. However, when I process the same MRM dataset using another LC-MS software, the QC values are within the expected range.

What could be causing this discrepancy in Skyline (background subtraction, noise threshold?), and which settings or processing steps should I check to improve quantification of low-intensity peaks?

do we have a rule of thumb for how many transitions per GB of RAM, given mass resolution, run length, etc that skyline can integrate without having to write to disk?

Hi Team, I have been dealing with an issue where I have set the sample names to Standards, Unknowns etc, given the light concentrations for the calibration curve samples that are marked as Standards, provided the Internal Standard concentration but the calibration curve (attached) has no values.

Not all Standards samples appear on the calibration curve and still it does not provide values, I assume that the ones on it have the peaks.

Could you please help me solve this issue? Thanks in advance!

I ran DIA-NN on 10 files. DIA-NN version is the latest 2.3
After that instructions said that click on Skyline and it should visualize the chromatograms, but i see this error:
[2026/01/16 04:39:47] Error: Failed importing the results file E:\Shubham\diaData\20250605_Stanford-Snyder_070_DIA_Slot2-5_1_4248.d.
[2026/01/16 04:39:47] Message: pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'E:\Shubham\diaData\20250605_Stanford-Snyder_070_DIA_Slot2-5_1_4248.d'.
The calculator fPop0059_1 requires at least 8 of its 10 standard peptides. The following 10 peptides are missing:

ADHASSY
AGGGRIDKPILK
AKPKAAKPKKAAA
DSTGHVG
EIEKFDKS
GSGGGGGGHGSY
LPPYVSNM
LSSNVTVSVTS
SAKKKNKKGKTISL
VHINAGPEI ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator fPop0059_1 requires at least 8 of its 10 standard peptides. The following 10 peptides are missing:

It is for some reason trying to look at the previous project. I wonder how to fix it?
I am attacjing the command line output that is printed when I click on Skyline button on DIA-NN.

Hi

I've been digging around a bit, and can't find a nice tutorial for TurnoverR like for several of the other Skyline functionalities. Is there one? I glanced at the one with the TurnoverR article (Supp file) and found it inadequate. Any info would be appreciated.

Regards,
Clark

Hi all,
I know you can export the AutoQC configs manually, but I'd like to automate the backup process. I'm hoping I can just grab a "AutoQC config file" with all of the necessary info. So far, my efforts at locating such a file have proven fruitless. Can someone let me know where it's located? Any other ideas are welcome.

thanks,
Peter

Hi

I ran some proteomic samples with PRM method to identify pyr-apelin-13 protein in them.
Then I ran them on skyline, some of them providing me truncated peaks but in some others are pretty good peaks,

I used same method for running them in the eclipse.

What could be the reason and how I can fix it?

Best,
Hadi

I tried to attached my prior proteins to the "Protein priority file" space, but I don't know if PRM conductor get the list or not.

I am using Skyline for proteomics analysis and am encountering an issue with a previously created document.When I attempted to reopen a Skyline document that I created approximately two months ago, the file failed to open. I then tried to recreate a new Skyline document by repeating the same workflow; however, I encountered an error during the peptide results import step.To minimize compatibility issues, I do not update Skyline frequently. I am currently using Skyline version 22.2.0.351 on Windows 11 Pro.I would like to ask whether there are any troubleshooting steps or alternative solutions to resolve this issue without reinstalling or upgrading to a newer version of Skyline.

Hi All,

I have posted the below message on the Panorama support page, but Josh has got back to me and suggested posting to Skyline support instead, with an example of a raw data file, as he thinks its an issue with importing the pressure trace in Skyline.

As an extra piece of information, the data is acquired from an Acquity UPLC (sample manager, solvent manager and column manager) attached to a Xevo ToF instrument. Acquisition computer was running Windows 7, with Masslynx 4.1.

Thanks,
David

Original post to Panorama support:
Hi all,
I am new to Skyline / Panorama (it was recommended by a collaborator), but ultimately the goal is to use it for monitoring system suitability data on a variety of instruments, mainly Waters QToF instruments, but eventually our triple quad, and maybe UV instruments too if it is possible to get that to work, but it is not high priority at the moment.
So far I have just been using our Xevo ToF instrument to acquire some test SST data (we work with small molecules), and have used Skyline (in molecules configuration) on the instrument computer to get a quick overview of the data, and then Panorama to have a more detailed look once the data is backed up to a server and can be accessed online. In both cases I have been using AutoQC to import the data, to make it as automated as possible.
I have to say I have been quite impressed with what I have seen so far, and think this could be a really useful solution for us to take our SST use to another level, and make it more proactive rather than being used for troubleshooting when things look wrong....
However, I was really hoping that I would be able to also use the column backpressure as one of my SST criteria, and it seems that I should be able to do that from what I have read. Unfortunately, when I follow the instructions how to add a new or custom QC parameter, Panorama says that there are no traces found. If I look in Masslynx I can clearly see the pressure traces, so the UPLC SST method is correctly recording the pressure (and some other parameters) as I asked it to, but this doesn't seem be getting into the Skyline or Panorama files....
Is there some trick or setting somewhere that I am missing so that the pressure traces are also loaded? Or is there a known problem with importing this data from a Waters .raw file?
Any suggestions would be gratefully received... I feel I am probably doing something wrong, but I have no idea what it is....!
Thanks,
David

Dear SkyLine Team,
I am trying to upload my Thermo data (From an exploris 480) as .RAW on skyline for peptide search (DDA), but they cannot load (it says it's not a valid imput file). I have also tried to convert them into a mgf and mzML but it gives me the same error. I can open those files on FreeStyle and Proteome Disc, so I do not think they are corrupted or invalid.

How can i proceed now?

Thank you for your help,

Lorenzo Tagliazucchi from University of Parma, Italy

Dear support team,

I am attempting to load post-analysis internally calibrated Bruker timsTOF .d data files into Skyline and am encountering an issue with the representation of calibrated data.

For the same acquisition, I have two differently processed datafiles:
ZTB11_Boltje-O-glycan-S1-2uL_GA2_1_1564_uncalibrated.d (no post-analysis calibrated)
ZTB11_Boltje-O-glycan-S1-2uL_GA2_1_1564_calibrated.d (post-analysis internal calibration applied in Bruker DataAnalysis)

In DataAnalysis, the calibrated file shows the expected improvement in mass accuracy, with peaks calibrated to approximately 2 ppm compared to ~23 ppm in the uncalibrated data (example shown for m/z 675.2466 in DataAnalysis_cali_vs_uncali.png). However, when loading both the calibrated .d file into Skyline, the calibrated file does not reflect the post-analysis mass calibration. The observed m/z values in Skyline appear identical to those from the uncalibrated data, suggesting that the post-analysis calibration is not being recognized. I have included the Skyline document and associated files Calibrated_vs_uncalibrated_file_2025-12-16_14-42-39.sky.zip for reference. This leads me to suspect that Skyline is accessing the original acquisition data rather than the post-processed calibrated data.

Is post-analysis internal calibration applied in Bruker DataAnalysis supported when importing timsTOF .d files into Skyline? What are the steps required to ensure the calibrated data are correctly loaded?

Thank you for your help.
Agnes

accidently posted this message orginally to PanoramaWeb, Brendan replied with the following message
2025-12-17 05:14:55
Hi Agnes,
Please post this to the Skyline support board:

https://skyline.ms/support.url

That reaches the Skyline team more directly and you will get your best response there. They will still be able to access your data on Panorama.

I understand your issue. Slides 22-27 of Skyline Tutorial Webinar 15 describe a case where I got a Bruker dataset without post-analysis calibration and eventually worked that out and managed to get the same dataset calibrated. That worked for me back then (2016), but it wasn't even timsTOF data. So, something may have changed more recently.

Unfortunately, the most obvious developer to look into this is on vacation until December 29. I hope one of us can still look into this, but it can certainly get attention within 2 weeks.

Thanks for taking the time to report your issue.

--Brendan

In Skyline's feature detection, using the wizard, there seems to be access to only 3 or 4 parameters for Hardklor and Bullseye. Is there a way to change any of the defaults?

Dear Skyline team,

My name is Samuel and I am currently developing a software for Quality control in my core facility. It is a streamlit application developed in python which actually detects when a ".raw" file is generated in my directory and automatically launches skyline. For that purpose a template is taken into account for searching albumin. Everything works correctly, my ".raw" files are automatically searched and results are stored with the information I seek but my problem arises when I try to launch the skyline GUI with the ".sky" file result I want to inspect. I manage to launch the skyline GUI but not with the .sky file loaded.

Could I achieve to executethe Skyline GUI with my .sky file loaded?. Here I attach the "launch_skyline.bat" script which is set to launch the GUI.

Kind regards,
Samuel

Hi,

I know that this question has been answered, but I am still having issues. I believe that to open the report-lib.parquet.skyline.speclib file in skyline I need to make sure I have the correct .tsv file with "Precursor.Id", "Q.Value" and "RT" (according to a previous post). However, I can't seem to find this file in the output folder. I'm not sure if maybe I did something wrong in DIA-NN or I'm not looking in the right place. Can you help me out?

Thank you!
Robyn

Hello!

I have dilutions of a molecule and I would like to use the slope of the molecule for other molecules of my data set too.
But Skyline calculates its own slope for every molecule (name).

I attached a photo to explain it better: I want to use the slope of Cer 34:1 (for which I made the dilutions) for Cer 32:1 too.

So I would like to be able to just Copy Paste my slope or use a slope selection tool like for the Surrogate Standards.

Best regards
Helena

FIXED workaround see my reply

Hello,
I have had great use of the possibility to export methods as .exp files to Masslynx. I have used the Skyline variables to adjust the acquisition times in masslynx:

Explicit_Retention_Time
Explicit_Retention_Time_Window

Usually this has worked but I have found that sometimes I don't get the expected values in the exported file. I have therefore investigated what triggers the problem. I could se that for the same parameters in my two skyline templates with same compounds one way of formatting resulted in wrong times in exported file.
I have created two simple compound tables of same analytes just different names and would appreciate of you could take a look at this and see if it can be fixed.

1. "MRM template to skyline CE.xlsx" the excel file that I use to insert transitions from one sheet with working format and one with the format that does not work
2. "CE working.sky" "CE not working.sky" the skyline dokuments I used for export
3. Masslynx EXP files has the export template and the export with correct times and the one with wrong times

Overall this has worked Fabolous so if this bug could be fixed it would be amazing!
I have tested the problem on two different computers with latest Skyline Daily installed (Skyline-daily (64-bit) 25.1.1.271 (67f3e15e7))

Regards
Per

Thank you guys for a great Webinar and a great feature. This addresses a lot of issues I've seen with peak detection/integration over the years.
I am now trying it on my dataset - it is over 700 Astral runs searched and processed through DIANN which I am visualizing in Skyline (for now a subset of ~120(2,000 proteins, 15,000 precursors, 137,000 transitions). I see that the imputation runs really slow (16 core/24 processors, 64 GB RAM Dell desktop). I wonder what would speed things up - more RAM, more CPU, run in background with SkylineRunner (if so what would be the command..). Or just be patient?

Thanks a lot for any advice.

Tomas

Hi! I am attempting to add molecules from a new library to an existing skyline document. I added the new library to the libraries list via the library explorer, and I can see all of the molecules and spectra in the library. When I select a molecule and "Add", I receive the message:

"The library 20251205_NSTD6_ISTD1 is not currently added to your document. Would you like to add it?"

When I select yes, nothing seems to happen. I attempted to save the document, close skyline-daily and reopen, and was then able to add a molecule (can see this in audit log), but then not another. I continue to receive the message asking if I want to add the library. Then I tried removing the library from the list and re-adding with a different name, but still encounter the same problem, and can't replicate the close-reopen fix. There is no indication the library is added to the document in the audit log, and it isn't listed in the Libraries tab of Molecule Settings. This is the document I've shared. So, I think there might be a problem with adding libraries to the document via library explorer.

Next, I added the library to the document via the Molecule Settings window, and I can see the library is added to the document from the audit log, and can add molecules to the document via library explorer.

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

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

Do you have any idea what is going on here?

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

I am very grateful for your help.
Wesley.

Hi,

I have been analyzing PRM data with skyline, and have had success when first searching with another software and importing the pepXMLs. I wanted to try the Feature Detection from raw files and have not been able to get it to work. Anytime I try to do the Feature Detection I get an error saying the search failed - this is with the same data that works if I search first and import pepXML. Does the skyline team have any troubleshooting suggestions or could you point me towards additional resources to setup the Feature Detection from raw files? I am using skyline 25.1.0.237 version on windows 11.

Thanks,
Nithesh Perumal

Hello,

Do you think you could implement z-ions. Those are really handy in EThcD to distinguish Leucine and Isoleucine.

Thank you.

Eric

Hello, I have run PRM with 2 peptides sequence in timsTOF, each 2ng of peptides, which should be enough. I can observe the MS1 but there is no transition information. Is there a problem with my setting? I can upload my skyline files if needed. Thank you.

Having trouble installing skyline on my work computer. Any ideas?

PLATFORM VERSION INFO
Windows : 10.0.26100.0 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.8.9181.0 built by: NET481REL1LAST_C
clr.dll : 4.8.9310.0 built by: NET481REL1LAST_C
dfdll.dll : 4.8.9181.0 built by: NET481REL1LAST_C
dfshim.dll : 10.0.26100.1882 (WinBuild.160101.0800)

SOURCES
Deployment url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application
Server : Apache
Deployment Provider url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application
Application url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Application%20Files/Skyline-daily_25_1_1_271/Skyline-daily.exe.manifest
Server : Apache

IDENTITIES
Deployment Identity : Skyline-daily.application, Version=25.1.1.271, Culture=neutral, PublicKeyToken=9286511f3362df93, processorArchitecture=msil
Application Identity : Skyline-daily.exe, Version=25.1.1.271, Culture=neutral, PublicKeyToken=9286511f3362df93, processorArchitecture=msil, type=win32

APPLICATION SUMMARY
* Installable application.

ERROR SUMMARY
Below is a summary of the errors, details of these errors are listed later in the log.
* Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application resulted in exception. Following failure messages were detected:
+ Downloading https://skyline.gs.washington.edu/software/Skyline-daily13-64/Application Files/Skyline-daily_25_1_1_271/Method/Agilent/MH12/Agilent.MSDrivers.LCQuadrupole.MethodResourceMgr.dll did not succeed.
+ The remote server returned an error: (403) Forbidden.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
No transaction error was detected.

WARNINGS
There were no warnings during this operation.

OPERATION PROGRESS STATUS
* [12/5/2025 12:00:13 PM] : Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application has started.
* [12/5/2025 12:00:14 PM] : Processing of deployment manifest has successfully completed.
* [12/5/2025 12:00:14 PM] : Installation of the application has started.
* [12/5/2025 12:00:15 PM] : Processing of application manifest has successfully completed.
* [12/5/2025 12:00:17 PM] : Found compatible runtime version 4.0.30319.
* [12/5/2025 12:00:17 PM] : Request of trust and detection of platform is complete.

ERROR DETAILS
Following errors were detected during this operation.
* [12/5/2025 12:00:26 PM] System.Deployment.Application.DeploymentDownloadException (Unknown subtype)
- Downloading https://skyline.gs.washington.edu/software/Skyline-daily13-64/Application Files/Skyline-daily_25_1_1_271/Method/Agilent/MH12/Agilent.MSDrivers.LCQuadrupole.MethodResourceMgr.dll did not succeed.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
at System.Deployment.Application.SystemNetDownloader.DownloadAllFiles()
at System.Deployment.Application.FileDownloader.Download(SubscriptionState subState, X509Certificate2 clientCertificate)
at System.Deployment.Application.DownloadManager.DownloadDependencies(SubscriptionState subState, AssemblyManifest deployManifest, AssemblyManifest appManifest, Uri sourceUriBase, String targetDirectory, String group, IDownloadNotification notification, DownloadOptions options)
at System.Deployment.Application.ApplicationActivator.DownloadApplication(SubscriptionState subState, ActivationDescription actDesc, Int64 transactionId, TempDirectory& downloadTemp)
at System.Deployment.Application.ApplicationActivator.InstallApplication(SubscriptionState& subState, ActivationDescription actDesc)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivation(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl, Uri& deploymentUri)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
--- End of stack trace from previous location where exception was thrown ---
at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)
--- Inner Exception ---
System.Net.WebException
- The remote server returned an error: (403) Forbidden.
- Source: System
- Stack trace:
at System.Net.HttpWebRequest.GetResponse()
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)

COMPONENT STORE TRANSACTION DETAILS
No transaction information is available.

Hello,

I am trying to install Skyline-Daily on a Windows 10 AWS instance and I am getting an error. I've attached a screenshot of the dialog that comes up with the error. I've also attached the insall.log file that is written to the Downloads folder.

The relevant lines at the end of the log file seem to be:

URLDownloadToCacheFile failed with HRESULT '-2146697208'
Error: An error occurred trying to download 'https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application'.

I have also tried this with the stable version of Skyline and I get the same error.

I was able to install the Unplugged version of Skyline-Daily in the mean time, but I thought it was still worth reporting this. Is there some sort of network setting that could be causing problems?

-Aaron

I am using Stellar MS to acquire tMS2 (PRM) data for a single compound at multiple HCD collision energies (e.g., 5, 15, 25, 35, 45). The output is a single RAW file containing MS/MS information at these different CEs. When I analyze the file in Thermo FreeStyle, I can view the MS/MS profiles for each CE and determine which collision energy works best.
However, when I try to analyze the same RAW file in Skyline, I cannot figure out how to visualize the MS/MS profiles at multiple CEs. A colleague suggested that to see fragmentation profiles in Skyline, I would need to acquire separate injections for each CE (i.e., five injections from the same vial, each at a different CE). I am wondering if there is a way in Skyline to view fragmentation profiles from a single tMS2 file that contains data acquired at multiple collision energies.

Looking forward for your response.

Regards,
Dilip Singh
CCHMC, Cincinnati

Hello,

I am running a PRM experiment using Heavy internal standards to quantify the L/H ratios of a peptide which I hope can provide me with relative quantification values across different samples. For accurate quantification, my understanding is that it is best to ensure your values are within the linear range of quantification i.e. above the LLOQ and below the ULOQ, and we can obtain this by generating a standard curve with different concentrations of heavy peptide spiked into our background sample.

My question therefore is when providing relative quantification values for PRM, is it essential to have calculated the LLOQ/ULOQ from a standard curve to ensure you L/H ratios are within this range or can you trust the L/H ratios without prior knowledge of the LLOQ/ULOQ?

Many thanks,

Sam

Dear Skyline Team,

In my phosphoproteomic PRM analysis, I used Proteome Discoverer for database searching and then imported the exported .MSF or .pdResult files into Skyline to build the spectral library. While the library was successfully created, I encountered an issue where many spectra that matched my target peptides were associated with multiple ambiguous peptide-spectrum matches (PSMs). These ambiguous matches were automatically filtered out, resulting in the loss of numerous peptides I need to target. Consequently, after importing my peptide list, many of them failed to match any spectra in the library. What should I do to resolve this?

As follows：
Skyline
Library successfully created. Spectra matching the following peptides previously had multiple ambiguous peptide matches and have been excluded:
AG[+79.96633]Saktalcavem[+15.99492]K
AG[+79.96633]Saktalkewmk
AGS[+79.96633]Vichtalcavem[+15.99492]Q
AGS[+79.96633]Wicktalcavem
Awkwatkandith[+79.96633]Tibbink
Avkoutkandit[+79.96633]Pink
Athaslattuvat[+79.96633]Spask
Adaslatvats[+79.96633]Basque
Ests[+79.96633]Spager
STX[+79.96633]Bakr
Akakkas[+79.96633]Kibaktubsappar
Akkak[+79.96633]Bishop
Akkax[+79.96633]Abcaper
Alex[+79.96633]sk
Alex[+79.96633]sk
Anisnikins[+79.96633]SPKPSK
Anisnigans[+79.96633]Pickups
The [+79.96633]Substituteist
ATS[+79.96633]BSTListxR
Awaaz[+79.96633]Spanchevk
Awas[+79.96633]Pansevak
Aws[+79.96633]Jindpivigiri
Avsnt[+79.96633]BIVKIBIR
Taktak[+79.96633]Hold on
DocTaxSPVT[+79.96633]Q
Delpt[+79.96633]DBsec
Tailft[+79.96633]Bisec
TikBekect[+79.96633]Setlock
TGBEKects[+79.96633]Edluck
DS[+79.96633]Gisvicker
DSS[+79.96633]iCaser
Ectex[+79.96633]Tcivic
Iktekpst[+79.96633]Chekvik
Ideas[+79.96633]sk
Ideas[+79.96633]KK
Itate[+79.96633]Wirpsup
Edaturbs[+79.96633]Bactic
ESP[+79.96633]DSlog
Eggpst[+79.96633]DSlock
EPS[+79.96633]AbsipaikspakupatBabbar
EfSubsipay[+79.96633]GSBayAbir
Afsopsipyks[+79.96633]Spakapir
ESAP[+57.02146]KKS[+79.96633]StandFinder
ESSP[+57.02146]GCS[+79.96633]TPint
ESSp[+57.02146]GCEST[+79.96633]PIN
Estes[+79.96633]
Estes[+79.96633]PK
Faket[+79.96633]Chevpinet
FGETS[+79.96633]Wifnettler
G[+79.96633]Saktalcavem[+15.99492]K
G[+79.96633]Chaktalkevm
KISS[+79.96633]Vichtalkevm[+15.99492]Q
KISS[+79.96633]Wicktalkevm
STST[+79.96633]Swift
Husts[+79.96633]Swithak
Hachtswd[+79.96633]Attic
HTCit[+79.96633]As
Hdisted[+79.96633]Oct
Eas[+79.96633]SVTPIK
IEASS[+79.96633]VTPIK
IEASSVT[+79.96633]PIK
IEDVGS[+79.96633]DEEDDSGK
IEDVGSDEEDDS[+79.96633]GK
IM[+15.99492]PDS[+79.96633]NDSPPAER
IM[+15.99492]PDS[+79.96633]NDSPPAEREPGEVVDSLVGK
IM[+15.99492]PDSNDS[+79.96633]PPAER
IM[+15.99492]PDSNDS[+79.96633]PPAEREPGEVVDSLVGK
ISPS[+79.96633]TDSLAK
ISPST[+79.96633]DSLAK
ISPSTDS[+79.96633]LAK
IT[+79.96633]SPLM[+15.99492]EPSSIEK
ITS[+79.96633]PLM[+15.99492]EPSSIEK
KMT[+79.96633]SPENDSK
KMTS[+79.96633]PENDSK
KPT[+79.96633]ETPPVK
KPTET[+79.96633]PPVK
LDNVSHT[+79.96633]PSSYIETLPK
LDNVSHTPS[+79.96633]SYIETLPK
LDS[+79.96633]TAGM[+15.99492]PNSK
LDST[+79.96633]AGM[+15.99492]PNSK
LIS[+79.96633]SENFENYVR
LISS[+79.96633]ENFENYVR
LLLDPS[+79.96633]STPTK
LLLDPSS[+79.96633]TPTK
LLLDPSST[+79.96633]PTK
LLLDPSSTPT[+79.96633]K
LS[+79.96633]SLVIQM[+15.99492]AR
LS[+79.96633]SLVIQMAR
LSS[+79.96633]LVIQM[+15.99492]AR
LSS[+79.96633]LVIQMAR
LTGQENIS[+79.96633]SK
LTGQENISS[+79.96633]K
M[+15.99492]T[+79.96633]SPENDSK
M[+15.99492]TS[+79.96633]PENDSK
M[+42.01056]ASGNDS[+79.96633]TTVK
M[+42.01056]ASGNDST[+79.96633]TVK
MT[+79.96633]SPENDSK
MTS[+79.96633]PENDSK
NQQEHGGEDS[+79.96633]SQESK
NQQEHGGEDSS[+79.96633]QESK
NSLS[+79.96633]DSADPFLIK
NSLSDS[+79.96633]ADPFLIK
QSPLVTPGS[+79.96633]TTK
QSPLVTPGST[+79.96633]TK
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Confirm

Hello there,

I am completely new to Skyline MS and am just trying to open a mzML file. I keep getting thrown an error message that I can only open skyline files, but that does not seem to be true? I should be able to open a mzML file? May someone please help me troubleshoot this?

I double checked that it was not an issue with the file (i.e. corruption) itself by trying again with a mzML file that my colleague was able to open in Skyline on their computer, and I got the same error message. I converted to mzML using ProteoWizard's msConvert with no filtering applied from the original .raw file, but that does not seem to be the main issue as the .raw file cannot be opened on Skyline either.

The version I am using was downloaded directly from the main page and is named as "Skyline (64-bit) 25.1.0.237 (519d29babc)" in the About section.

I have tried both File --> Open and File --> Import --> Document and Open File from the Start Menu.

My .mzML file is able to be viewed on ProteoWizard's SeeMS and looks as expected, but I want to open on Skyline to be able to analyze it for PFAS.

My computer (PC Dell Desktop) is running Windows 11. Processor: Intel(R) Core(TM) Ultra 7 265 (2.40 GHz). 64-bit operating system, x64-based processor.

I have attached a file of the error text I am receiving and an example .mzML file I am trying to open.

Please let me know if you can help! I am very confused.

Thanks!

Since Skyline is not yet suitable for the identification and quantification of intact glycopeptides. I plan to use MSFragger-Glyco to search glycoproteomic data, simultaneously export a Skyline document, and utilize Skyline to construct a PRM method containing precursor ion and product ion information based on the Skyline document . Next, I will apply this PRM technology for targeted quantification of glycopeptides. However, I have encountered some difficulties: MSFragger-Glyco search was successful, but the Skyline document failed to export. Could you please recommend a strategy for establishing a PRM method for intact glycopeptides using Skyline or relevant team members to help me resolve this issue?
Thank you very much! I’m looking forward to your reply.
Best regards,
Lian Shu

Hi all,

Could we please the Peak Imputation Window that was available for the special build Skyline-daily (imputation) v24.1.1.445 or something similar? This window was great for checking what Skyline was actually choosing for imputed peak boundaries and it provided a very transparent view on correcting boundaries one analyte at the time.

The support for checking this in the most recent Skyline-daily is not great compared to that mentioned version. My labmates and I keep reverting back to the special build version only because of that feature.

Please let us know if there is anything we can do to bring back that feature.

Sincerely,
Juan C. Rojas E.

Hello,
I have a question about the use of the idotp value in the small molecule interface within Skyline (daily). To determine the detection limit and quantification limit, we typically use a 3x or 10x signal-to-noise ratio. My question is whether the IDOPT value (>0.9) is a good substitute for determining whether a peak has been reliably measured.

Thanks already.
Best,
Pim

Hello,
For my targeted methods in lipidomics I am at the lower limit of dwell times for instrument. Exact data acquisition windows would be very beneficia but can be difficult to set for a big MRM table this could be solved (if it is not already):

1. having a reference sample run before each new sample set (can be split over several injection if needed)
2. Peaks are integrated over the retention time that I wish to use for my data acquisition. If there is a wide (double peak) i will use wider integration window.
3. A scheduled method is then exported that use the integration window, set by me dragging under the chromatogram.

This would allow me to very quickly set optimal scheduled MRM data acquisition every time a run a new batch. Currently there is the feature use measured RT with a fixed time window around the RT but I would like the feature use integrated RT to allow different windows (widths) for different compounds.

A work around is to export Start Time and End Time in the document, write a script that calculates the explicit retention time and explicit retention time window based on these. Explicit times can be used for the export method :-)

Dear Skyline Development Team,

I am currently working on a calibration using only heavy peptides (without light peptides) for my analysis. However, I am encountering an issue when extrapolating the calibration curve (CC) for unknown samples. The software seems to extrapolate the CC based on the area of the heavy peptides, rather than using the light peptides.

Could you please advise on how to resolve this issue or if there is a setting I might have overlooked that would allow the CC to be extrapolated using the light peptide areas, even though I am only using heavy peptides for calibration?

Your help would be greatly appreciated.

Best regards,

Dear Skyline Team,

I am currently trying to import some FragPipe results using heavy/light labelling into Skyline (25.1.0.237). The import itself, using the interact.pep.xml files, following the workflow suggested by FragPipe, works, however, when I take my result list from FragPipe to filter the Skyline document, most of those peptides are not present (i.e., out of 1189 peptides 1050 are not present). I tried setting the score threshhold really low, but that did not change the outcome.
I checked and those peptides are present in the interact.pep.xml so I'm not sure what I'm doing wrong that they are not included in the document.

Best regards,
Tanja

Dear Skyline team,

I am using the small molecule interface in Skyline 25.1.0.237. I am trying to see the top three isotopic peaks (M, M+1, M+2). I changed the settings according to the Adding Isotopic Peaks section in the Hi-Res Metabolomics tutorial.

• Transition Settings - Full Scan: Peaks is set to 3, Retention time filtering set to "Include all matching scans".
• Transition settings - Filter: Precursor adducts: [M+H], ion types: p. Auto-select all matching transitions is checked.

I am still not seeing the isotopic peaks for all molecules (some molecules have all three isotopic peaks, some only have one or two). Any suggestion is appreciated!

Thank you,
Shimin

Hello,

I'm trying to run Expert Review external tool from Thermo via CLI (command line), but I'm not able to get the results and send back to skyline in order to obtain the correct integrated peaks.

The external tool works pretty well on the graphical interface. Is there a way to run it via command line ?

I tried to use:
--tool-add="Thermo\Expert Review" ^
--tool-conflict-resolution="overwrite" ^
--tool-program-macro="ExpertReview,1.0" ^
--tool-program-path="C:\Users\workstat\AppData\Local\Apps\2.0\CWMKAT46.3P7\60MOKXQP.7YG\skyl..tion_9286511f3362df93_0019.0001_e71402c3ddc399fe\Tools\Sky3432\Thermo.TNG.PeakPick.SkylineTool.exe" ^
--tool-initial-dir="C:\Users\workstat\AppData\Local\Apps\2.0\CWMKAT46.3P7\60MOKXQP.7YG\skyl..tion_9286511f3362df93_0019.0001_e71402c3ddc399fe\Tools\Sky3432" ^
--tool-command="$(ToolDir)\Thermo.TNG.PeakPick.SkylineTool.exe" ^
--tool-arguments="$(DocumentPath)" $(SkylineConnection) "$(InputReportTempPath)" ^
--tool-report="Thermo_ART_Integration" ^

but it didn't run the tool.

Thanks in advance.

Hi,

I have a question using a targeted-MS2 method from an Astral mass spectrometer. I would like to set an isolation offset to shift the quite narrow isolation with of the Astral to get a second isotope into the isolation window, without increasing the isolation width.

In Transition Settings - Full Scan - MS/MS filtering I can set PRM, but this will exclude most of the peptides, as the precursor mass in the tMS2 scans get a different precursor mass. If I try a workaround and set the scan filter to DIA, skyline ignores the peptides selected from the library, and tries to match ions to all possible peptides in the library.

Is there a way to use isolation offset in PRM experiments?

Thanks for any help!

Johannes

Hi Nick,
I'm trying to export a custom report that contains RTs and intensities for all transitions, for all samples. I've included a screenshot of my current report template. I'm confused by the fact that "Interpolated Times" and "Interpolated Intensities" do not contain the same number of points. For example, if I export this report, one of my transitions has 2389 times and 1246 intensities. I've attached a small version of this report as well. I've noticed the same thing when I export "Raw Times" and "Raw Intensities". I'd like to obtain matched pairs of RT, Intensities for each transition. Is this possible?

Also, I'm confused as to what the "Interpolated Number of Points" field is telling me. For the example I mentioned, this number is 406....I can't figure out what this corresponds to.

Hi Team,

Is it possible to change the display format for the LOQ on the calibration curve?
Is it linked to any particular table?

Best,
TY

Hello,

I would like to quantify peptides at the MS1 level in Skyline using identifications that were generated by an external tool (PNovo3).

Is there a way to import a peptide list with associated retention times without going through the iRT predictor workflow? The iRT module read the retention times already be present in the Skyline document, which require peak picking beforehand, something I cannot reasonably do manually.

Thank you for your help.

To whom it may concern,

I have a problem with the imported data from SCIEX. I haven't seen it before and I've been using Skyline for 2 years now. In SCIEX files the chromatogram looks normal and there is the small peak at 6.7 min but in Skyline it's just a flat line (see the attached file). I tried to download the newest version and it didn't help. I tried to re-import files and it didn't help either. Could you help me?

Kind regards,
YK

Hi there!

I'm hoping you can help me with a problem that's hopefully somewhat straightforward. I'm trying to filter the skyline document for N-modified residues resulting from a FragPipe Glyco-N-DIA search (with a DDA library). To do so, I'm going to Peptide Settings > Filter > Exclude peptides containing: NXT option. However, I'm noticing this doesn't function as expected since my peptide/precursor/transition numbers stay the same.

Overall, I'm hoping to find an ability in Skyline to filter my target list to those with an N-modification (doesn't have to match the motif). Please let me know if there are any questions!

Thanks again for all the work you do for the greater community!

Hi everybody,

I am using Skyline for identification of tri and oligo peptides. That is working perfectly.
I was wondering if there is a way to predict the transitions of cyclic dipeptides (diketopiperazines) for LC-MS as well. I used the modification option to subtract one water molecule from the dipeptide but unfortunately that didn't work properly.

I am looking forward to your responses.

Best regards,
Mike

I am using Skyline (64-bit) version 25.1.0.237 on Windows 11

Hi Skyline team,
I am unable to import Waters SONAR file as a result file into Skyline. Attached is the message which pops up when import is initiated.
Please resolve.

Thanks.

Hello, Can I use a peptide containing cystine for heavy labeling? If so, would the process be the same as for a regular peptide? Thanks

Hi Skyline team,
We have a mixed sample with 18 plex TMT, now we want to use this sample for PRM, and in the subsequent experiments, we want to quantify the same peptide in this sample, different tags, we can know the peak area of different tags of the same peptide through the skyline?How can it be, please let me know how.
Thank you and have a great life.

Hi,

I have a question about the peak areas by replicates window display:
when I select a peptide I have the option to also select precursor/fragment/all to show peak areas by replicates. On the other hand for a protein selection, only all can be found on the menu and can only visualize the sum of all (fragments + precursors). Is there a way to show ms1/ms2 intensities.

Thank you,
Janos

Hi,
Sorry for bothering again, we have 11 different acids and aim to have internal standards for each one of them. I was wondering, whether it would be possible, to have different analyte concentration for each of the acids in one document? Right now if we look at the calibration curve, it looks funny/wrong for some of the acids, due to the different values. Is there a way see for each acid the right concentration that we actually spike in?
Thanks very much,
Best Sophia

Dear Skyline team,
First of all,
Thank you very much for making skyline great! Its fantastic and I am adapting more and more workflows in Skyline.

I have an issue when I am trying to overlay two peaks (DDA mode). The chromatograms are then not displayed correctly and are distorted.

Is there a way how to fix it? Am I doing something wrong?

I am sending corresponding chromatograms attached.

Best,
Adam

Hi Skyline team,

We are using Skyline to analyze PRM data to build a peptide calibration curve and calculate LOD and LOQ. We spiked a constant amount of heavy into serially diluted light peptide. We also acquired two blanks in which only the heavy peptide is present at the same amount as the serially diluted samples. Our quantification settings are as follows: Regression fit = Linear in log scale, MS2 level, LOD = blank + 2SD, LOQ: Max LOQ bias = 20%, Max LOQ CV =20%. We are using the ratio to heavy from the Peptide Ratio report to build calibration curves in R and calculate LOD and LOQ using blank and regression methods. The LOD/LOQ values that we obtain with our script differ from those calculated by Skyline. Is there a document we can refer to that describes the steps Skyline uses to build peptide calibration curves and assess LOD/LOQ (is skyline using all fragments, are the fragments used always the same in different samples, are ratio L/H ratio calculated first for each transition and then summed, etc)? In case LOD are negative values what is the recommended approach? Many thanks for your support, Floriana

I am trying use the "Copy Data" feature to copy the data from the "Full Scan" viewer that appears when you click on a chromotagram.

In general I think it is really useful to extract the underlying data from the visualizations in Skyline, however I am finding the way the output is formatted difficult to understand. What I would like to do is plot the data from the scan in R, but the format makes that difficult without moving a lot of things manually. At this point I am questioning whether the formatting is a feature or a bug.

I'm pretty sure the "Copy Data" feature is simply broken for scans with ion mobility data. I've attached a csv with the output that I am seeing when copying the data from a scan from a timsTOF .d file. The columns for m/z and 1/K0 (Vs/cm^2) are repeated multiple times. It looks like the values for intensity are there but it not clear which rows they apply to. In row 3, 1/K0 (Vs/cm^2) is repeated in 57 columns.

I would think the best way to format the output would be a simple table with a single header and 3 columns: m/z, 1/K0 (Vs/cm^2), and intensity.

The first thing I tried to do was to see what the output for a 2D scan without ion mobility data looks like. I think it a bit more comprehensible but still not very R/python friendly. There are 3 separate set of headers for each ion annotation.

Again I would think the best format would be a simple table with 3 columns: m/z, intensity, annotation.

Thanks,

Aaron Maurais

Hello,
I'm doing the PRM analysis using the skyline.
If a specific transition is not clear, we are excluding that transition. (Rt. click -> remove peak: then the peptide turns to Red X mark)
My question is,

1. if I want to reinclude a transition I've excluded, can anyone tell me how to do that?

2. When removing a specific transition, it seems that the total ratio value (in the Export-Report-Peptide ratio results, not the total ratio in the screen view) is the same whether I remove only the transition of the light peptide or whether I remove only the heavy or both the light and heavy. Is that correct?

Hello there!

Would it be possible to define an additional attribute for modifications to define them as different isomers? This is applicable for glycoproteomics, but it should also be applicable to other modification types based on some of the talks I saw in Skyline Online 2025.

In glycoproteomics, you commonly see glycopeptide structural isomers due to different arrangements of the connectivity between glycan monomers. We can resolve these chromatographically in many instances and would like to quantify them independently.

We have found a workaround for this by creating multiple entries of modifications where the only different thing is the name of the modification. Example:

H6N5S3_a
H6N5S3_b
H6N5S3_c

Here the letter suffix just indicates the elution order. This allows us to create different targets separated by the "Peptide Modified Sequence Three Letter Codes" column in the Document Grid reports.

However, we are having issues with Skyline recognizing these modification isomers when it comes to re-importing peak boundaries and defining independent entries in an iRT library since modified peptides are written with respect to modification mass.

For example for the peak boundaries case, all boundaries of the isomers are fixed to the boundaries of only one isomer after importing a "Peak Boundaries" report.

I am not sure what is the best support for this in Skyline, but we thought maybe an additional attribute written in the Skyline XML files and displayed in the GUI indicating "Isomer = TRUE/FALSE" + choosing which (e.g. user defined as "a", "b", "c", etc.) could work?

We are happy to provide some example data if it would help.

Looking forward to hearing back from you.
Sincerely,
Juan C.

Their Skyline support team,

is there a possibility to apply different integrations (min and max rt for integration) for light and heavy without changing the transition list, that the quantification is performed with a surrogate?

Our department uses Skyline a lot for quantification, however, often different internal standards are used that require a different integration. We struggle with the use of the surrogate option, as this does not allow us a layout, where the peak can be shown in the same window together with the internal standard.

Thank you very much for your help,

Greetings from Zürich

Hi Nick:

Thanks for your reply. I can see ion mobility information in library explorer. But when I export results, no matter what ion mobility items I choose, the value is always empty. I attached an example here.

I uploaded the file to the dropbox you mentioned. The name is "fragpipe_skylib-EGFR-DIA.sky.zip". Please have a look.

By the way, when I did search with MSfragger, there is an option to create a skyline document. So the skyline document was created in Fragpipe.

Haiyan

Dear Skyline team,

I am trying to import nucleosides NLS data from Agilent in Skyline, but Skyline gives me an error message Failed importing results file. The sample contains no usable data. It seems that the spectra is not read by ProteoWizard. I cannot upload here the data since they are not yet published, but I can send it to you if you need them to check, or look for a published equivalent to post here.

Thank you for any help you can provide!

Best regards,
Cristina

Since version 2.0 DIA-NN no longer generates speclib files for its spectral libraries but instead only parquet files. I used to be able to import the speclib libraries into Skyline but since they are no longer generated by DIA-NN I wonder how I can import the parquet libraries into Skyline. The direct link from DIA-NN to Skyline (Skyline button in DIA-NN) never worked for me but importing the DIA-NN generated spectral libraries worked well until DIA-NN version 2.0 when the speclib libraries are no longer generated. The DIA-NN developers told me that Skyline will soon have parquet import capability - is this already a function that is available in Skyline Daily? If, so where can I find it?
Thanks much,
Dietmar

Dear Skyline support,

according to some opinions, in silico spectral library generation is good enough and experimentally generated spectral libraries are no longer needed.

Do you agree?

Mehul

Dear Skyline team,

As the title of this request suggests, the default value that is set by Skyline for Collision Energy in an isolation list (“Export – Isolation List”) for Bruker timsTOF equals zero (i.e. 0eV). I believe that this behavior is unintended, since the timsControl software used for method generation for Bruker instruments interprets this value literally, meaning that there will be no fragmentation (though 'useless' MS2 scan will be present in the data file). To use appropriate collision energy as intended by Bruker, this CE value should default to a "-" hyphen symbol (ASCII 45). I have forgotten a few times by now to change this value in a spreadsheet manually, resulting in some wasted samples…

Since I'm already writing a post here, I would like to ask for a feature for timsTOF in Skyline specifically. It would be immensely useful if the "Retention Time - Scheduling" window would show the number of precursors per TIMS ramp and the separation of targets in RT and mobility dimensions, when ion mobility separation is utilized as selected by values in Transition Settings > Ion Mobility (I have attached a screenshot of this exact feature from Bruker software). Since ion mobility separation has an impact on the number of precursors that can be isolated and measured in a prmPASEF method, this would be much informative than a “Concurrent Precursor” plot. To illustrate for example, assuming two methods with the same number of Concurrent Precursors measured in unit time, their measurement will result in more points per chromatographic peak if the mobility of precursors is dissimilar and less points per peak if mobility of these precursors is nearly the same. That’s why this would be really helpful.

Thank you for your help in advance!

Dear Skyline team,
I'm creating libraries for nucleosides compound with different collision energies with Skyline. Could be nice to have the relative abundance information of the library and ideally have the option to remove noisy transitions, for example transition with Relative Abundance less than 5%.
Attached you’ll find an Skyline document with some nucleosides and the corresponding library, if you need something else just tell us.
Best regards,
Guadalupe

Hi Skyline Team,

When I try to import the Skyline library, I get an error saying that the .parquet file is missing the column Global.Q.Value. I’ve attached a screenshot of the error message. Could you help me figure out what I might be doing wrong?

I have also attached the parquet file and the lib file. I used DIANN 1.9.1

Hello, I am having an issue uploading a .raw file containing PRM data to Skyline. The error states "A device attached to the system is not functioning. :iostream error". Can anyone give me a clue as to what this might mean?

Dear Skyline Support Team,

I am trying to build a quantitation workflow for intact proteins, where I wish to obtain XICs for my "naked" protein, and the protein with a glycosylation.
My protein has an average mass of 76132 Da, and the most abundant charge states are between 50-55+. Following some other helpful advice, I defined my protein as a long peptide and created a modified and nonmodified version. My issue is, that I record the data with a Q-TOF and since I do not have isotopic resolution, I need to use the average mass to get Skyline to pick the correct peaks. However, when I navigate to Settings>Transition settings>Prediction and switch 'Precursor mass' to Average I am met with the following message: "High resolution MS1 filtering requires use of monoisotopic precursor masses". Does anybody know how to select average masses for this type of analysis? Thank for your help! Version is Skyline-daily (64-bit) 25.1.1.206
Best
Soren

Hello,

I am writing to ask whether there is a plug-in or other option that enables more reliable automated peak picking in Skyline for large datasets.

In our workflows, Skyline frequently selects the highest-intensity peak, which is often incorrect. Even when I manually select the correct peak and apply it to all subsequent the alignment remains inaccurate for a substantial number of cases. This is particularly challenging in degradation assays, where we need Skyline to prioritize peak selection at the expected retention time—even when the signal is weak or largely background. At present, this does not appear to be feasible with the available settings.

If there are recommended approaches, plug-ins, or configuration strategies that would improve automated peak selection and retention time adherence at scale, I would greatly appreciate your guidance. Reviewing hundreds of samples across multiple peptides individually is highly time-consuming, and a more robust one-click solution would significantly improve efficiency.

Thank you for your assistance.

Dear Skyline team,

I hope you're doing well. I've previously performed a small molecule collision energy equation optimisation on an analyte in an older version of Skyline but now that I've tried to re-export the optimised CE values, they have used the precursor m/z as the intercept instead of the specified intercept in the equation itself (e.g. instead of the CE being 50, it's now 909.3 for my 909.3 m/z precursor). No matter what the intercept is, it defaults to the precursor m/z instead of the specified intercept. Maybe something has changed in how this is calculated?

This is the case in both standard Skyline (Skyline (64-bit) 25.1.0.237 (519d29babc)) and Skyline-daily (Skyline-daily (64-bit) 25.1.1.271 (67f3e15e7)). I've attached the Skyline document, and I'm using the custom CE equation of "Flat" as defined in the document.

Cheers,
Chris

Hello,

I couldn't download Skyline software on MacOS. If there is a link for MacOS, can you please send?
Thank you in advance.

Best regards,
Sudaba Ismayilova.

Hello,

I am hoping to figure out the best column to add to my document grid to display the value of the surrogate standard area used in normalization for each replicate. If there is a simple way to display this unique value for each replicate that would be great.

Thank you,
Zach

Hello,

I am attempting to import data from waters_connect using the remote account. I have set up the remote account and the connection tests OK. Then, when I click File - > Import -> Results -> Remote account, I can see the folders and data files within the waters_connect database. However, when I try to import/open those files I receive an authentication error (attached). Could you tell me please if there is a resolution for this or if there is a setting I am missing?

Thanks

Dear Skyline Team

Viewing the retention times as the alignment scores seems not to work in the latest -daily release. To reproduce:

1. Open any skyline document with an iRT predictor enabled.
2. Right-click in the View/Retention times (or chromatogram) view and select "Show [iRT predictor] Score"

Expected result:
The retention times (or chromatogram) is plotted in iRT dimension.

Observed result:
Skyline shows no data on the [iRT Predictor] Score version of the Replicate Comparison retention times plot, and does not update the chromatogram view to show indexed time (still just shows retention time)

Cheers

Will

Hello,dear Skyline team
Since I targeted to do 4D before, so the generation of offline data is mzml, many attempts have failed, the attachment is my screenshot and data, I hope to solve this problem, thank you very much。
Zhou Yanan

Dear Skyline Team,

I am experiencing an issue with the calibration curve in Skyline. It seems that the program is using peak area values instead of the total area when generating the calibration curve. As a result, the regression line is counterintuitive: the signal decreases as the concentration of the standard increases, even though the total area is actually larger for higher concentrations.

Could you please confirm whether this behavior is expected, or if there is a setting I should adjust to ensure the calibration curve is based on total area values?

Thank you very much for your support.

Best regards,

Dear Skyline Team,

I am wondering whether there is a SkylineRunner command to set the RT boundaries for a peptide across all my 96 runs?

Basically I am asking for a CLI version of this right click UI option of "Apply Peak to All", as shown in the attached screenshot.
(By supplying my own low-RT and high-RT boundaries to the CLI)

Thanks a lot. Kind regards,
Bingwen

When trying to do the peptide import for the tutorial, the box with "cutoff score" is not showing up on my screen, so I am unable to import the peptides and continue the tutorial. I was wondering how to get that box to show up.

Hello,

Please could you explain the difference between "Normalise to Total Ion Current" and Normalise to "Total" when viewing Peak Areas in replicate comparison view.

Couldn't seem to find this in your manual.

Many thanks!

Shaan

Hi, I recently updated my Skyline to V25.1. It can open my old project or create new project with no issue. But when I tried to import raw MS data (from Thermo Quantiva MS). I got an application error and the program quits. Please see the attachment for the screenshot of the error message.

Thank you.

Hello,

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

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

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

Thank you very much for your help.

Best regards,
Daria

Hi Skyline team,

I am trying to extract MS1 information from an enhanced dynamic range (eDR) full scan on the Orbitrap Excedion Pro. This scan is multiplexed at the MS1 level and combines 2 Orbitrap subscans to form a single full scan. The ion packets have variable injection times. I am getting the following error when trying to import the data…

[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=1": [SpectrumList_Thermo::getMultiFillTImes()] Unexpected fill time format: IT=25;35;22;4;3.6;0.21;IT=28;28;28;7.2;0.96;0.72

I am using the latest version of Skyline daily (v. 25.1.1.174). Is this type of data, eDR full scan, data supported yet?

I will upload the .raw file. Please let me know if you need any more information. Thanks!

Thank you,

Wes

Hi Skyline team,

Is it possible to export metrics such as FWHM as the full number rather than two significant figures when using the Skyline command line?

Also, is it possible to export FWHM, FWB and RT in seconds rather than minutes.

Thanks

Dear Skyline team,

I am trying to create a PRM PASEF method. For this, first i measured some samples on DDA and searched the raw file in Fragpipe. I uploaded that file to create library in Skyline and used the same result files to create mobility library . Now I want to filter the peptides, I saw most of them just have precursors ions no transition ions even though I have selected p, y, b ions in the settings.

Can you please have a look and let me know if I have done something wrong.

Best,
Susmita

Hello,

Given that the software requires you to save a file before importing data, there should always be enough information to automatically set the working directory to whatever dir the file is saved in. I don't know how much effort this would require, but it would be very welcome. As is, every time I'm ready to export a report, the wd is set to the last directory I saved a report in, which is almost never where I want the file to be by default.

Thanks for you consideration!

Nik

Dear Skyline team,

I am trying to create a PRM PASEF method. For this, first i measured some samples on DDA and searched the raw file in Fragpipe. I uploaded that file to create library in Skyline and used the same result files to create mobility library . Now I want to filter the peptides, I saw most of them just have precursors ions no transition ions even though I have selected p, y, b ions in the settings.

Can you please have a look and let me know if I have done something wrong.

Best,
Susmita

Hi all,

We are conducting targeted metabolomics analyses and have observed some expected retention time shifts in our data. I was wondering if Skyline includes functionality that utilizes relative retention time to assist with automated peak detection?

Thank you for your time and assistance.

Chloe

Hello,
I am trying to install the 64-bit Skyline setup, but I'm getting the "An error has occurred writing to the hard disk" message. Could you possibly help fix this issue? I'm using a 64-bit operating system. There is enough space on the hard disk (C), and write permissions are enabled. I have tried running it as an administrator, but it still gives the same error.

Hello,
I could not find anywhere else to pose this question so if there is a person I should contact please let me know. I am an employee at a commercial biotech company and our team wishes to use Skyline as part of our R&D for proteomics development. Our legal team found two components that cannot be used commercially, thus right now it appears we can't legally use Skyline. These are part of the Apache license:

1. Thermo-Scientific MSFileReader Library
2. Waters Raw Data Access Component Library

I wanted to ask if there is any way to remove these components and use the remaining components of the Apache license?

Thanks,
George

Dear Skyline-Team,

I would like to analyse crosslinked peptides for quantitation using a spectral library that already contains all the necessary information, including decoy fragments. I have tried this using the assay library import. Unfortunately, I failed to import the csv file because the peptide mass difference was too much from the calculated one by the sequence. That would be expected since crosslinked peptides are larger (two peptides is one sequence) and the automated calculation would naturally fail. But how can I fix this issue? Is there a way to still import my library? Maybe by converting the sequence to strings like that: GQKNSR-GQKNSR-[PhoX@3,3]

Or is there another way to do it?

Thank you very much for your help.

Best wishes,
Franze

To reduce storage needs and speed up processing, we acquired data for seven small molecules in tSIM on an Orbitrap IQ-X.

Settings:

Multiplexing: Off

Isolation window: 1.2 m/z

Orbitrap resolution: 120,000

Data type: Profile

Polarity: Negative ionization

Two compounds are isomers, so we entered five precursor m/z values in the table.

When I load the data into Skyline (25.1.0.142), the extracted chromatograms look wavy compared with Thermo FreeStyle (see attached). I’ve spent several hours trying different filter options in Skyline and MSConvert but haven’t been able to fix it.

Has anyone run into this and found a solution?

Thanks in advance—cheers,
Gerrit Hermann

Hi

I am using DIA NN 2.0 to search a set of files and generated a file called Human-Serum.parquet.skyline.speclib, I also put the report file into the same folder, called Human-serum-report.parquet. Because from DIA NN 1.18. I know the report should have similar name to the speclib file. But when I tried to import to Skyline, I got an error "parquet file does not have a column called Global Q value). I was wondering if somebody can help me with this? I have not been able to import any spectral lib since I used DIANN 2.0 and everytime is because of the same error although I certainly can find Global Q value column myself.

Dear Skyline team,

I appreciate that there is probably a post/tutorial that could help me but I could not find one.

I have created a MRM method that contains 120 transitions, I have 12 different peptides and each peptide has 5 transitions. I also have heavy labeled peptide as internal standards.

I have tried to import the transition list using Edit>import>transition list. In there I copied the sequence of the peptide (i.e DYSLFSYATK), added the name of the protein associated with that peptide, the precursor ion, product ion, RT, collision energy etc... Hoewever, it does not import it as it says that the modified peptide sequence has not been found. Am I doing something wrong?

Also side note, for my heavy labeled peptides (I did add the isotope modifications in the settings) how can I name them? Or can I keep the same name and have it as "heavy" in my label type?

Thank you very much for your help,

Best wishes,
Cecile

Hi,

I analyzed Stellar GPF-DIA runs in fragpipe. When I tried to create the library from the Fragpipe search results it failed. I have attached the error.

One more thing I noticed - Fragpie search of GPF-DIA with and without adaptiveRT results in very different ID numbers - I do not know why.

Thanks,
Maithy

Dear Skyline Team,

When generating a method for an Altis (Thermo) instrument in Skyline, which instrument type should I select: "Thermo" or "Thermo TSQ"? For future reference, what is the difference between these two options?

Thank you,
Qin Fu

Dear Skyline team,
I am trying to import peptide search results from Fragpipe outputs but am encountering a format mismatch error between files.
My results are from Fragpipe v23 and am importing into Skyline (64-bit) 25.1.0.142 (7401c644b4).

I have tried multiple times with two different files to follow the Fragpipe tutorial on importing data into Skyline. During the first step, once I select the interact files and click next, I encounter the following warning (attached 1): "[SpectrumList::find] mismatch between spectrum id format of the file (scan=1) and the looked up id (controllerType=0 controllerNumber=1 scan=726)"
This warning seems to be there for every spectrum (attached 2) and thus nothing is imported into the Skyline document. If I click more info, the first line is "System.IO.IOException: WARNING: Could not find native id or title 'controllerType=0 controllerNumber=1 scan=759' in ../250128_Hela_100ng_DDA_3ms_R1_uncalibrated.mzML."

Based on the comments in the submission 'opening.d files on skyline' https://skyline.ms/announcements/home/support/thread.view?entityId=e28a2047-ead0-103d-b4e5-22f535560118&_docid=thread%3Ae28a2047-ead0-103d-b4e5-22f535560118 containing similar mismatch issue discussed in the comments, I tried moving either the raw, uncalibrated mzml or mzml into the folder but all produce the same error.

Is this a file formatting issue from Fragpipe or a Skyline issue?
Thanks for you work,
Best,
Naomi O'Sullivan