Hi,

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

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

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

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

Thank you

Jean

Hi,

I am trying to build a spectral library with .msf files but it keeps showing an error:
"This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it"

However I cannot find where I can set this cut-off score. Could you please help me find?!
I am using Skyline 22.2.0.255; in older versions there was a small window where we could set the cut-off score...

Thanks

Skyline (64 bit) 22.2.0.255 (c99206313)

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

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

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

Thanks,
-Remco

Hi Skyline team,

I have been trying to insert the custom transition but the edit insert transition opens to a blank tab instead of the tabular layout that used to be in previous version. How can i insert transition in the Skyline 22 (newest veersion)

cheers
bharath

Hi,

I have a small issue when pasting peptides via "Edit -> Insert -> Peptides". My system is Windows 10, Skyline-daily 64-bit, 22.1.9.252.
When I try to copy a few peptides with descriptions from excel into the Skyline document, description is not included. The excel table looks like this:
EAGHLVFDR protein 1 description 1
IEAGHLVFDR protein 2 description 2
LIEAGHLVFDR protein 3 description 3
Just taking these three peptides from the Excel file into the clipboard and going to Edit -> Insert -> Peptides results in the third column empty in the table (see the attachement). I would expect to paste also the “description…” part.
Pasting only the description column into the third column of the grid did not work as well.

Thank you for your help.

Best regards,
Pavel

Hi guys,

The command line documentation lists some options for MS1 filtering that can be changed, under the Document Settings. What do you think about adding the MS/MS filtering options? These are: Acquisition Method, Product mass analyzer, Resolution, and even Isolation Scheme for the DIA acquisition method. A work around could be for an application to store Skyline file templates with the desired settings, but this is not as flexible as programmatic access.

Thanks
Philip

In reading about the SkylineCmd, you mention various ways of knowing where it is, which require the user to do a special kind of install, either as an Administrator or with the Unplugged installer. Would it make sense to provide Tool creators a macro that resolves to the SkylineCmd path? This way a Tool would have easy access to SkylineCmd without relying on the user to download SkylineRunner and put it in a particular place, or for the tool to package up the Skyline runner in its installation zip file.

Thanks
Philip

Hello,

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

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

Thank You,
Gabriela

Hi All,

I tried to build spectral library using MaxQuant msms.txt file and modifications.xml files. But I got the following error message. Would you please help to solve the problem? Please suggest next steps. Thank you.

More info (Error message):

System.IO.IOException: Error occurred running process.

Command-line: C:\Users\reddys49\AppData\Local\Apps\2.0\580TP24P.9OP\H3L3QW2E.RPJ\skyl..tion_e4141a2a22107248_0016.0002_1c0fe1ba9c975222\BlibBuild -s -A -H -o -i Urine_QEHF -S "C:\Users\reddys49\AppData\Local\Temp\tmp2031.tmp" "C:\Users\reddys49\OneDrive - Bristol Myers Squibb\Desktop\SPR_BMS\Sudhir Reddy\Database\From_XQ-AD_MQtxtfiles_UrineQEdata\txt\txt\Urine_QEHF.redundant.blib"
Working directory: C:\Users\reddys49\OneDrive - Bristol Myers Squibb\Desktop\SPR_BMS\Sudhir Reddy\Database\From_XQ-AD_MQtxtfiles_UrineQEdata\txt\txt
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_22_2\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_22_2\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 412
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161

Hello,

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

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

Thank You,
Gabriela

I have an external tool configured according to the nice documentation in https://skyline.ms/_webdav/home/software/Skyline/@files/docs/Skyline External Tools-2_1.pdf. The custom report has the Raw Times and Raw Intensities columns defined, which use comma delimited lists. It is more convenient to parse this report with tab delimiting, allowing to parse the Raw Times lists easily: the alternative I've found is to use regular expressions to tell the difference between the commas for columns and the commas in the Raw Times, and this is orders of magnitude slower than a simple line.Trim().Split('\t'). Therefore I'd like to request some way to specify the delimiting of these external reports, either in the tool.properties files, or else in the custom report .skyr file itself.

Thanks
Philip

Hi, I'm a new user for skyline. I have a more general question as I browse previous post and can't find the answer. I'm running targeted msms using Q-exactive. When I try to set up those transitions, for example, I can't find the instrument listing as QE. Shall I choose orbitrap instead? Also how do you define those values (such as library-ion matching tolerance, instrument-method matching tolerance)? Where to define the dot color (red/yellow/green) next to the peptide fragment? Thanks!

Hi Skyline team,

I am trying to use Skyline to track the performance of our System Suitability (DDA run, 15 peptide standards mix). The goal is to monitor MS1 and MS2 intensity change across the time.

Below is what I did: I searched the raw data in PMI and generated the mzid file that can be imported into Skyline. I used this mzid file to build the spectrum library. Then I set the peptide setting and transition setting based on the method edit tutorial. Then I imported all 15 peptides that I would like to monitor. Once everything is set up, I used File>Import>Result to import the raw data and try to view the results in Skyline. One thing I do not understand is that I can see all those product ion on the spectrum library, but why most of those product ions were labelled red in the Skyline? For example, if you look at the second peptide “GISNEGQNASIK”, I can see y7, y8 and y10 in the spectrum library, but these product ions were all labelled as red in the list (screenshot attached). The raw data I imported directly into Skyline is the same file I searched with PMI, so the fragment should be exactly the same. I have attached my Skyline file in case you want to take a look at the setting.

Another question is that I set in the transition setting that to monitor y, b, p ion types, why I only can see the precursor ion? I cannot find the way to display the fragment ions.

Hope I have my question expressed clearly, if you have any question or need any further information from me, please let me know.

Thanks,

Hi,
I was using Skyline small molecule to quantify this molecule ran on Shimadzu LCMS 9030 QTOF. The data was run using DDA method then raw data was imported to skyline for quantification.
I had a standard curve and a few actual samples that are run in triplicates
However, for some of the replicates, Skyline just stopped extracting/integrating in the middle of the peak (red box).
I went back to the Shimadzu's software and try manual extracted ion chromatogram of that peak and saw it's there in full.
I've never seen anything like this before, hopefully somebody can help,
Thank you very much,
Duc

Dear Skyline team,

I am struggling to import wiff2 from X500R files in Skyline Daily. Please find attached the error reported by Skyline and the wiff2 file.

The same file converted to mzml using MSConvert imports fine in Skyline.

Would you have any suggestions ?

Thank you very for your help,

Kind regards,

Sarah

First, thank you for offering this software, I’m new to Skyline and I am excited to use it.

My data files were collected from another lab using SCIEX triple Quad (5500). I am looking at small molecule data (around 200 metabolites). I was given a .wiff file containing the multiple runs and the .wiff.scan. However I am unable to upload either(file-->import-->results). I’m not sure if this is a file issue, software or something on my side. I have tried using the newest version of Skyline and Skyline Daily.
Attached are the errors that I receive for the .wiff file and .wiff.scan
Also attached is the .wiff and .wiff.scan files
Thank you for your time

I am trying to find an easy way to print out a list of small molecules that will fail a scoring model on account of having less than 3 isotopes. I can find the molecules by going to edit current scoring model, highlighting the row in the "Available feature scores" grid and then hovering the mouse underneath the "unknown" bar in the graph on the "Feature Scores" tab.

I can push the binoculars button and Skyline will fill in the "Find Results" window with the list of small molecules which are missing that score. However, I cannot find a way to easily print out that list of small molecules. I have tried using the menu item:
File > Export > mProphet Features
and exporting all of the feature scores to a CSV file and then look at it in something like Microsoft Excel.
The "PeptideModifiedSequence" column will contain the name of your molecule, and you can look for "#N/A" in the corresponding score column.

However, the issue is that I am using a dummy dataset to just find out which molecules will be excluded from the scoring model on account of having less than 3 isotopes. It looks like if it is not a hit from the dummy dataset I still get a lot of "#N/A" in the corresponding score column and these might have 3 or more isotopes. What would be ideal is if there was a way to just export the compounds ID’ed in the “find results” window with the list of small molecules which are missing the score from the scoring model. Any help would be greatly appreciated.

Dear Skyline support,

I've used LC-IM-MS/MS to separate small molecule isomers. The isomers have the same m/z, retention time, and MS/MS spectra, they could be srperated by diffrernt drift time(CCS). I hope to calculate the ratio of these isomers according to their LC peak area, not the IMS spectra intensities or area. Could skyline extract the LC area of isomers which separated by ion mobility?

Looking forward to hearing from you!

Thanks,
Anne

Hi Skyline Team,

Today I noticed that when I try creating an IMS library for XL-peptides with "Use Results" and multiple files a different value is stored for each precursor for each file. This does not happen for "normal" linear peptides.

I know that multiple Ion mobility values might be stored to represent multiple conformers but I am not sure that is what is identifying here. So I am wondering if this was intended to allow for multiple conformer annotations or is it a glitch for only XL-peptides?

Instrument: timsTOF Pro
Skyline-daily version: 22.1.9.208

As always, thanks for your time and the great work!
Sincerely,
Juan C.

Hey, Skyline team,

I've encountered a rather odd error while trying to programmatically access a report. When I try to access a report that contains either the Raw Times or Raw Intensities column using GetReport() the program throws System.IndexOutOfRangeException: 'Index was outside the bounds of the array.'

_toolClient = new SkylineToolClient(args[0], "Skyline Tool")
_report = _toolClient.GetReport("Tool Report");

Including either one of the following lines in my tool's report file causes the error. I can programmatically access any report that doesn't contain these two columns.

<column name="Results!*.Value.Chromatogram.RawData.Times" />
<column name="Results!*.Value.Chromatogram.RawData.Intensities" />

Viewing the report in the document grid works as intended, and it's specifically when the tool calls GetReport() that I reach the error. My only guess as to the cause is that the IReport object being created is trying to store too much information, since each of these arrays are over a thousand elements long. Do you know why this is happening?

I'll also attach the report file in case it's helpful.

Thanks,
– Tommy

Hi all,

first off, I love Skyline and I'm a long time user. I really appreciate all the hard work for such a great (free!!) product.

This pertains to the latest version of Skyline with Sciex data.

I am currently using Skyline to assess some DIA runs. I use the term "some" lightly. I've loaded a smidge over 700 runs and they all seem to load okay. But when I go to review the data it is quite clear that I cannot view all the files. If I go to Manage Results, all the files are listed. If I remove a good number then I effectively remove the files I can browse freely. I assume I'm hitting some limit here. If this is the case, can you tell me what that number is, and if not, can you suggest what may be happening?

I, and others in our group, have seen similar issues when loading very large numbers of files in Skyline.

thanks,

Peter

Hi Skyline team,

I am struggling to perform absolute quant using Linear fit and 1/x*x weighing using Skyline. And the reason for this is that I am using 1 amino acid (1AA) K/R and 2 amino acids (2AA) (K/R + amino acid after K/R) heavy peptides to establish calibration curve. so I have three peptides os same sequence but three different masses (Endo light, 1AA heavy and 2AA heavy).

I am using 1AA heavy peptide with varying conc. and 2AA heavy peptide as internal standard (fixed conc.) to establish the calibration curve. This is due lack of surrogate matrix for our sample types. I am analysing the sample (unknown) by spiking same conc of 2AA IS and taking the ratio of Light/2AA to calculate the peptide conc. in unknown. All these works I am doing is in excel by exporting the sum transition peak area from Skyline separately and plotting the calibration curve.

The problem on Skyline is:
Skyline is not giving option to select 2AA heavy as IS and taking ratio of 1AA/2AA heavy to plot the curve. How can I modify the setting to achieve 1AA/2AA heavy ratio for calibration curve and Light/2AA ratio for Unknown analysis in Skyline?

Kind regards,

Hello Skyline team,

The recent change in the way the rdotp is calculated since Skyline v21.2 Released on 1/4/2022 causes us big troubles with data integrity.
We already published papers that used this value as a filter for the identification of the endogenous peptides. We are currently in a process of a publication and we cannot upload the Skyline files on a public repository because the rdotp values are not the same between the manuscript and the skyline file.

Could you at best re-introduce the original rdotp in skyline, or at least provide a way to convert the new values to the old values? This is really problematic for publication.

Thanks

Antoine

Hello Skyline team,
Thanks for making this awesome software. This has help me a lot!
I have a question about the spectral library and dotp value (in small molecule mode). I have 4 different targets in one spectral library and then I import this library into my skyline document. The spectral librayr was made using the same skyline document but importing the high concentration standard and then export to "spectral library" and then load it back into the skyline document for data analysis. However, dotp only shows up in one of my target. I would like to see dotp show up for all 4 targets. I am not sure how to address the question better, but hopefully with the attached Skyline zip this can let you understand my question and help me.

Thank you

Hi support team,
I am working on an isotope tracing experiment where we used a DIA method on a Bruker Impact II instrument to record the data.
In general I am very satisfied on how skyline handles the data but I have a problem with some ion traces.
For example sample QC-pool_2: I have two Isomers listed in the Molecule List "MGDG". One traces for the second Isomer is shown, for the first it is not. Also, and this occurs for multiple molecules and samples, the ion trace is shorten on both sides. You can also see this in the example of MGDG and sample QC-pool_2.

I uploaded the skyline document with the name 'MS012_share.sky' to the share folder. Attached you find a screenshot from Bruker DA with the EIC of the MGDG and the raw data file of QC-pool_2 in case you need to reimport this particular sample.

I hope you have an idea what is wrong here.

Best,
Dennis

Hey, Skyline team,

As the title suggests, I've been developing an external tool (in C#) and have run into a roadblock with implementing some of its functionality. I'm currently trying to write data generated by the tool to the Skyline file itself, data that can be displayed in a column of a custom report in the document grid. However, I can't quite figure out what I need to access in order to assign the data to its intended column/location in the document grid.

I've made the connection from my tool to Skyline with the SkylineToolClient object, and I can read information from it as well as an IReport object using a custom report. The goal of this tool is to read data, generate a flag based on some criteria, and then write the results back into the Skyline file (which are displayed under a column that the custom report specifies). This last part is what I've been having issue with, as the arrays that the IReport objects expose to the user don't seem to have the functionality to update the underlying data file. The values can be easily set by accessing IReport.Cells, however I have indeed realized that the array is not accessing the data itself, only a copy that's presented to the user.

Am I able to have a tool write data to a Skyline file in this way, with it eventually being displayed in the document grid? And if so, what do I need to access to do it?

Thanks for your help, and if I need to provide any more clarification then please let me know.
– Tommy

I am using the 'Import DDA peptide search' wizard to import results from a dataset generated by msfragger/fragpipe. The dataset contains peptides from human and a couple of viruses, with all the detected proteins included in the fasta library that I used during loading of the dataset into skyline. All viral peptides are in the generated spectral library, but none of them - and none of the proteins - made it into the target list, only the human proteins/peptides are there. I am wondering if I overlooked a setting that may have this effect. Any help would be greatly appreciated.

I am using skyline-daily v22.1.9.208.

Thank you,
Janos

Hello,

I just download the Skyline beta, based on a thread for reading .lcd files so that I can open my LCMS data. These data concern assessing the purity (e.g. in the UV trace) of samples from organic reactions and checking whether small molecules I synthesised in the lab have the expected mass. Is Skyline beta the right software to use for looking at my data?
After installing and running the software, I chose "molecule interface" and clicked on "Blank Document". Then I tried opening my data, either by dragging it in the Skyline or through "Open" but doesn't open it. Probably I'm doing something wrong so any help on how to easily open my data would be greatly appreciated. For convenience in case someone wants to test it, I have attached the lcd file.

Thanks a lot.

Best,
Dimitris

Hi Skyline Team,

In Skyline Proteomics interface, theoretical isotopic precursors with m/z values lower than the monoisotopic precursor were displayed. My understanding is that the monoisotopic species is of the lowest m/z (I could be wrong though as I am not familiar with the various ways of determining the isotopic envelope). I attached a screenshot here. The Skyline zip file was uploaded to https://skyline.ms/files.url (file name monomer.sky.zip).

I was trying to understand this to help interpret a related Skyline analysis in Molecule interface.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address: bobxiong@joinn-lab.com

Hi Skyline team,

When using the Retention Times - Scheduling graph I always get a totally different picture of how many transitions are concurrent in skyline compared to when I export that transition list to my Thermo method and make the same plot in this software.
For instance with a 4 min window I get max 100 concurrent transitions in skyline but in the Thermo method software I get 350...

Am I missing something or is this normal?

Greetings,
Erik

Hi,

I'm unable to install the latest version of skyline-daily on my computer. I've tried running the setup using "run as administrator" but still get the same error message "Unable to retrieve application files. Files corrupt in deployment". I have also tried re-downloading the setup file, but without success.

This is the information I get in the log:

PLATFORM VERSION INFO
Windows : 10.0.19044.0 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.8.4270.0 built by: NET48REL1LAST_C
clr.dll : 4.8.4515.0 built by: NET48REL1LAST_C
dfdll.dll : 4.8.4270.0 built by: NET48REL1LAST_C
dfshim.dll : 10.0.19041.1 (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 Files/Skyline-daily_22_1_9_208/Skyline-daily.exe.manifest
Server : Apache

IDENTITIES
Deployment Identity : Skyline-daily.application, Version=22.1.9.208, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil
Application Identity : Skyline-daily.exe, Version=22.1.9.208, Culture=neutral, PublicKeyToken=e4141a2a22107248, 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:
+ Exception occurred loading manifest from file protobuf-net.dll: the manifest may not be valid or the file could not be opened.
+ File protobuf-net.dll is not a valid Portable Executable (PE) file.
+ PE file does not have enough data.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
No transaction error was detected.

WARNINGS
There were no warnings during this operation.

OPERATION PROGRESS STATUS
* [8/25/2022 3:46:28 PM] : Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application has started.
* [8/25/2022 3:46:28 PM] : Processing of deployment manifest has successfully completed.
* [8/25/2022 3:46:28 PM] : Installation of the application has started.
* [8/25/2022 3:46:28 PM] : Processing of application manifest has successfully completed.
* [8/25/2022 3:46:40 PM] : Found compatible runtime version 4.0.30319.
* [8/25/2022 3:46:40 PM] : Request of trust and detection of platform is complete.

ERROR DETAILS
Following errors were detected during this operation.
* [8/25/2022 3:47:31 PM] System.Deployment.Application.InvalidDeploymentException (ManifestLoad)
- Exception occurred loading manifest from file protobuf-net.dll: the manifest may not be valid or the file could not be opened.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.Manifest.AssemblyManifest.ManifestLoadExceptionHelper(Exception exception, String filePath)
at System.Deployment.Application.Manifest.AssemblyManifest.LoadFromInternalManifestFile(String filePath)
at System.Deployment.Application.DownloadManager.ProcessDownloadedFile(Object sender, DownloadEventArgs e)
at System.Deployment.Application.FileDownloader.DownloadModifiedEventHandler.Invoke(Object sender, DownloadEventArgs e)
at System.Deployment.Application.FileDownloader.OnModified()
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.IO.IOException
- File protobuf-net.dll is not a valid Portable Executable (PE) file.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.PEStream.ConstructFromFile(String filePath, Boolean partialConstruct)
at System.Deployment.Application.Manifest.AssemblyManifest.LoadFromInternalManifestFile(String filePath)
--- Inner Exception ---
System.IO.IOException
- PE file does not have enough data.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.PEStream.PEComponent.ReadData(FileStream file, Int64 position, Type dataType)
at System.Deployment.Application.PEStream.ResourceDirectoryEntry..ctor(FileStream file, Int64 address)
at System.Deployment.Application.PEStream.ResourceDirectory..ctor(ResourceSection resourceSection, FileStream file, Int64 rootResourceAddress, Int64 resourceAddress, Int64 addressDelta, Boolean partialConstruct)
at System.Deployment.Application.PEStream.ResourceSection..ctor(FileStream file, SectionHeader sectionHeader, Boolean partialConstruct)
at System.Deployment.Application.PEStream.ConstructPEImage(FileStream file, Boolean partialConstruct)
at System.Deployment.Application.PEStream.ConstructFromFile(String filePath, Boolean partialConstruct)

COMPONENT STORE TRANSACTION DETAILS
No transaction information is available.

Hi,

I hope you're well

I'm having trouble training a mProphet reintegration model that can separate targets from decoys. I have followed the advanced peak picking tutorial, but I am uncertain if there is an error in my Skyline document settings (or my spectral library) that may be contributing to this.

I had set my peptide and transition parameters in Skyline and then imported a spectral library generated by protein pilot (for approx. 8000 human proteins including PepCalMix iRT peptides) and generated decoys peptides (same number as target peptides) using either the shuffle or reverse sequence method. After importing DIA data, I trained a mProphet reintegration model but it does not separate the target peaks from the decoys. Also, I want to integrate peaks with a q-value <0.01, but most of the peaks in the model have large q-values

I noticed that multiple features were greyed out (retention time difference, library intensity dot product, etc) - I deleted the unknown peptides to use these features, but that did not improve the model.

If I integrate peaks with the default method, I get approx 1500-2000 proteins at q-value <0.01. I can identify a similar number of proteins (q-val <0.01) in my samples using the same spectral library with other software. I'm aware that the default method works quite well, however as I have a large target list, I was hoping to use the mProphet model for more accurate peak picking. Any support to understand and rectify this situation would be much appreciated.

Kind regards,
Susannah

My lab is just starting to collect DIA data on a Lumos and we've run into an issue while testing. After we do a DIA run and attempt to build a Skyline document there is an error (ERROR: No spectra were found for the new library) at the end of the DDA Search step of the import peptide search process. Our assumption is that there is some issue with the instrument's DIA settings, but we can't find an actual issue. I've attached the error message, the instrument settings, FASTA file used, the .raw file (collected on a sample of BSA tryptic digest), and the .mz5 file that Skyline made.

Hello!

We're having a recent problem where the import of MRM data specifically from our Agilent 6495 system seems to be slower than normal. Specifically data import quickly reaches 99% then hangs for about 15-20 seconds per data file before completing the import. I'm running Skyline Daily 22.1.9.208 on a Windows 10 workstation. Import speeds for equivalent data generated with a Sciex QTrap 6500 is normal. Any assistance would be greatly appreciated.

Thanks very much,
Vincent

I'm working with the latest Skyline daily on a Targetted PRM on a Fusion Lumos. The Retention Times - Replicate Comparison pane is showing a single data point in the graph instead of the usual start-end time bar information. The Retention Time Peptide Comparison pane (see images attached) shows bars starting form 0 time. This is not as easy to visualize as the start-end of integration boundaries which allows a quick validation of integration process. Was this changed for any reason??

Hi there!

I am working with the Bruker timsTOF Pro instrument and have collected DDA-PASEF for a set of lipid standards. After using LipidCreator to create a transition list, replicate data from a dilution series was analyzed.

When adding "Ion Mobility MS1" to the Document Grid, the column shows up as NAN for every row. I'm not sure if I've missed something in the document setup?

Any help would be appreciated and thank you in advance!

Dear Skyline developers,
Thanks for developing and continuing to update this great tool.

We would like to be able to identify scans in which particular diagnostic ions occur, regardless of the precursor. Our modifications are Lysine linked ubiquitin or Sumo type peptide remnants. The mods are 4-6 amino acids long. We are running an Q exactive HF in standard TopN DDA mode. The modification itself is a peptide, and upon HCD fragmentation produces a predictable pattern on neutral losses (which remain attached to the parent peptide) and released diagnostic b ions. I've attached an example spreadsheet of one such tryptic remnant, from tryptic digest of the human sumo-like protein UFM1.

The spectra for these modified peptides is normally quite complex and we don't get many good matches using PD or maxquant. If possible I would like to use skyline to identify what scans contain these diagnostic b-ions. Once I know that, I can make an inclusion list to re-run the same samples, acquire better quality spectra, enabling us to get and ID and modification site assignment.

Can you please tell me if I can use skyline to analyze transitions only, without considering what precursor they came from?

Thanks!
Janelle

Long story short: We recently got a Lumos with Faims source, and I run our BSA samples we run as QCs using CV of 40, 60 and 80 in DDA mode. I created a skyline document using the Import DDA Peptide Search, using the .raw files and CRAP Fasta file (Contains BSA) and Fragger (I tried Amanda too). I get the expected proteins and peptides, but the XICs appear rugged, which I suspect corresponds to the traces for the different CVs.
I tried the Ion Mobility library correction (as suggested in previous post), but this doesn't seem to fix the issue. Basically, I'd like Skyline to filter the CV with the best signal for each precursor. Could this be done? Attached my skyline file.

Cheers

Alejandro

Hi,

I got an error after I imported the MRM results file, "chromatogram information unavailable" for only some of the specific heavy labelled peptides of all replicates, all light peptides and other heavy peptides are fine. I've attached the screenshot, could you please let me what to do to fix it?

Thanks and best wishes,
Channing

Dear Skyline Team,

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

Regards,
Arttu

Hello Skyline Team,

We started using InstantPC to label released mAb N-glycans to study and analyze N-glycans. For this purpose, a Waters QTof system and an MSe method were used. We could analyze our data using Skyline small molecule interface. However, this analysis was based on the precursor ion and its multicharges. As you know, some of the glycans have the same masses and using precursor ions will not let to differentiate these glycans. We are interested in using MSe and MS/MS data to correctly identify N-glycans. It seems that N-glycat library is the one that can help for this purpose. I could not find this library in your website. Not sure if it actually exists. If not, can you help us know how to use MSe data for correct ID? Is there any other library that can use MS1 and MS2 data to assign detected N-glycans? Most of the N-glycans that we are trying to identify is neutral or monosialylated.

Thank you,
Mary

Hi,

I am trying to update the gene field for proteins. I was following this posting #25357, clicked 'populate' and 'genes' and then followed the steps described. After the procedure finished, I got a new name - instead of the original e.g. NP_000012, now it is PSEN1. I'd like to keep the original name but add PSEN1 to the gene field. Is there a way to do this?

Thank you,
Janos

I tried to export the method to MassLynx using Skyline, but the number of channels for peptides containing two or more modified amino acids was limited to five.
In such a case, is there a way to add 5 or more channels?

Kind regards,
Ryuta

Dear all,
Why in some cases when I do the group comparison with scope protein some proteins are not display while in the scope peptide yes?
Best regards

Hi Skyline Team,

Is there a way to manually set the ID marker in the chromatogram? I would like to explore the exported peak area data based on the manually "identified" peaks rather than what Skyline picked out. Screenshot attached.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address: bobxiong@joinn-lab.com

I'm trying to install MSstats tool. I installed R-3.6.1 in Documents\R\R-3.6.1, then added the tool of MSstats, it automatically installed R-4.1.2 in Documents\R\R-4.1.2, then it installed some files and stopped with error message of "Unknown Error installing packages. Output logged to the immediate Window".

Immediate Window:
A version of this package for your version of R might be available elsewhere,
see the ideas at
https://cran.r-project.org/doc/manuals/r-patched/R-admin.html#Installing-packages
Error in loadNamespace(x) : there is no package called 'BiocManager'
Calls: loadNamespace -> withRestarts -> withOneRestart -> doWithOneRestart
Execution halted

It looks like "BiocManager" is missing. Could you help?

Thanks,

Dear Skyline team,

I hope you are doing well. When I have set up the small molecule iRT calculator and begin integrating peaks using the "Show XXXX Score" display, I'm encountering a few issues after successful calibration:

1. The windows are not updated from "Retention time" to "XXXX Score" for both the file-specific chromatogram window and the "Retention Times - Replicate Comparison" window. This appears to happen the first time I click on each molecule, but after I click back and forth, Skyline then displays the right window.
2. The first time I integrate inside the file-specific chromatogram window when it is set to "XXXX Score", the display bugs out and the integration appears to be different from what I selected in the window file-specific chromatogram window. I think it is setting the iRT value used for integration as the retention time instead of the iRT score (e.g. integration from 1 to 10 score instead integrates 1 to 10 minutes in the retention time view).

The Skyline file used for this is attached. Happy to give more detail.

Cheers,
Chris

Hello,
I am trying to open .sky or .skyd on Skyline v21.2.0.568 and it throws an error - see attached. This same file would open on someone else's PC. Are there dependencies that I need to install to be able to open Skyd file? I am having hard time understanding the error message? I have the most updated skyline version and the person who made these files also has the most updated version, so in theory the file should be compatible.
Please let me know,
Shankha

Hello,

I use the Bruker timsTOF Pro, and wanted to export an isolation scheme through Skyline. I created a scheme under Transition settings and did the following:

File --> Export --> Isolation list --> selected Bruker timsTOF and set the run duration

After saving the .csv file, no file seems to be created. I'm not sure if I missed something on my end. Any help would be appreciated!

Best,
Premy

Hi all,

I am during an experiment with target proteomics with non labelled peptides. My targets are fungal proteins in infected plant tissue. The goal of my study is to detect and then to quantify the proteins. I am trying to find the most sensitive way to detect them. I try to determine the retention time of the proteins of interest (350), but as I expecten the range of the retention time is really big, so I don't know if something like this can load it in the instruments. So, my question is , do you beleive that there is a limit of proteins when you want to predict retention time using PRM?? The other alternative is DIA running method. Does anyone has the same issue?

Thank you in advance for your response,
Best
Tasos

Hi there,

I'm on Skyline-daily version 22.1.9.208, using the small molecule interface and ion mobility filtering. I am trying to rearrange the tab order of my chromatograms to match the order of the results. However, when I click and drag a tab, it does not stay in the spot it is dropped. Instead it jumps two tabs to the left of where I tried to move it. This is really just a minor inconvenience, but it would be nice if the click and drag rearranging worked as expected.

It would be even nicer if there were an upgrade so that when you rearrange your results in Edit > Manage Results, the tab order automatically changes to match.

Thanks!
Anna

Hi. A group comparison bar graph plots the Log 2 fold change with error bars. If I right-click to "Copy Data" from the bar graph and paste in to excel, I get column headers Protein, Log 2 Fold Change, and StdErr. I have multiple comparisons so I made a custom report to export the information I want. I added a column to export "Standard Error"; however, "Standard Error" does not equal "StdErr."

What is the difference between the 2 standard error values?

Thanks in advance,
Caroline

"Copy Data" results:
Protein Log 2 Fold Change StdErr
ALDOA 0.317333532 0.097111714

Report export results:
Protein Log2 fold standard error
ALDOA 0.3173 0.049317949

Hello!

I was wondering if there was a way in Skyline to display the TIC with labeled peptides. I'm new to the software, so as I'm progressing through the tutorials (specifically, the Full-Scan Filtering one), I'm trying to see if I can label each peak with the peptide that it matches to and then display the entire chromatogram (instead of clicking on each peptide and seeing a portion of the chromatogram).

Thank you so much!

Hello,

I am using the molecule interface of Skyline in conjunction with the ALIS system to perform RNA/protein screens looking for novel chemical matter. Since I will be screening for novel binders, I need to have a peak integration method that identifies the correct peak. I cannot go through each chromatogram to perform a manual integration as that will not be feasible due to the sizes of the screens. Instead, I have to rely on applying different filters in the exported CVS file to filter out nonspecific binders from specific binders and trust that the initial peaks identified are the correct ones. I have tried to optimize the peak integration method by spiking in known "weak" binders into pooled compound wells of 250-300 compounds per well and seeing if the correct peak is integrated. I am doing this as we don't expect to identify very strong binders in the initial screening process.

However, in some cases, it identifies the wrong peak and a peak with an idotp score much lower than the correct peak with the much higher idotp score. I have tried to look under the "refine" tab and the advanced section and modify the parameters, especially the minimum idotp value. However, changing the minimum idotp value seems in mess up my method and results in the wrong compounds being identified as the peak in the respective chromatograms. I will typically know the chemical formula of all the compounds in the well and would expect an idotp score of 0.80 or greater. I have also tried to set the amount of transitions to 3 (M, M+1, M+2) and would expect to see at least main precursor and the C13 isotope. I also don't see a feature to isolate peaks based on the mass error (typically less than 10ppm) and in general, changing the parameters under the "refine" tab and the advanced section does not seem to have much of an effect. It would be super helpful if people who have experience could point me in the right direction and ways to optimize the peak integration methods so that only peaks with high idotp scores, low mass error, etc etc are identified from the chromatogram.

Thank You,
Tony Considine

I have just updated to version 21.2.0.565 and now can't access the prosit server.
Server:
131.159.152.7:8500 Server unavailable

Then in the library match window it says:
Status(StatusCode=Unavailable, Detail="failed to connect to all addresses").

I have seen another error similar when skyline 21.1 was released in 2020.

Hello,

I am using Skyline (64-bit) 21.1.0.536 for small molecules where I have isotopically labeled and natural abundance molecules. I have noticed for many of my Blanks the Light/Heavy ratio is very high due to differences in the baseline, however no actual peak is present. This large difference in baseline intensity results in a LOD larger than most of my actual standards (where a peak is visibly present & integrated).

The only potential solution I've come up with is to exclude the blanks, however I cannot calculate an LOD without them in this case. Are there any potential solutions to this problem? I'm also, worried how this will affect samples (rather than blanks) where there no peak present, but a larger baseline difference between the Light/Heavy resulting in a high calculated concentration when there is really no peak present at all.

I have attached an example image here.

Thank you,
Brianna Garcia

Hi all,

I tried creating an IMS library of cross-linked peptides with the "Use Results" option. After creating the library and re-importing the results I noticed that no IMS filtration was applied.

Closer inspection of the library shows that the name in the library does not include the appropriate notation to be recognized as a cross-linked peptide or the site of the cross-link and is only taking the "left peptide" sequence. Are there any extra steps I need to take or is this a bug?

I have provided some example files (raw data and Skyline project) in our private Panorama "MLU - AGSinz - Data Sharing" folder for testing in case it is a bug.

As always, thank you for your time and support.
Sincerely,
Juan C.

Dear Skyline Team,

We are trying to create a Skyline library using a .pep.xml file (3.31MB) generated with PEAKS, the related Spectrum file .mzXML has a size of 928MB. It takes more than 1h to create the .blib file. In case of bigger files it takes all the night. We have noticed that BlibBuild is using only one CPU, so we want to know if it is possible to improve processing speed incrementing the number of CPUs used by BlibBuild.exe which is the bottleneck.

We have installed SkylineDaily in two computers and we have the same problem in both:
-Computer 1: processor: Intel(R) Core(TM) i9-9900K CPU @ 3.60GHz 3.60 GHz, RAM: 64,0 GB (63,9 GB usable), Windows 10 Pro 64-bit operating system, sockets: 1, cores:8, logical processors: 16
-Computer 2: Intel(R) Xeon(R) Silver 4214 CPU @ 2.20GHz 2.20 GHz (2 processors), RAM: 512 GB (511 GB usable), Windows 10 Pro 64-bit operating, system, sockets: 2, cores:24, logical processors: 48

Thanks in advance for your help

Nieves Ibarrola

I'm in the habit of using the Associate Proteins... feature of skyline, which correctly combines protein names that are grouped together. A problem seems to arise when I then upload the Skyline file to Panorama. Panorama seems to have an upper limit of 500 characters for a protein name, but when many proteins are grouped together I can get very large names (from large numbers of proteins in the same group). I can manually delete some of the characters to bring the size below 500, but maybe Panorama should set an upper limit of several thousand characters. Here's an example of the error message from within Panorama:

14 Jul 2022 11:46:18,743 ERROR: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
org.labkey.api.pipeline.PipelineJobException: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:275)
at org.labkey.targetedms.pipeline.TargetedMSImportTask.run(TargetedMSImportTask.java:63)
at org.labkey.api.pipeline.PipelineJob.runActiveTask(PipelineJob.java:841)
at org.labkey.api.pipeline.PipelineJob.run(PipelineJob.java:1077)
at org.labkey.pipeline.mule.PipelineJobRunner.run(PipelineJobRunner.java:40)
at java.base/jdk.internal.reflect.DirectMethodHandleAccessor.invoke(DirectMethodHandleAccessor.java:104)
at java.base/java.lang.reflect.Method.invoke(Method.java:577)
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: org.labkey.api.query.RuntimeValidationException: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
at org.labkey.api.data.Table.insert(Table.java:751)
at org.labkey.targetedms.SkylineDocImporter.insertPeptideGroup(SkylineDocImporter.java:1319)
at org.labkey.targetedms.SkylineDocImporter.importSkylineDoc(SkylineDocImporter.java:405)
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:248)
... 23 more
Caused by: PreferredName: Value is too long for column 'PreferredName', a maximum length of 500 is allowed. The supplied value, 'A2AE18_MOUSE/tr|Q6PEE4|Q.../tr|L7N1Z0|L7N1Z0_MOUSE', was 1231 characters long.
at org.labkey.api.query.RuntimeValidationException.<init>(RuntimeValidationException.java:36)
... 27 more

Hi team,

The most recent version of Skyline-daily (21.2.1.53) is now preventing me to create a spectral library from a PTM search from the export created for Scaffold (.mzID and .mgf files). I have been using this since creating a spectral library with the export for Skyline (.pep.xml and .mzxml) simply takes too long for timsTOF data.

As Matt pointed out (https://skyline.ms/announcements/home/support/thread.view?rowId=56180), it seems to be an issue with PTMs. I can create a spectral library when only Cys carbamidomethylation and Met oxidation are considered (PEAKS DB attached search). But when I try to create it with the results of all default PTMs withing PEAKS (PEAKS PTM attached search) then I receive a warning that does not allow me to finish.

What could be the problem? Let me know if I can help in any other way.
Sincerely,
Juan C.

Hi Skyline Team,

I have an odd question about computer configuration in support of Skyline usage. I tested a manually prepared transition list of 4000 transitions on a 3G mzML DDA data file using the Molecule interface. While importing the transition list and the result mzML data seemed slow, it was not responding to any kind of clicking to view the data afterwards. The computer hung up. Please see the attached for my current computer properties.

Assume that I have generous funding, what kind of computer resource configuration should I go for? I do not want to be bottlenecked by a computer to perform Skyline work. For a disulfide linked tri-peptide molecule, the transition list can easily reach 50K transitions to consider a combination of multiply charged precursor and product ions. I expect it to become even more demonding if multiple replicates of mzML data are loaded in Skyline.

I would greatly appreciate any suggestion/recommendation.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address: bobxiong@joinn-lab.com

Hi Skyline developers,

I am using Skyline version 21.2.0.425 for DIA files and typically add all library peptides into the document. Recently we have decided that our data analysis works just fine with maximum of 10-20 peptides per protein, and additional peptides only work to slow down file import. Thus I setup a blank document with these settings update before adding library peptides to document:
Peptide Settings>Library: Limit 20 peptides per protein
Peptide Settings>Filter: Auto select all matching peptides

I still get more than 20 peptides for some proteins whether I select "Pick peptides matching: Library" or "Pick peptides matching library and filter". The only way I can get the limit of nPeptide/protein enforced is to go to "Refine>Advanced" and tick "Auto-select peptides". This refinement takes some time to run and results in repeating sequences within proteins (but still unique to their respective proteins) being introduced as duplicate peptides, and the rest of the peptides within the top 20 rank going missing.

In this example using rank 1 GTYSTTVTGR in the protein APOA_Human, only the first instance of this sequence is present in the document when no limit on nPeptides/protein is applied. Ranks 2-20 are occupied by other peptide sequences, with many more other peptides. At this point I had checked for duplicate/repeated peptides in the document and found none. However, when the limit of 20 peptides is enforced using "Refine", each occurrence of GTYSTTVTGR in the protein is re-ranked and counted as separate peptides.

How do I get Skyline to only keep one instance of GTYSTTVTGR as rank 1 and the other peptides ranked 2-20 in the document? Is there also a way to limit the peptides when the library peptides are being added to document, instead of adding all and then removing excess peptides?

I'm uploading the document "20220712_template.sky.zip" on the filebox.

Best regards,
Liyan

Hello,

I'm running a method which combines scheduled MSX MS1 scans with scheduled non-MSX PRM scans and the MS1 scans don't seem to import properly. In the attached image the MS1 data cuts out partway through the peak, this corresponds to where the scheduled acquisition window of one target ends and so the scan filter in the raw data changes. The data is there as extraction works fine in Xcalibur.

This is running on a QE+ with xCalibur 4.0. Skyline 21.2.0.536. Let me know if there is s private location I can upload the raw and project file to.

Thanks,
Simon

Hello,
I am working on bacterial samples and want to use skyline to process my data.I am trying to normalize my samples using the optical density readings.Is there a way I can import these OD readings into skyline for normalizing my data before processing?

Thanks in Advance,
Keerthi.

I have iRT values predicted for several standard peptides by prosit in my spectral library. I would like to add them as iRT standards with iRT values predicted by prosit (see attached screenshot). My questions are:

1. is there a way to do this in 1 click, i.e. to copy iRT values from the library?
2. if there a way to export the iRT values from the library with corresponding peptide sequences, so I can copy and paste the table?

Hi Skyline Team,

I am using Skyline as a visualization tool to display chromatograms for verification of data analysis results produced by vendors' DDA data analysis software. While going about this, I discovered that the chroma graph does not show any peak when my transition list contained say 1000 entries. Is there any way to not show the ion names in the chroma graph? My guess is that the ion names took all the space leaving no room for showing peaks. I have attached a screen shot on 40 entries in the transition list to help explain my experience in Skyline.

Thank you so much,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
E-mail address: bobxiong@joinn-lab.com

Dear customer service,
I am running product ion(MS2) experiment on thermo orbitrap. I want to process data with skyline. However, I can only process the SIM and MRM data but don't know how to process MS2 data for quantitation. Could i view spectrum by skyline and pick the strongest product ion to quantitation with skyline? Is there any video or manual can help with this?
Thank you a lot.
Xue(Katie) Shi

Hi there,

We've been using the Sciex TripleQuad7500 in our lab, which generally generates .wiff and .wiff2 files. However, during more complex analyses (e.g. positive and negative mode combined) only .wiff2 files are generated.

So far, I have not been able to import these .wiff2 files into Skyline. Is there a way to fix this, or could you possibly include this in a new update?

Thank you!

Hello all,

We have some DIA data generated from digestions with chymotrypsin. We were wondering if Skyline supports chymotrypsin search for DIA.

Best,

Yadira

I’m trying to generate a mobility library, so far I have curated my target peptides and transitions. I have also made the following changes shown in the screenshots attached.

Hi,

Coming from nanoflow QE area and recently adapting to a timsTOF, one thing that is pretty handy is the early-run calibration. Briefly, a loop is filled with calibrant (e.g. sodium acetate/formate + agilent tunemix) and is pushed into the MS while in the dead volume (e.g. 0.1-0.3 min window). This allows through the Bruker software "dataanalysis" to perform a post acquisition calibration for m/z and IMS (tims), which is really nice to reach sub 2 ppm mass and ~ 1 % CCS precisions. This is also implemented for nanoflow with different procedures.
Are you considering adding this kind module to Skyline which would be awesome for both proteomics and small molecule analysis ?

Thanks again for developing Skyline.
With kindest regards,
Vivian

Hi, I am using SkylineRunner to automate the processing of different dataset. I am wondering are there any commands to import a peak boundaries file and analyte concentration for each replicate?

Hi Brendan,

During a conversation recently you mentioned that there were 'buried' some calculations in Skyline related to predicting CE migration time based on solution charge and size. I'm just placing this message here as a formal feature request to discuss exposing those predicted migration times.

Cheers

Will

How does Skyline calculate the "score" that appears on library match MS2 spectra, and why are many scores 0.000000 when the score of the MS2-sequence match is not zero? Is the Skyline "score" used in any way? Thank you.

Why are the displayed mass errors incorrect? Please an example in the attached PowerPoint file. Thank you.

Hi Skyline developers,

We run DIA on plasma and serum samples in our lab. We have an in-house library generated from fractionated DDA data that gets updated several times a year when we have a new cohort. This has been done on Skyline 19.1 for past two years. The library searches were done on Proteome Discover 2.3 or 2.4.

We are now setting up Skyline 21.2.0.425 on a server and we also have a new cohort to add on to the library. We know that the library PD search on each of our cohorts has around mid 1000+ proteins, and the concatenated library would have 2000+ proteins at most. However one of the library files is giving me an additional 5000 proteins when I add it to the document. This happens whether I append .blib files or select multiple libraries under peptide settings.

Could I send you the problematic library file to take a look?

Hey all!

Does anyone have any experience comparing an instrument-automatic optimisation process to select the best voltage settings vs Skyline's CE optimisation? Does Skyline also optimise other voltage settings (such as interface voltage, DL bias, Qarray RF, Q1 and Q3 pre-rod bias)?
I'm using a Shimadzu LC-MS/MS 8060NX (QQQ).

I've been using skyline for a proteomics project to quantify very-low abundant proteins in very rare samples.. So I'd need to have my instrument settings as optimal as can be. Just don't know if it's best to use Skyline or the vendor-specific optimisation tool.
I already have my MRM transitions selected and I know the retention times of my peptides, so it's just the final step of finetuning the triple quad before running all samples, but I'm running low on heavy standard..

If anyone with experience on a Shimadzu 8060 could help me with this, would be much appreciated!

Many thanks,
Jan

Dear Skyline Team,

I am trying to build a Skyline document using proteomic DDA data, but am running into an error at the DDA search step at the very end of the import peptide search process:

Search failed: [RawFileImpl::ctor()] Instrument index not available for requested device
Parameter name: instrumentIndex

It seems like an issue with the raw files themselves, since Xcalibur won't open them and MSConvert gives the same error as Skyline. I've seen a similar error in other support threads but none with "ctor()" as the designated cause of the problem. I've attached a screenshot of the error message. The raw file itself is too large to post, though I'd be happy to provide it any other way if you'd like. Is there any solution to this issue?

Best,
Evan

Hello Skyline team,

I have a question about the large RT shifting issue among chromatograms. I am trying to find peaks in each other chromatogram for the presence of each RT peak in a chromatogram. I have developed an automated isomer detection algorithm in Skyline and mostly, it works very well but in some cases, it doesn't work because of the large RT shifting issue. By any chance, are there any features in Skyline to realign chromatograms? Or do you know any solutions to fix the large RT shifting issue between chromatograms?

Thank you,
Manhoi

Dear Skyline Team,

I do targeted mass spectrometry for phosphopeptides. Since the same peptide can be phosphorylated at different positions, it is crucial to have a specific fragment ion for site localization. Is there a function in Skyline to somehow highlight a selected fragment ion in a peptide sequence? For example, highlighting amino acids with another colour or a rectangle (see attachment). This would allow a quick visual assessment of the role of the fragment ion for PTM site localization. Now I'm counting with my eyes and moving a pencil on the monitor, both suffer :)

I really appreciate any help you can provide.

Kind regards,
Nadzeya

Dear Skyline Team,

I started to use Skyline recently. How can I export information about amino acids with a particular modification and their position in the protein? For example, I want information about phosphosites in the following format: protein, AA in one letter code and position in the protein.
I found the First position and Last Position (number) of the peptide in the protein. I can calculate the phosphosite position in Excel: find AA before [+80] and then add its number to the "First position" reported in Skyline. Undoubtedly, it would be nice to get information about phosphosite from Skyline directly.

Many thanks for considering my request.

Kind regards,
Nadzeya

Hi everyone!

I have a simple question: I am working with compounds that react covalently to Cys on targeted proteins and I would like to ask if there is a way to automatically selected peptides containing Cys, similarly to the "Exclude peptides containing" in the Peptide Settings.

Thank you very much!
Stella

Skyline daily was not able to update, so I uninstalled it, downloaded the setup.exe file and double clicked. At some point a window said that skyline couldn't be installed and that I should contact the administrators of the software. There was a 'details' button that provided the following information:

PLATFORM VERSION INFO
Windows : 10.0.14393.0 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.8.4290.0 built by: NET48REL1LAST_B
clr.dll : 4.8.4526.0 built by: NET48REL1LAST_B
dfdll.dll : 4.8.4290.0 built by: NET48REL1LAST_B
dfshim.dll : 10.0.14393.0 (rs1_release.160715-1616)

SOURCES
Deployment url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application
Server : Apache

IDENTITIES
Deployment Identity : Skyline-daily.application, Version=21.2.1.514, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil

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:
+ Value does not fall within the expected range.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
No transaction error was detected.

WARNINGS
There were no warnings during this operation.

OPERATION PROGRESS STATUS
* [6/17/2022 8:26:54 AM] : Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application has started.
* [6/17/2022 8:26:55 AM] : Processing of deployment manifest has successfully completed.

ERROR DETAILS
Following errors were detected during this operation.
* [6/17/2022 8:29:33 AM] System.ArgumentException
- Value does not fall within the expected range.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.NativeMethods.CorLaunchApplication(UInt32 hostType, String applicationFullName, Int32 manifestPathsCount, String[] manifestPaths, Int32 activationDataCount, String[] activationData, PROCESS_INFORMATION processInformation)
at System.Deployment.Application.ComponentStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.SubscriptionStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.ApplicationActivator.Activate(DefinitionAppId appId, AssemblyManifest appManifest, String activationParameter, Boolean useActivationParameter)
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)

COMPONENT STORE TRANSACTION DETAILS
* Transaction at [6/17/2022 8:29:32 AM]
+ System.Deployment.Internal.Isolation.StoreOperationSetDeploymentMetadata
- Status: Set
- HRESULT: 0x0
+ System.Deployment.Internal.Isolation.StoreTransactionOperationType (27)
- HRESULT: 0x0

I created a transition list with the species name, formula, charge state, adduct, CCS, and drift time but not able to observe the drift time and CCS values when I load in a datafile. I can observed drift times from the transition settings and use results to build a CCS database but that does not include isomers found where multiple conformers are observed for a species. Any suggestions?

hello Team,

After generating a speclib. file (from DDA data) and comparing my .raw data files (DIA and DDA, in separate Skyline studies) with it on left panel I am getting a list of proteins (and associated peptides).
But while exporting through peptide ratio results I am getting blank sheets, even view > document grid looks empty.
Even after doing Refine > Associate proteins, it doesn't look promising.

I am interested in
Peptide
Protein
Replicate
Precursor m/z
Charge
Product m/z
Charge
Fragment ion
RT
Area
Background
Peak rank

these column heads for this analyzed data.

Please assist me with this.
Regards.

Hi!

I want to explore the MS1 signal of some samples in which proteins were phosphorylated with heavy ATP. I know that the gamma phosphate of my heavy ATP has two Oxygen-18 atoms. Hence when I type in the formula in both Maxquant and Skyline, I get a mass of 83.9748. I did my database search using Maxquant and now I am trying to import the results into Skyline to explore the MS1 signal.
For some reason, it seems that Skyline is not recognizing the Phospho-O18 (STY) modification I've created, since the wizard for importing the search results doesn't suggest its addition (in contrast to when I do the exact same thing using results from a sample with light ATP, meaning the regular Phospho STY (80) is added). But most importantly, after the wizard finishes importing the raw files, the 18O modified peptides are not detected.
To me it seems like Skyline is filtering these peptides out during the import of the database search results, hence they are not in the target list and the chromatograms are not extracted.
I have already confirmed that the 18O peptides are there (they are detected by Maxquant and I've seen their MS1 signal in Qualbrowser)
Any ideas on how to fix/solve this?
Thanks in advance for the help!
Best regards,

Juan M. Valverde

Dear Skyline developers,

We are currently working on a Waters Synapt G2-Si instrument in IM-MS mode. We have been searching for a way to analyze the samples using Skyline, including CCS/drift time values, but we haven't found a tutorial on that (perhaps we haven't looked well enough?)

Could you please direct us to a tutorial on converting the Waters .raw files with MS and Ion Mobility information for their use on the Skyline software?

Thank you in advance for your time.

Best regards,

F. J. Díaz-Galiano

Hello,
Multiple members of our lab have started to use SkyLine and have posted questions here in the forum. Each time we appreciate the answers and the help you have provided. Thank you. Generally, we work on small molecules so the recent additions to SkyLine in that realm are helpful.

In a current project I have three sets data for each molecule: unlabeled, 13C labeled, and D5 labeled. I have 13C- and D5-labeled compounds for different standard curves (in the same sample). Hence, this is a use case not within the standard use of SkyLine and I have setup a specific set of molecule names so I can process peak areas that I export out of SkyLine (as raw peak areas, not normalized, no ratios, etc.). We do our quantification on MS1 peaks. However, we use the MS/MS peaks to verify the identity of our known compounds. Hence, even if there is only one MS2 scan with a transition, we find it useful. To make this work I have taken a few extra steps. Thanks to the help you have provided here on the forum (e.g. here, here, and here) I have set the transition settings/full scan to DIA and blanked out the explicit retention times when importing the transition settings.

This gets me the MS1/precursors for the unlabeled, 13C labeled, and D5 options. However, I am still missing some transitions. In the PowerPoint file I show an example with tryptophan where I only see one fragment (shown in MZmine and SkyLine) and a second example with tryptamine where I see the two fragments I am expecting. The missing MS2 peak is the lowest intensity, but it still appears in 6+ MS2 scans so I know it is there.

Any thoughts on additional settings I may try to have SkyLine show me all (or more) of the MS/MS peaks?

Thanks again for your help.
Krista Longnecker
Woods Hole Oceanographic Institution

One detail - I also uploaded a zip file (from File/Share) that is Longnecker_LowIntensityMS2.zip, and the two *raw files to the FTP site.

Hello

Are there any online skyline courses planned in the near future, or is everything going to be in-person from now on?

Thanks!

Ronen