support

Welcome to the Skyline support forum. If you have a question about using Skyline, or if you encounter a problem, you can post your questions here.

It is likely that your question has already been asked and answered.  Please use the search box in the upper right corner of this screen before posting a new question.

Support is provided by the creators of the software, as time allows, though we hope others will share their experience as the user community is now quite large.

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

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When you post a question, please include the following information:

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

If you are including text output from a tool, please attach files to your message, rather than pasting in long text.

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that .sky.zip file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
Generating a mobility library
(7 responses) alan mckenzie-coe 2022-06-06

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.

 Skyline_Error.docx 
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Peptide format from IMS library not recognized
(5 responses) Juan C. Rojas E. 2022-06-27

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.

 IMS_library_error.PNG 
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Early-run m/z + IM calibration (timsTOF / any Q-TOF ?)
(6 responses) v delcourt 2022-06-30

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

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Command line for importing peak boundaries and analyte concentration
(4 responses) jianqiaoshen 2022-06-29

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?

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Error when creating spectral library from .mzID and .mgf export from PEAKS
(3 responses) Juan C. Rojas E. 2022-06-30

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.

 Cytochrome_C_PASEF-DDA_PEAKS_PTM_20logP_mgf.zip  mzid_error.pptx 
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feature request, predicted 'score' for predicted migration time for CE in Skyline
Will Thompson 2022-06-29

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

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How to improve BlibBuild CPU usage
(6 responses) nibarrola 2022-06-22

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

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Library search result "score"
(1 response) mmr6q 2022-06-29

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.

 Library Match example.JPG 
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Mass error
(1 response) mmr6q 2022-06-29

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

 Mass error example.pptx 
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Adding library peptides to document giving impossibly large number of proteins
(13 responses) Liyan 2022-05-24

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?

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Skyline Tuning optimisation vs instrument software Tuning optimisation Shimadzu 8060?
(1 response) jxg918 2022-06-27

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

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Strange ppm mass error averaging
(10 responses) jrenders 2022-06-24
Hi there,
I am observing some strange ppm mass error averaging for a signal in my document. Basically, when I choose to integrate an entire peak, the average mass error is calculated as 0 ppm. I understand this to be an average of all precursor scans across the peak (from previous related questions). However, if I integrate any individual point in the peak, there is no section that has a ppm mass error under 8ppm. Additionally, when I browse the raw data in Freestyle (Thermo) there are no MS1 signals that are within 25 ppm of the mass in question.

This leads to three questions for me:
1. Why does averaging several signals that all have ppm mass error's greater than 8, lead to an average of 0?

2. How can two different software platforms have such a different interpretation of of what the ppm mass error is when the theoretical mass is the same in both cases. There is only one formula for ppm mass error right (delta mass/mass * 10^6)? I see this discrepancy between mass error calculations often in Skyline (vs Thermo's Freestyle) and in some cases it actually leads to quite a few false positives based on exact mass.

3.Is there any way to have skyline filter out MS1 signals that are above a certain ppm mass error threshold? For example, based on my instrumentation and mass accuracy I don't care to see signals that are greater than 5 ppm at all in the MS1 dimension.

I have attached screen shots and my skyline doc. Let me know if you need more info. Thanks.
 Picture1.png  Picture2.png  ppm mass error.sky.zip 
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Is there any way of setting a transition with an m/z >10,000?
lam5672 2022-06-23
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Error when Importing .raw Files: "Search failed: [RawFileImpl::ctor()] Instrument index not available for requested device"
(8 responses) ehubb004 2022-06-21

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

 error message.PNG 
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Re-align RT peaks in chromatograms
(2 responses) mhhur 2022-06-17

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

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Highlight fragment ion in peptide sequence
Nadzeya 2022-06-19

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

 specific_fragments_phosphoisoforms.jpg 
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Export PTM site
Nadzeya 2022-06-19

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

 PTM site.jpg  PTM site_calculated.jpg 
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Cys occupancy and peptide inclusion
(1 response) Stella 2022-06-17

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

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Can't install skyline daily to Crucios's PC
(1 response) rj8 2022-06-17

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

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Import drift time and CCS from transition list
(1 response) rebecca glaskin 2022-06-16

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?

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Document grid looks empty reg...
(3 responses) amey shirolkar 2022-06-16

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.

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Custom made modification [Phospho-18O (STY)] is 'filtered out' during import of peptide search results
(9 responses) jmvb885 2022-06-13

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

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Waters Synapt G2-Si data
(18 responses) diaz-galiano 2022-05-23

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

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Is SkyLine filtering MS/MS peaks with low intensities?
(4 responses) klongnecker 2022-06-15

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.

 Longnecker_SkyLine_question.pptx 
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Strange error when making Library from mzML files from TimsTOF
(11 responses) mnt 2022-06-07
Hi Skyline

I am attempting to use skyline on a dataset of mine, however in the process of creating a library, the following error is generated.

---------------------------
Skyline
---------------------------
ERROR: No spectra were found for the new library.

Command-line: C:\Users\au297068\AppData\Local\Apps\2.0\V13GT215.OHN\V48609XA.R6H\skyl..tion_e4141a2a22107248_0015.0002_38ac599f018d3163\BlibBuild -s -A -H -o -c 0.95 -i IMTest -K -S "C:\Users\au297068\AppData\Local\Temp\tmpC0B2.tmp" "C:\Users\au297068\Desktop\IMTest-xiSEARCH\SkylineOUT\Compressed\IMTest.redundant.blib"
Working directory: C:\Users\au297068\Desktop\IMTest-xiSEARCH\Data\Compressed
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\au297068\AppData\Local\Apps\2.0\V13GT215.OHN\V48609XA.R6H\skyl..tion_e4141a2a22107248_0015.0002_38ac599f018d3163\BlibBuild -s -A -H -o -c 0.95 -i IMTest -K -S "C:\Users\au297068\AppData\Local\Temp\tmpC0B2.tmp" "C:\Users\au297068\Desktop\IMTest-xiSEARCH\SkylineOUT\Compressed\IMTest.redundant.blib"
Working directory: C:\Users\au297068\Desktop\IMTest-xiSEARCH\Data\Compressed
   ved pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) i C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:linje 149
   ved pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) i C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:linje 201
   ved pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) i C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:linje 157
---------------------------

I am unsure of what exactly this means, however the purpose of the test is to investigate if we can use mzML files that are converted like this. The orriginal data is from a TimsTOF, the converted mzML file is with ScanSumming in order to collapse the IM. Thus my question is if this is an error, or if this approach is not compatible with Skyline.

Best
Martin
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Online Skyline Courses
ronen gabizon 2022-06-14

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

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The document format version 22.12 is newer than the version 21.2 supported by Skyline (64-bit) .
(2 responses) aa16518 2022-06-13

Hi Skyline Team,

I am trying to open a Skyline file, but I am receiving this message as stated, "The document format version 22.12 is newer than the version 21.2 supported by Skyline (64-bit). 21.2.0.425 (e653b4c5e)." This is not my own file - I downloaded it through Dropbox.

I read a previous post with a similar issue, but this is different because I cannot seem to find a 22.12 version?

Thank you in advance for your help,
Audrey

 opening_file_log.txt 
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Required third party software
(2 responses) taperez 2022-06-09

Hello Skyline Team:

Our IT (Information Technology) department is evaluating our PC’s and software’s to determine what is pre-requisite for your instrument/test equipment product(s) to run properly. The goal of this exercise is to minimize our agencies security exposure. If possible, can you give us a list of pre-requisite software needed for your products to run properly? some examples of third party software: 7-zip, Dynamic Web TWAIN, Java, etc.

thanks!

Tatiana

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Observed transition mass
(3 responses) dan15060 2022-05-30

Dear Skyline support

I was wondering if it is possible to get the measured, observe transition mass from skyline for a specific scan in a specific sample/raw file. So something in analogy to "Raw Spectrum Ids" or "Raw Times". I'm after the transition mass(es) that I see when I look at a fragment spectrum in xcalibur/freestyle.
I'm was not able to find this in the Export Report options, I apologize if I just missed this option.

I'm running Skyline 21.2.0.369 on win10, the raw files were acquired on a Thermo Exploris 480 and the measured transition masses were accessed using Thermo Freestyle 1.6.

Thank you for your help

Dan

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Chromatogram Graph Properties
(2 responses) bobxiong 2022-06-10

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

 screenshot_bx.pdf 
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Error when inserting cross-linked peptides into document
(2 responses) Juan C. Rojas E. 2022-06-09

Hi Skyline team,

So I am trying to insert a set of cross-linked peptides into Skyline through Edit -> Insert -> Peptides... by copying a list of crosslinked peptides that I parsed from a MeroX output into Skyline format. I loaded a .txt file (attached) with these sequences in Excel and attempted to copy-paste the whole list into the window in Skyline as I usually do for other "normal" peptides, but keep getting a "This peptide sequence contains invalid characters".

Strangely, when I click inside the cell of "Peptide Sequence" and paste a single sequence from the parsed list it accepts the entry. Any ideas what is going on?

Attached is also the Skyline document with the required mods and some pre-loaded peptides.

Hope ASMS was fun!
Sincerely,
Juan C.

 MeroX_skyline_identifiers.txt  ribosome_timsTOF_publ_share.sky.zip 
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Error when inserting cross-linked peptides into document
Juan C. Rojas E. 2022-06-09

Hi Skyline team,

So I am trying to insert a set of cross-linked peptides into Skyline through Edit -> Insert -> Peptides... by copying a list of crosslinked peptides that I parsed from a MeroX output into Skyline format. I loaded a .txt file (attached) with these sequences in Excel and attempted to copy-paste the whole list into the window in Skyline as I usually do for other "normal" peptides, but keep getting a "This peptide sequence contains invalid characters".

Strangely, when I click inside the cell of "Peptide Sequence" and paste a single sequence from the parsed list it accepts the entry. Any ideas what is going on?

Attached is also the Skyline document with the required mods and some pre-loaded peptides.

Hope ASMS was fun!
Sincerely,
Juan C.

view request
Windows upgrade-to windows 11
(3 responses) paul.derbyshire 2018-10-08

Dear Skyline support team,

I am running Skyline on Windows 7 operating system but we are due for upgrade to Windows 10. Will Skyline still perform the same after the upgrade and will my Skyline documents be unaffected?

Thanks

view request
Retention Time Black Arrow
(1 response) taperez 2022-06-01

Hello:

When we exported this data, there was low peak area, we believe its because Skyline is automatically selecting the wrong "peak". Could you explain how we can automatically have skyline select the "true peak" which I believe seems very obvious in the first top box. It seems that it does not recognize the true peak because the peak boundaries are off. When we manually moved the peak boundaries, the correct peak was selected. Is there a way to set these boundaries so that the right peak is recognized? We imported multiple runs into the same file, and if you see image attached, the correct boundaries are applied on some runs, but not all runs (i.e. first top left box). Is there a reason for the file not to universally use the same boundaries? Do we have something switched on/off incorrectly?

Thanks,

Tatiana & Team

 20220531.JPG 
view request
QuaSAR and AvantGardeDIA
(2 responses) stephenaw777 2022-05-26

Hello Skyline,
Are the plugins QuaSAR and AvantGardeDIA still being used and updated? I was unable to load either plugin into Skyline. I had no issue with all the other tools. I noticed the plugins were from 2012-13 time, so I am wondering if people no longer use them since our EIS specialist could not get either plugin to load? Better alternatives? Please advise.
Thank you,
Stephen

view request
Issues importing *.wiff files in 21.2.0.425
(6 responses) matt albertolle 2022-05-27

Running on Windows 10:
This is the error I encountered

Failed importing results file sample Blank3.
[ExperimentImpl::getSIC()] Could not load file or assembly 'OFX.Core.Contracts, Version=1.0.0.0, Culture=neutral, PublicKeyToken=50cfc49bfcc958d0' or one of its dependencies. The system cannot find the file specified.

view request
Are all peptides incl. iRTs used for normalization?
(1 response) roberthardt 2022-05-31

Dear Skyline-team,

I am just curious to know if Skyline actually uses all peptides/precursors present in the document when I select "normalize medians" in the quantification settings? Or does it only use the target peptides and omits the iRTs? I am asking because I currently rely on the CiRt peptides, of which not all are present in all my samples at the same amounts. Thus taking also this peptides into account for normalization could potentially skew my results.

Best

Robert

view request
Failed to check for an update
(3 responses) Joerg 2022-05-28

Hi,
since the latest update, my Skyline Daily version fails to check for an update. It does the checking automatically during starts, but the error occurs also when I try to update manually. Am I the only one experiencing this?
Best Regards,
Joerg

view request
ERROR: Failed to find required column named 'file'.
(6 responses) zzhu248 2022-05-26

Dear Developers and all researchers,

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

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

I would be very appreciated for your help!

Best Regards,
Peter

 Screenshot 2022-05-26 143119.png 
view request
Crash when importing raw files
(5 responses) erin weisenhorn 2022-05-25

I am running Skyline Daily with version 21.2.1.485 on Windows 10 and am trying to import MRM raw files from a Xevo G2-XS. When I import the files Skyline crashes and gives no error. I have replicated this on multiple computers. Under transition settings I have the mass analyzer set to 'TOF' and not centroided as another user reported causes error. I would really appreciate help with this and would be happy to provide the raw files.

 TransitionSettings.PNG 
view request
Deconvoluted Spectra - Intact Proteins - timsTOF
Diego Assis 2022-05-24

Dear Skyline Developers,

I would like to quantify relative abundance of intact proteins isoforms from data acquired on timsTOF (mobility ON and a deconvoluted spectrum). Is possible to import this deconvoluted mass spectrum (or some file extension) and perform quantification on skyline? If yes, which Bruker's file format would be compatible with this workflow?

Thank you so much

Diego Assis

view request
DIA Analysis through command line tool.
(11 responses) jingyangzhang0222 2022-05-17

Hi Skyline Team,

I am trying to implement a Skyline workflow through the command line, referring to this document(https://skyline.ms/files/c90e7bac-8854-102e-9e41-e08d31b9c205/%40files/docs/Skyline Command-Line Interface-4_2.pdf?renderAs=DEFAULT&_docid=dav%3A%2F_webdav%2Fc90e7bac-8854-102e-9e41-e08d31b9c205%2F%40files%2Fdocs%2FSkyline Command-Line Interface-4_2.pdf).

It is very useful to open, modify and save the existing Skyline files through the command line, but there seems to be no "new Skyline Project" in the command-line options, and how can I set the work mode to "DIA"? If there is a general DIA workflow for Command Line Tool, it would be very helpful, thank you!

view request
SureQuant Method export
(3 responses) klemens froehlich 2022-05-19
Dear Skyline Developer Team,
I am currently trying to export a SureQuant method from Skyline for an Exploris system.

I get the following error message:

System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found. ---> System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found.
   at pwiz.Skyline.Model.ThermoMassListExporter.EnsureLibraries() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 1065
   at pwiz.Skyline.Model.ThermoSureQuantMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 1992
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1939
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2232

I checked the Thermo.TNG.MethodXMLFactory.dll and found it here:
C:\Thermo\Instruments\Exploris\3.1\System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll

Is Skyline looking for this dll somewhere else (where I could copy it to)? I have tried to copy the dll into older Exploris software version eg:
C:\Thermo\Instruments\Exploris\3.0\System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll
but it still gives me the same error.

Any help would be much appreciated, as the method export for sureQuant would be extremely convenient!!!

Best, Klemens
view request
Can I set up Skyline to exclude quantification results when scan numbers are too low?
(1 response) wangn2 2022-05-20
Hello there!
I got a question about the scan numbers (or also called "points across peak"?) in Skyline.
When the concentration of a target peptide is too low in a sample, usually the scan numbers of the chromatograph peak would be very small, say 3-6.
Can I exclude such peaks for quantification, meaning, ask Skyline to report intensity as N/A or 0, for peaks that have scan numbers less than a specific number, e.g. six?

Thanks a lot!
 skyline_scan_numbers.JPG 
view request
Relative quantification with surrogate standards
(3 responses) clay davis 2022-05-20

I am trying to quantify different lipid classes using several isotopically labeled surrogate standards. Once the labeled lipid internal standard is chosen as "surrogate standard" and set the "Normalization Method" to the respective standard for the other lipids, the normalized area reports n/a. I’ve looked at several ratio outputs in the report option but only the “Total Area Normalized” (Molecule list – Molecules – Precursors – Precursor Results and Area Normalize (Molecule list – Molecules – Precursors – Transition Results) seems to output a value as a percent and not necessarily the ratio or ratio x IS concentration.

  • Clay
 Skyline.JPG 
view request
Collision Energy Optimization Small Molecules
(2 responses) klemens froehlich 2022-05-18

Dear Skyline Developers,
First of all thank you very much for your continued support and an amazing software package!

We are currently starting to use Skyline for small molecule projects.
I know that Skyline can do collision energy optimizations for SRM type experiments using transition list method export as described in a tutorial.

However, we only have high resolution instruments (orbitraps) in our lab and would like to do PRM of small molecules.

Accoding to this thread:
https://skyline.ms/announcements/home/support/thread.view?rowId=55461
Skyline cannot do collision energy optimization for PRM data.

Now my question is:
Wouldn't it make sense to optimize the collision energy for small molecules even if we are using PRM? For example if I go to a publication where a compound was measured on a machine that uses a different type of fragmentation and I do not know in what range my collision energy would be optimal?
Or if only one transition is reported in a SRM experiment and I want to make sure to get the best sensitivity from my orbitrap machine for this transition?

Of course I can use more transitions in PRM but for small molecules I think that collision energy optimization is more important as you often only use one or two transitions as compared to pptide quantitation where you use more transitions.

Would it be possible to integrate PRM collision energy optimization somehow in Skyline?
Maybe by shifting the transition mass also by 0.0001 instead of 0.01 as for SRM experiments?

Best, Klemens

view request
Integration of single masses in a DIA experiment on Thermo Scientifi Orbitrap ID-X Tribrid Mass Spectrometer
(7 responses) harald schoeny 2022-05-12

Dear Skyline Team,
I am trying to integrate a single mass (184.07332 Da) on MS2 level of a DIA experiment, in which I selected a specific precursor mass range (670-900) and measured a specific product mass range (180-190). I want to obtain a sum value for a lipid class and I have already applied this method on a Thermo QExective HF (see attached publication- AIF method).
Now I have measured the same on an ID-X instrument and the MS2 was recorded in an ion trap instead of the orbitrap.
Unfortunately, I am not able to get anything in skyline although these settings worked for the HF experiment. I have tried to set the product mass analyzer in the transition settings to QIT (but also every other option) but nothing worked.
Do you have any idea, what I can try?

Thnak you very much! All the best,
Harald

 Schoeny 2022 Anayltical Chemistry.pdf  Capture.PNG 
view request
Can I specify the retention time when exporting quantification results?
(6 responses) wangn2 2022-05-18
I am using skyline 21.2.0.425.
I found that skyline doesn't always pick the right retention time peak for quantification, possibly due to ion interference from the background (I am using cell lysate as background). Since I have already got retention times of my peptide by running neat standards, I was wondering if I could set up skyline so that for a specific peptide, it only export the quantification result at a fixed R.T.
For example, by running neat standard sample of peptide A I found its RT to be 60.28 min, then by running a sample with peptide A in cell lysate, I would like skyline to export the quantification of peptide A only at the R.T. of 60.28 +/- 5 min, is this possible?

Thanks a lot for your help!
view request
Installation Skyline-daily
(1 response) dima kovalerchik 2022-05-17

Hi Skyline team,
I am trying to install Skyline-daily. I enter the link https://skyline.ms/project/home/software/Skyline/daily/begin.view? but it is written:

"Oops! An error has occurred.
User does not have permission to perform this operation.
Please contact this server's administrator to gain access."

How can I get the permission?
Thanks Dima

view request
Average Mass Error PPM
(4 responses) sarah.lennon 2013-11-27
Hi Brendan,

I'm currently using skyline Daily vr. 2.1.1.5169 to do MS1 quantification using Maxis Impact Bruker datas. In order to apply a second filter, I wanted to export the average mass error ppm. Here, it appears, that the number reporter in the .csv file is not exactly the same than the one on the top the peak indicated in the skyline file.
Do you have an explanation? Maybe I'm not exporting what I'm thinking.

Enclosed, you will find the skyline file as well as the .csv file.

Thanks for your help,

Regards,

Sarah
 20131028 6 bandes vr2rs 40000 br.csv  20131028 6 bandes vr2rs 40000 br.sky.view 
view request
Optimization of injection or fill time in Exploris 240
(1 response) danielacgranato 2022-05-16

Dear,
We are performing PRM in Exploris 240 and we would like to optimize the best injection time/fill time for each peptide, especially in the case of peptides that compete in the same scheduled window. Can this optimization be performed in Skyline? Can Skyline extract information of best injection time or acquisition time? Can Skyline help us evaluate what peptides need to improve injection time?

Thank you very much.
Best, Daniela

view request
DNA and mRNA oligonucleotide mapping using Skyline
Alok 2022-05-16

Hello Skyline Team,

In addition to protein/peptide therapeutics, there is a quite a lot going on with regards to DNA and mRNA therapeutic development. As far as I am aware, one can analyse a small number of DNA or mRNA using 'molecule interface' of Skyline. However, this is not possible when a long DNA (e.g. full length plasmid DNA) or mRNA need to be studied using mass spectrometry based workflows. Similar to bottom-up proteomics, the long DNA or mRNA needs to be digested by endonuclease(s) followed by either MS1 based mass fingerprinting or MS2 based sequencing analysis. I wonder if these kind of full length DNA or mRNA MS-based analysis features are available on Skyline. If not, then is there any plan to develop such features in future?

Thanks,
Alok

view request
Bad DIA Result, is something I set was wrong?
(12 responses) jingyangzhang0222 2022-05-03

Hello Skyline Team,

I am a university student trying to reproduce some results from this paper(https://pubs.acs.org/doi/10.1021/acs.jproteome.1c00490?ref=pdf). I have used the same setting(including Isolation Scheme, Instrument, etc.) as the paper did, but the result was not good enough to show the data's validity.

Only a few peaks show reasonable shapes, which can be observed in the official DIA tutorial. Besides, I was trying the acquire quantification data from the software, but it was not reliable, either. I have uploaded the project file(.zip) to the server, you can download it here(https://skyline.ms/_webdav/home/support/file sharing/%40files/blib_narrow_50fmol.sky.zip), is there any advice on how to adjust the settings, or to correct my operation? It would help a lot for my project if any guidance should be given.

Best,
Finn

view request
13C phenylalanine and second isotope abundance of unlabeled phenylalanine
(1 response) Sam110 2022-05-10

Hello, I have a dataset with phenylalanine and isotopically labeled 13C phenylalanine which is only one mass unit from the unlabeled phenylalanine. I wanted to know if Skyline can subtract the value for the second isotope abundance of the unlabeled phenylalanine from the 13C phenylalanine?

view request
Error message when trying to open Bruderer.bcfg on Skyline batch
(1 response) lutho 2022-05-10

Hi there,

I am trying to open the Bruderer.bcfg files in preparation for the online course currently running at the May Institute. I have installed Skyline batch and downloaded the Bruderer.bcfg file. I cannot open the file on Skyline batch and I have attached a screenshot of the error message that I am getting.

 Screenshot 2022-05-10 161435bruderer error.png 
view request
SureQuant Tutorial for Custom Method
(1 response) comtewal 2022-03-31

Dear Skyline Developers,

Would it be possible to create a generic tutorial to use when acquiring data on a Thermo instrument using a SureQuant custom method (as opposed to using the commercial kits of reagents/peptides) supplied by Thermo.

I have been trying to create a Skyline method to quantify our peptides of interest using Skyline v. 21.2.0.425 but the transitions for my peptides (heavy and light versions) are importing but I am not able to visualize them.

Thank you so much,
Susana Comte-Walters
MS Redox Core Facility
Medical University of SC
comtewal@musc.edu

view request
Support for SureQuant custom panel
(1 response) edf4n 2022-02-14

Dear Skyline,
I read in your release notes for Skyline 21.2 the announcement:
“New! Improved SureQuant support with "SureQuant" MS/MS acquisition method and method export for Exploris.
o Also added new PRM acquisition method (an improvement over the old "Targeted" method – now deprecated).”
I’m wondering if you can point me to using these features in your tutorials? I have been working my way through the skyline Targeted Method Editing tutorial. Eventually I would like to create a transition settings report for a custom SureQuant panel. I’m not sure that I am doing it correctly, especially for the export settings.
I have an Orbitrap Eclipse instrument and am using version 21.2.0.369 of skyline
Thank you!

view request
Prosit score export
(1 response) nifr 2022-05-04

Dear Skyline team,

is it possible to export the dot products from matching of my spectral library with Prosit via the document grid?

Best
Nicola

 Screenshot 2022-05-04 171832.png 
view request
Protein group, razor peptide and issue #861
ho-tak lau 2022-05-06

Hi Skyline Team,

I have been hoping to be able to remove repeated peptides in certain ways in Skyline for a long time. Recently, I saw the discussion in issue #861, and I would like to chime in.
I wonder if Skyline can integrate ProteinProphet or to read the prot.xml from ProteinProphet, and then have an option to remove/export the repeated peptides/empty proteins. Alternatively, Percolator also does protein inference now. Percolator wiki Maybe this can be an option for MSAmanda search.

Regards
Ho-Tak

view request
Which formula to choose for combining peptides intensity into protein intensity ?
(2 responses) nicolas pierre 2022-05-05

Hello Skyline Team,

I have measured endogenous peptides with their heavy counterpart by SRM. I have also spike a protein in my sample for normalisation.
I have some questions for translated results at protein level.
When I divide the intensity of endogenous peptide by their heavy counterpart it become a ratio and in many cases this ratio totally change the ponderation of each peptide for their contribution to the protein intensity. Typically, peptides representing the highest intensity (the one which would contribute the more to protein signal with the addition of peptide intensities) for the protein can, when divided by its heavy counterpart, be the one with the lowest ratio among the other peptide of the protein. In this case this will totally change the ponderation. I am not very confident with this phenomena because usually signal with the lowest intensity are those with the lowest quality (near LOD, lowest level of precision, lowest quality of the peak...). It means that with a division by the heavy peptides I will give more importance (not systematically) to signal with the lowest quality.
In fact their is different solution to combine the signal of peptide into protein signal. For instance:

-Signal of protein A= (Signal of endogenous peptides 1/ signal of heavy peptide 1) + (Signal of endogenous peptides 2/ signal of heavy peptide 2)
-Signal of protein A = (Signal of endogenous peptides 1 + Signal of endogenous peptides 2)/(signal of heavy peptide 1 + signal of heavy peptide 2)

As explained, the first formula can change the ponderation of the peptides contributing to the protein signal, the second one will not. We could imagine other solutions...Also, I have spiked a protein for normalising my signal and I do not know where I can put it in the formula, where would be the best place..
Do you have some advises for this problem ? In there some Skyline solutions ?
Thank you in advance for your help.

Nicolas Pierre

view request
Zeno MRM Wiff 2 file
(1 response) bkr 2022-05-04

Dear,

I am currently working on Zeno MRM data and I am not able to import wiff2 data into skyline.

I get the following error

"At 13:10:
Failed importing results file 'F:\20220504\GA_Zenooff_q1_open_50ms_MRMHR_01.wiff2'.
[WiffFile2Impl::ctor()] Object reference not set to an instance of an object."

Kindly help me with this.

It would be also great if you can suggest transition settings for this type of data

regards,
Bharath

view request
Collision Energy Optimisation on TimsTOF Bruker
(2 responses) karweens jean 2022-05-03

Dear Skyline Team,
I am using a Bruker TimsTOF for proteomic analysis in PRM-PASEF mode and I would like to use the skyline software to optimize the collision energies of the produced ions. I have created a file taking into account the different collision energies to test per ion.

The acquisition went well and I managed to import the file on skyline.

However, skyline refuses to display the full histogram for the different collision energies tested.

I would like to know if it is possible to optimize collision energies with a TimsTOF file on skyline.

Thank you very much for your help,
Kind regards,

Karweens

 Collision Energy Optimisation on TimsTOF Bruker.docx 
view request
Generating mass list
(1 response) tdpatsch 2022-05-03

Hi, I am quite new in the field of proteomics and therefore my request might be rather trivial. I have a database containing hypothetical proteins in FASTA format. I would need a program that imports these FASTA sequences, does the tryptic digest in silico and provides me with an MS list that I can use as an inclusion list on a Thermo QExactive Plus to search specifically for these proteins. Somebody told me that skyline can do the job. Would be great if someone could explain how to do it or refer to the proper tutorial.

view request
ERROR: No spectra were found for the new library - since updating to v 21.2
(10 responses) Francis Beaudry 2022-04-28
Hi,
 
Since I updated to 21.2, I have a very difficult time with Skyline. Sometimes it works, but most of the time it doesn’t work. Currently, we are trying to process DDA datasets and use the option DDA Raw to search and build the library. Attached, you will find the message we are getting 9 times out of 10 trials using the same raw files and search setup. Weird … Can you help ??
 
Regards,
Francis
 
---------------------------
Skyline
---------------------------
ERROR: No spectra were found for the new library.
 
Command-line: C:\Program Files\Skyline\BlibBuild -s -A -H -o -c 0.95 -i 07ADT_27avr_std_3 -S "C:\Users\francisb\AppData\Local\Temp\tmp8B1F.tmp" "C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22\Skyline\07ADT 27avr std 3.redundant.blib"
Working directory: C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.
 
Command-line: C:\Program Files\Skyline\BlibBuild -s -A -H -o -c 0.95 -i 07ADT_27avr_std_3 -S "C:\Users\francisb\AppData\Local\Temp\tmp8B1F.tmp" "C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22\Skyline\07ADT 27avr std 3.redundant.blib"
Working directory: C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22
   à pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) dans C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:ligne 149
   à pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) dans C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:ligne 201
   à pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) dans C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:ligne 157
---------------------------
view request
Normalizing Unknowns?
(5 responses) whale 2022-04-29

Hello:

I am running a peptide quant experiment and spiked the same amount of ISTD into each unknown. . However, according to the BCA assay, a few of the unknowns had a very small amount of protein (likely due to treatment) so I was unable to inject the same amount of total protein for each sample. Is there a way to normalize these small amounts (i.e. I injected 10x less material for unknown 1 vs. unknown 2)?

Many thanks!

view request
Mass Error Plots
(2 responses) philip remes 2022-04-30

When I attempt to view the Mass Error plots for PRM QIT data, no data are displayed. I don't know if this is because mass error plots are not supported for this type of data, or the errors are larger than what the tools are anticipating, or something else. It would be useful to have this feature working for the fragment ions. Ask Lilian if you need some data or a skyline document. It would let the user know what mass tolerance is feasible. A larger request would be if you could implement Jarrett's Fine Tune algorithm to calibrate the mass scale. https://pubs.acs.org/doi/abs/10.1007/s13361-012-0482-z.

Thanks
Philip

view request
Import DIA peptide search
(1 response) dilip singh 2022-04-28
Dear Team,

I am using tool "Import DIA peptide search" and getting error at last step, which is mentioned below. Kindly suggest the solution for it.

---------------------------
Skyline
---------------------------
Error
---------------------------
OK More Info
---------------------------
System.ComponentModel.Win32Exception (0x80004005): The system cannot find the file specified
   at System.Diagnostics.Process.StartWithCreateProcess(ProcessStartInfo startInfo)
   at System.Diagnostics.Process.Start(ProcessStartInfo startInfo)
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 73
   at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 162
---------------------------

Regards
Dilip Kumar Singh
view request
idotp filtering
(2 responses) lisa walrond 2022-04-26

Hello,
I am trying to filter my data based on idotp threshold (0.8). It appears when I apply this filter that peptides from all samples are deleted if peptides from any of the samples do not meet the idotp threshold. As opposed to just filtering out the specific samples that do not meet the threshold and leaving the good result. I am looking for a faster way to remove peaks from peptides that aren't real. I hope that is clear.
Thank you

view request
"Error getting score type for this file" (.mzid)
(3 responses) anita liu 2022-04-27

Hi,

I have been encountering errors whenever I try to build a spectral library by importing ".mzid" files generated from Byonic (Protein Metrics). The error message is as follows:

"Error getting score type for this file.
ERROR: Unknown exception
ERROR:
ERROR: reading file test.mzid"

This happens even when I try to import files that have previously been imported in the past (when my skyline version did not have the "score type" and "score threshold" columns). Could you please advise on how we currently import .mzid files to build spectral libraries?

Thank you!

view request
Import Sciex 7600 Zeno TOF data (wiff) - get error
(1 response) heather eastwood 2022-04-26

Hi,
I am trying to import Sciex 7600 Zeno TOF data (wiff file) but am getting the attached error. Do you know how I can fix this?

Thanks,
Heather

 MicrosoftTeams-image.png 
view request
the error messages when importing result file into Skyline
(6 responses) mei chen8686 2022-04-07

Hello,
I have two questions related to the error messages when importing PD result files. I am using Proteome Discover 2.1. The questions are in the attached file.
Thank you,
Mei

 Sklyine questions 04062022.docx 
view request
How to make skyline pick the peak with highest dot product?
(1 response) pavel shliaha 2022-04-22

I am not sure how skyline picks which peak corresponds to the peptide from library (I use extremely high RT tolerance) but it picks a peak which has a low dot product (0.3-0.5), but there are other candidates which have dot product as high as 0.95 (which I think is my actual peptide). Is there a way to specify skyline to select the peak with highest dot product?

NOTE: I am NOT asking about filtering on dot product score. If skyline selects a peak with dot product of 0.4, while there is a candidate which is 0.95 and I specify the threshold of 0.9 in refine > advanced > min dot product skyline simply removes the selected peak, but it does not help identify the peak with 0.95 dot product

view request
Retention Times Score to Run Regression questions
(2 responses) roman sakson 2022-04-22

Hi Skyline Team,

I am exploring the accuracy of various RT predictors wenn applied to my experimental data. Skyline is (as always) extremely useful here! Normally, I am using the residuals plot. I would like to understand the visualization a bit better and have some questions:

When applied within a document with several runs I can additionally get a score to run regression for all replicates or for the "best" one. I assume that these replicates have nothing to do with possible condition replicates annotated in my document and that Skyline just takes all present results as replicates here? Is the empirical retention time for each peptides just being averaged if I choose to plot all replicates or how are those combined? How is the "best" one chosen?

Thanks a lot,
Roman

view request
Import MS2 spectra from Waters Xevo TQ-XS (Survey Scan)
(7 responses) gioele visconti 2022-03-07

Hello,

I am using a Waters Xevo triple quadrupole (TQ-XS) instrument that contains MRM and MS2 scans (Survey Scan, which is the equivalent of the full scan).
I would like to use Skyline to search for specific m/z, so created an import list with the name of the precursor, its m/z and the polarity. When importing the RAW file, I can see the preview of the imported spectra (so the import step was fine) but when i select the precursor I don't have any spectral information. By looking the details with SeeMS, the Survey Scan spectra are settled as MS level "2". How can I extract this information in Skyline? Should I modify something into the transition list?

I also tried to convert the full scan spectra usign ProteoWizard but the issue was still present (and spectra are settled as MS level "2").

Thanks in advance and best regards,

Gioele

view request
Peptide Negative Ion - Transitions
(2 responses) dawn dufield 2022-04-18

Hi Guys,

Is there a way to use skyline to predict peptides in neg ion mode (precursors and transitions?)

Thanks

view request
How I can retreive the TIC?
(1 response) karin yanagi 2022-04-18

I am try to retreive the total tic current over time in TOF experiement as a normalization method and take a look to see the machine siganl over time. In the result, there'is an option to select TIC column, but I dont have any numbers in the result. Is there anything did i did wrong? I attached the wiff file that I am working with.

 StdA_30um.wiff  StdA_30um.wiff.scan 
view request
Tutorial on Prosit use within Skyline?
(4 responses) wxxx 2022-03-26

Hello,
I would like to be able to submit a list of proteins to Skyline and get an:

  • in silico trypsin digest
  • predicted RT of each tryptic peptide
  • predicted ms2 spectra of each peptide

What I would like to be able to do is to identify 'best' transitions from eac protein (ms1 and ms2) - i.e. those with highest intensity.

Via googling I figured out Prosit can do it. But I cannot find any tutorial which would discuss how to use it within Skyline (either by googling or searching this page). I would need a step-by-step guide - I rarely use Skyline and am not very familiar with it!
Or maybe I should do this by downloading a spectral library?

Could you please point me to any document that mentions prosit?

Thank you.

view request
How to extract result from two different product ion experiement from QTOF, same precrusor, same product, different collision energy
(2 responses) karin yanagi 2022-04-16

This is a run to look at free fatty acid and I have two collision energy set for each mass (for example 253.22 m/z for palmitiec acid, i setted CE as -25 and -15) I want to look at the product ion as same mass as the prevcursor in different collision enegy. They should have different intensity, but no matter how i define the collision energy in the transition list. It seems to lead me to same chromtagram.

 StdA_20um.wiff  StdA_20um.wiff.scan 
view request
Library Build from MaxQuant Results Error Message
(4 responses) brian mcdonagh 2022-04-13

Dear Skyline,
Having trouble building a spectral library from MaxQuant results and don't know the best way to solve it. Error message is attached but is an unexpected closing parenthesis found in sequence.......NETVVK(NEM)EK (line 84).
ERROR reading file msms.txt.

Any advice or help would be greatly appreciated.
Thanks,
Brian

 Skyline error in library build.PNG 
view request
Synchronized Integration - remove peak issue
(2 responses) halwaseem 2022-04-14

Firstly I have to say I LOVE the synchronized integration feature. It has reduced peak picking time tremendously!!!

I have been having this issue with all of my integrated peaks being removed when I click "remove peak" for a sample that was NOT checked under synchronized integration. I usually select all of my biological samples for synchronized integration and leave out neat standards (QC std) because I only use them for RT reference (peak areas too high in comparison to bio samples so I don't want to integrate them). Because I have explicit RT's in my transition list, the QC stds always end up getting integrated. When I click on remove peak for any QC std, this is applied to all of my samples. This happens when I have all my bio samples selected and NONE of the QC std files selected under the synchronize integration option.

I am able to get around this by unselecting all the samples, selecting the QC std files and then clicking remove peak. This only removes peaks that were selected and so I am able to selectively remove all the QC stds. I can then go back and select all my bio samples and integrate them. This is a quick thing to do with a short list of metabolites but with >100 transitions, it adds up.

Is there a way to selectively remove peaks of samples that are not checked under synchronized integration?

Notes: I am using the latest version of Skyline Daily but have had this "issue" with previous versions. I am using the molecule interface mode. This is not specific to certain raw files or transition lists. I do not have my samples grouped.

Thank you for you help!

view request
Skyline for GC-MS data
(1 response) Furuhashi Takeshi 2022-04-13

I would like to ask if there is a Sklyline software,
which can do quantification of GC-MS data (SRM, SIM and XIC)?

view request
Added capabilities for import/export of notes?
(2 responses) apickard 2022-04-13

Hi all,

Thank you again for all of the work that you're doing on this. We have started using skyline to help annotate our LC-MS methods (we're a large metabolomics core facility), and have found that adding molecule list, molecule, precursor, and transition notes and colors are extremely helpful. I'm not sure if I'm missing this, but it would be very useful if we were able to export the note color with the note so that when we get a very large (~300 compound) data table out of skyline, we could sort by note color to know what is good to go and what we need to double-check or exclude. For now, we are working around this by writing the note color at the beginning of the note, so that it exports in the notes field.

We were also wondering if it is possible to have added import functionality for notes. Right now it seems the only field we can import is "Note", which goes to the transition note, pretty far down in the tree, but we are doing a lot of high-res work, so we are typically using notes on molecules and precursors. Right now I just use the document grid to copy these notes over to those other fields so that we can see the notes easier on the UI, but sometimes we will have different notes for molecules and precursors it would be helpful to import the notes into separate fields. This again would be something great if we could also import note colors as well, but right now we go through the list anyway to check everything.

We plan on using these skyline documents for other projects, so these notes are important to alert us of things to lookout for with certain molecules in many more projects down the road.

Thanks again,
Amanda

view request
Input data
(1 response) asamaras 2022-04-11

Hello,

My name is Tasos and I a new user of Skyline. I have a problem with a protein list input. My goal is to insert a protein list with 854 proteins in a fasta file. The goal is to find the predicted peptides of these proteins after trypsin digest. The problem that I have is that when I insert my protein list, I can see predicted peptides only to the first 5 proteins in the list, but if you click to the other proteins you can see them. But if you just see the list you can't see them. This could be a limitation of the software in the number of the proteins?
Thank you in advance for your help.

Best regards,
Samaras Tasos

view request
Import NIST_2020_MSMS_HR
(1 response) abrahampe 2022-04-08

Hello,

I am trying to import the NIST20 HR MSMS library into Skyline. The most obvious file made available by NIST to upload into Skyline is the .msp file. There are multiple collision energy MS/MS spectra per compound that I'd like to have available, but it seems that after import only one CE version is available. Please advise on how to proceed.

Cheers,
Paul

view request
issue with chromatogram information unavailable from DDAsearchMS1
(1 response) diletta piana 2022-04-12

Hi, I am Diletta Piana a PhD student and I did a DDAsearch to find a specific protein and transition.
I don't know why I have identified the protein and it shows me the peptide and precursor but the chromatogram information is unavailable.
Do you know what the answer to this problem is?
How can I see the chromatogram information of this peptide?
Do you suggest me to do other search ?
Looking forward to your kind reply,

Kind regards,

Diletta Piana

 chromatogram information unavailable..jpg 
view request
Spectra became dotted lines but are still marked Quanitative
(1 response) steve drake 2022-04-08

Probably a newbie issue but I can't figure it out or find it in the support section. My spectra were working wonderfully - picking peaks, adjusting peak widths, giving dotp values and retention times (Great Program). I imported a raw data file and everything went to dotted lines and added precursor ions in my transition lists. I read that the dotted lines means they are now marked as non-quantitative but I checked and they are still marked quantitative (see attached). I started a new file, loaded a single peptide and the older libraries (without the raw file) and it is still dotted with no dotp or peak picking. I tried removing the precursor ions from the list and Refine/Reintegrate but no luck. What did I do wrong and how do I fix it?

Skyline 21.1.0.278

 skyline dotted lines.jpg 
view request
No data import when PROSIT generated library is activated
(3 responses) pavel shliaha 2022-04-08

I am trying to load in some data against a library generated in PROSIT. However I get an empty chromatogram with RT from 0-1.2min, as shown in slide 1 (my runs are 0-80 min). If I then Go to peptide settings > library and untick the library the runs load normally, as shown in slide 3, but of course dotp cannot be calculated. Any suggestions why? Note in the library the predicted RT are all over 80 min (slide 4). Could this be a problem?

 2022_04_08_skyline_issue.pptx 
view request
Cannot download or install Skyline Daily
(2 responses) mattkarasu 2022-04-07

Ive tried to install Daily here at UWMC this morning and it has thrown up this error:

Cannot download the application. The application is missing
required files. Contact application vendor for assistance.

DETAILS:

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 : 4.8.4470.0 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_21_2_1_455/Skyline-daily.exe.manifest
Server : Apache

IDENTITIES
Deployment Identity : Skyline-daily.application, Version=21.2.1.455, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil
Application Identity : Skyline-daily.exe, Version=21.2.1.455, 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:
+ Downloading https://skyline.gs.washington.edu/software/Skyline-daily13-64/Application Files/Skyline-daily_21_2_1_455/BaseCommon.dll did not succeed.
+ The remote server returned an error: (404) Not Found.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
No transaction error was detected.

WARNINGS
There were no warnings during this operation.

OPERATION PROGRESS STATUS
* [4/7/2022 10:10:13 AM] : Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application has started.
* [4/7/2022 10:10:14 AM] : Processing of deployment manifest has successfully completed.
* [4/7/2022 10:10:14 AM] : Installation of the application has started.
* [4/7/2022 10:10:14 AM] : Processing of application manifest has successfully completed.
* [4/7/2022 10:10:16 AM] : Found compatible runtime version 4.0.30319.
* [4/7/2022 10:10:16 AM] : Request of trust and detection of platform is complete.

ERROR DETAILS
Following errors were detected during this operation.
* [4/7/2022 10:10:24 AM] System.Deployment.Application.DeploymentDownloadException (Unknown subtype)
- Downloading https://skyline.gs.washington.edu/software/Skyline-daily13-64/Application Files/Skyline-daily_21_2_1_455/BaseCommon.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: (404) Not Found.
- 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.

view request
Issue with importing Skyline document
(1 response) Juan C. Rojas E. 2022-04-07

Hi Skyline team,

I tried merging the results of two skyline documents by using File -> Import -> Document...

However, this is giving me duplicate replicates that I can't figure out how to remove from the "Manage Results..." window (see attached PowerPoint).

Why are the files duplicated? Any suggestions for leaving only one?

In advance, thanks!

Juan C.

 Additional_replicate.pptx 
view request
Small Molecules - Possibility of adding specified peak rankings or ratios between transitions within a precursor
(3 responses) Allison Haase 2022-04-05

Hi MacCoss Lab,

My lab is currently using Skyline for GCMS processing.
It would be helpful if there was some way to specify the predicted peak ranking for some of the transitions within a precursor for peak identification/picking, or if there is a way to specify the ratio between two transitions within a precursor. We are dealing with chiral molecules that have very similar ions, but the ratios between the ions differ.

I have attached my Skyline document which has my current method.

Allison

view request
signal to noise calculation
(3 responses) xue shi 2022-04-05

Hi Skyline team,
I am trying to calculate signal to noise. Does skyline have signal to noise calculation option?
I also found skyline has background area calculation.
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tip_peak_calc
However, I don't know how skyline calculate background area for this case. Please find attachment for the figure.
Could you please let me know which choice skyline to calculate the s/n?
Thank you,
Xue

 skyline questions.docx 
view request
Import a large # of peptides with heavy isotope labels on c-terminal residue
(1 response) wangqingok 2022-04-03

Hi Skyline Expert Team,

I am trying to import a large number of peptide sequences. After the import, I then chose the "modifications" to have a heavy K/R, and then all the K/R are highlighted in blue. But I only want to have the C-terminal K or R to be heavy labeled, but leave the internal K/R to be just light. Is there anyway to specify a modification position, not only on a certain residue but also a location of it? Such as "C terminal".

Or is there a way to allow me to modify the peptide sequence that will be imported to have a label such as [C13N15] near by the residue that I am going to specify as heavy and can skyline recognize such "C13N15" string and take is as a dedicated modification on this peptide? Thank you so much!

view request
Import Peptide Result Issue in 21.2.1.445
(3 responses) philip remes 2022-04-01

Use File - Import - Peptide Results, and choose a PDResult file. Presumably other types of peptide result would also cause an issue. The new layout here apparently allows a Score Type and Score Threshold instead of the old method where there was no Score Type, and the Score Threshold was global for all the files imported. Here there was no ability to change the Score Type or Score Threshold, so it wasn't possible to create the spectral library.

 skyline_import_peptide_search.png 
view request
Library download
(1 response) r benoni 2022-04-01

Hello everybody,
I have a question regarding the download of library for the identification of an unknown protein. I have a pulldown of protein from the membrane of human cancer stem cell (CSC), I isolated one band and analysed, after digestion, with Orbitrap with DDA method. I try to download few library from some website in internet recommended in some tutorial but were not well read from skyline when I try to do Import peptide search. Can you recommend some website or if you have some library of human proteome (membranome) suitable for the protein identification.
I hope I was clear enough.
Thanks in advance
Roberto

view request
Prosit mirror dotp broken
(2 responses) Tobi 2022-03-25

Dear skyline team,

the library match with a direct prosit mirror is a great feature,
it just sadly takes all peaks on both sides and matches them blindly against each other,
incl. precurors and fragments which simply do not exist in the opposite spectrum leading to false dotp.

I cant even disable the dotp so for slides etc. you always would need to cover or hide it somehow.
The option not to display the dotp would be nice, or have it calculated after excluding peaks which are null on at least one side of the mirror.

Already pictured in Issue 831

Best wishes,
tobi

view request
Creating a Spectral Library from Existing Data Set
(1 response) madeline colley 2022-03-30

Hello,

I have a series of DDA-PASEF experiments, but I am confused on how to start making a spectral library for these small molecules (some lipids and metabolites). I can see there is an "export spectral library" function, which I am assuming saves your transition list into a spectral library format.

I have used lipidcreator to give me a few transition ions for some lipids I am interested in, but when I load that, I am not sure how to then look at that with ion mobility or then add the ion mobility from this specific data set into the library.

Is there a tutorial on how to do this?

Best,
Maddie

view request
Export Report Extremely Slow
(5 responses) graham delafield 2022-03-29

Hi there,

My current Skyline project includes signal extraction from a large proteomics spectral library in order to do MS1 and MS2 LFQ. The projects complete successfully, even if it takes time to chug through the library.

However, when I wish to export a report (~10-15 columns),it takes an extremely long time - sometimes 12 hours for only 50,000 rows. I have applied filters to reduce the number of rows, but there is always a significant stall when the report is 75-90% complete, regardless of the number of rows.

My questions are 1) any tips on why this may be happening? and 2) is the skyline data accessible any other way? For example, can I write some custom SQL to access file where the data is stored?

Skyline Version: 21.2.0.425

System: Windows 10 x64, i9-10940x, 64 GB RAM

Thank you!

view request
Creating spectral libraries from Proteome Discoverer result files
(1 response) johannes voshol 2022-03-25

Hi
I am trying to create spectral libraries from PD results, using Skyline's 'Import Peptide Search' functionality. When I import from the .pdResult file, the scores are not recognized and all set to 0. When I use the corresponding .msf file, the scores are imported, even though - obviously- they are exactly the same and have the same column name (PercolatorqValue). Is that intended behavior or is there something wrong?
A second question is about the SpecIDinFile parameter. This ends up being a strange looking float like -1602.53037. In all cases the number before the decimal point (here 1602) corresponds to the WorkflowID from PD, but the rest has no obvious relation to file ID or scan number as one would maybe expect. Do you have any info on how this parameter is calculated and/or if and how you could retrieve the actual scan number from it?
Thanks a lot for providing these great tools to the community!
Hans Voshol

view request
Suggestion
(1 response) carlos penno 2022-03-29

Dear all,

During importing transition list for small molecule work would it be possible to include a column to determine if a transition is qualitative and quantitative. not sure if that is already possible?

Best, Carlos.

view request
Fill-in Transition List Table Gone?
(4 responses) kchiti 2022-03-23

Hello,

I am new to Skyline and have just been using the PRM Small Molecules mode. I usually just fill in the transition list table after going to edit>insert>transition list but after updating to the 64bit 24.2 when I go to insert my transition list the only option I have is to paste it in. Is there a way to get the table back so I can directly fill in?

Thanks,
Kat

view request