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.  

In order to post to the forum, you'll need to sign-in or if you don't yet have an account sign up. Forgot your password? You can reset it using the "(forgot password)" link on the sign-in page.

You can also follow the Skyline support board through email updates after you sign up.

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
Fix for QuaSAR download error
(2 responses) jung165 2020-03-02

Hello, Skyline team

I have one question about downloading QuaSAR.
I am currently using Skyline 20.1.

When I try to download QuaSAR, it automatically downloads R 3.0 and continue to download QuaSAR related package.
However, the error message appealed with mentioning I need R 3.5 or higher version to download the packages.
So, I downloaded R 3.5 but, error message still appeals and it seems that those packages were still trying to be downloaded in R 3.0 folder.
May I ask your help for this issue??

I appreciate for your help

Skyline

The following packages failed to install:

bitops
reshape
gtools
ggplot2
gplots

view request
DIA-NN .speclib support
(9 responses) Tobi 2020-05-27

Dear Skyline Team,

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

https://github.com/vdemichev/DiaNN

With best regards,
tobi

view request
Import PRM results from Xevo QTof
(9 responses) taleb sedighi 2020-05-29
Dear Skyline Support Team, I have a problem in importing PRM result files from Masslynx (acquired on a Waters Xevo G2-XS QTof) into the Skyline. The PRM data includes one MS scans and 5 MSMS scans. Importing completes without any error but Skyline only shows the precursor chromatograms, not the product ions. Because it only reads the MS scan, not the MSMS ones. Here, I have attached the selected transition settings as a ppt file. In the full-scan tab, "Targeted" is selected as the acquisition method; so Skyline should expect multiple MSMS scans but it does not read any of them. I would appreciate your help! Taleb
 PRM_transition_setting.pptx 
view request
Boxcar support
(13 responses) DiegoB 2020-05-13

Hi Skyline team
I was wondering if Skyline support MS1 filtering on data acquired using Boxcar on the Exploris.
Thanks a lot
Diego

view request
Unable to Import Result Files due to multiple chromatograms
(3 responses) stephanie 2020-05-28

Hi,

I am trying to import results (from a byonic search) for full scan filtering. I created my library and a background proteome. I have a custom labeling system with labeled R/M and methyl groups. My samples are fractionated. I have tried adding samples as multiple replicates with multiple injections, single injection, and one new replicate but I get a similar error message (see below).

I was working with three libraries and importing multiple replicates. I am going to go back and re-build my library as a single library. Not sure if that will help.

Do you have any suggestions? If there is one peptide like this, there will be more.

Best,
Stephanie

Error Message:
At 8:44 PM:
Failed importing results file 'C:\Users\slynn\Documents\3Plex_Fraction_Skyline\ADMA Fraction\20200113_SML_ADMACombo_HILIC_10.raw'.
Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.

Inner exceptions:
Exception type: System.InvalidOperationException
Error message: Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.
Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.
at pwiz.Skyline.Model.Results.PeptideChromDataSets.AddDataSet(ChromDataSet chromDataSet) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 838
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Add(PeptideDocNode nodePep, ChromDataSet chromDataSet) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 794
at pwiz.Skyline.Model.Results.ChromCacheBuilder.AddChromDataSet(Boolean isProcessedScans, ChromDataSet chromDataSet, PeptidePrecursorMz peptidePrecursorMz, IDictionary2 dictPeptideChromData, ICollection1 listChromData, ChromFileInfo fileInfo) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 670
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CalcPeptideChromDataSets(ChromDataProvider provider, List1 listMzPrecursors, HashSet1 setInternalStandards) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 510
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 371
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

view request
Selective peak integration based on idotp
(4 responses) Fabian 2018-05-29

Dear all,

i would like to have the option to integrate only peaks above a certain idotp threshold.
At the moment, the option [refine_advanced_results] Min idotp: decides if a peptide is kept or not.
I would like to have the same option based on the peaks not on the peptide.

Did I miss sth. or are there workarounds (peak training model did not convinced me).

Best regards

Fabian

view request
LOQ CV requirement iterates from low to high concentration, but high to low concentration would be better
(1 response) philip remes 2020-05-28

Hi guys,

I'm trying to use the Calibration Curve functionality, and have set the LOQ CV requirement to 20%. I notice some strange stuff going on, where sometimes a compound will have crap CV's at low concentration, then randomly have a level with a CV less than 20% when there's just noise. This level will get picked as the LOQ, even though some higher concentrations have worse CVs. Looking at the data, it's clear that the true LOQ should be at much higher concentration. To me, this looked as though the algorithm was proceeding from low concentration to high concentration, and taking the first level that meets the CV requirement as the LOQ. I would suggest that the LOQ would be more accurate if you proceeded from high concentration to low concentration, and picked the last level that meets the CV requirement. The peptide IFYNQQNHYDGSTGK is one example of this.

Thanks
Philip

view request
Moel Method for Scheduled PRM
z-proteomics 2020-05-28

Can any skyline experts with experience of using QE HF machine provide me a model PRM (Scheduled) method with detailed parameter setting?
Greatly appreciate your helpn in advance.
--- OmicSky

view request
unable to import HD-MRM result files into skyline in the last release version/daily version
(2 responses) simon daled 2020-05-28
i am unable to import my HD-MRM runs into skyline. It works fine in the 19.1 release version, but not in the newest version, there must be some bug somewhere. The runs are partially imported but the shown results are completely wrong.
Is it possible to download the previous version somewhere?
Thank you!
view request
Support for RDkit isotope aware
(1 response) landerson 2020-05-26

Does Skyline support RDkit isotope aware molecular formulas? RDKit uses nomenclature that a heavy carbon is [C13]. For example, if 2 carbons in glucose were heavy you might use C4[C13]2H12O6.

I know of the apostrophe " ' " method e.g. C’C5H12O6 that is embedded in Skyline. The RDkit nomenclature feature would make the synergy between a postgresql database more streamlined.

view request
Some small molecules don't support calibration curve
(1 response) philip remes 2020-05-27

Hi guys,

I have a small molecule document that is being used to analyze a dilution curve. Of 79 molecules, 2 of the calibration curves give the message "Error: All of the external standards are missing one or more peaks". This would appear to be similar to other reported issues.

Other support requests that mention this error, but don't seem to help me

Similar Issue 1
Similar Issue 2
Similar Issue 3

However, there is no internal standard normalization, the Analyze Concentrations are set, and the Sample Types are set to standard. Additionally it's kind of weird that just two of them do this, when they both have reasonable looking Peak Area plots vs Analyte Concentration.

Thanks
Philip

view request
method export error
(3 responses) dawn dufield 2020-05-22
Hi.

do you know what would cause this error. We have the analyst configuration in simulation mode and seems to be able to open the methods properly. This same skyline file can be exported on a different computer with analyst. So it feels like an analyst config issue, but seems like it should work.

any suggestions

---------------------------
Skyline
---------------------------
An error occurred attempting to export.
ERROR: Unable to cast object of type 'Sciex.MultiQuant.AnalystImprs.ApplicationObject' to type 'Interop.Analyst.ApplicationClass'.

Command-line: Method\AbSciex\TQ\BuildQTRAPMethod -s -m "X:\Analyst Data - BioPharma\Projects\Peptide\Acquisition Methods\Trap Pepcal 10 min.dam"
Working directory: X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods
Standard input:
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\~SK14E.tmp
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\test.dam
1752.439942,1930.069112,5,sequence1.xremoved this sequence info....
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: Unable to cast object of type 'Sciex.MultiQuant.AnalystImprs.ApplicationObject' to type 'Interop.Analyst.ApplicationClass'.

Command-line: Method\AbSciex\TQ\BuildQTRAPMethod -s -m "X:\Analyst Data - BioPharma\Projects\Peptide\Acquisition Methods\Trap Pepcal 10 min.dam"
Working directory: X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods
Standard input:
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\~SK14E.tmp
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\test.dam
1752.439942,1930.069112,5,sequence removed....

---> System.IO.IOException: ERROR: Unable to cast object of type 'Sciex.MultiQuant.AnalystImprs.ApplicationObject' to type 'Interop.Analyst.ApplicationClass'.

Command-line: Method\AbSciex\TQ\BuildQTRAPMethod -s -m "X:\Analyst Data - BioPharma\Projects\Peptide\Acquisition Methods\Trap Pepcal 10 min.dam"
Working directory: X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods
Standard input:
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\~SK14E.tmp
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\test.dam
1752.439942,1930.069112,5,sequences removed.....

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 57
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 44
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3906
   at pwiz.Skyline.Model.AbiMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 2397
   at pwiz.Skyline.Model.ExportProperties.<>c__DisplayClass119_0.<ExportAbiQtrapMethod>b__0(IProgressMonitor m) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 523
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\UtilUI.cs:line 125
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 246
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1907
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 194
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2022
---------------------------
view request
Exporting user-defined reports from command line
(2 responses) fcsigloch 2020-05-27

Hi,
I am trying to export a custom report from command line. I defined the report previously out of the GUI and can access it from there. However, when I try to run
SkylineCmd.exe --in=someFile.sky --report-name="MyResults",
I get the error: Error: The report MyResults does not exist.

I tried to fix the problem by running SkylineCmd.exe --in=someFile.sky --save-settings, but that did not help.
(However save-setings seems to have fixed a similar problem with a user-defined mProphet model. This can now be used for reintegration.)

How do I correctly export the report?

Greetings, Florian

P.S.: Sorry for posting at the wrong place at first!

view request
Missing transitions in timsTOF data
(1 response) matt 2020-05-26

Hi

I have some timsTOF Pro PRM data which I am having problems with. For some of my targets Skyline does not find the MS2 fragments but I can clearly see them using Bruker DataAnalysis software. It only seems to be selected targets, some work fine.
Any suggestions?
Thanks!
Matt

view request
Pivot Editor Median Feature
(4 responses) roman sakson 2020-05-20

Dear Skyline Team,

first of all, thank you for adding the pivot editor capability to the report grids, making Skyline even more versatile! I was wondering whether it would make sense to add the option median to what the editor can do, as at the moment only the mean seems available. I think that the median could be helpful at least in some cases as it is more robust against outliers than the mean.

Thank you for consideration,

Roman

view request
How to use CIRT to predict retention time?
(3 responses) caixue 2020-05-20

Hi,
I am trying to use the peptides selected by myself as CiRT to predict the retention time. The irt value of each CiRT peptide has been calculated and saved as a new Retention time predictor. Please see the attachment. However, when I chose the predictor, the document could not import the cirt peptides I selected, so I could not import the result. What should I do?
Looking forward to a reply. Thanks!

 Skyline cirt problem.pdf 
view request
MaxQuant spectral library
(1 response) xzhong9 2020-05-20

Hi Skyline Team,
I would firstly like to thank you for enabling Skyline for DIA data analysis.
I have a question about using MaxQuant msms.txt file to construct a spectral library in Skyline. The two samples are labeled with two mass-defect tags with a mass difference of 18 mDa. To construct the spectral library, the mixed samples were acquired in DDA mode, searched in Proteome Discoverer or MaxQuant, and import the .msf or msms.txt file into Skyline. I want to compare the results difference between PD and MaxQuant. I am able to get great results using .msf from PD, but have no clue why Skyline is not able to resolve the light and heavy fragment ions using msms.txt from MaxQuant as shown in the attached files. I would appreciate it if you could help me resolve it.

Thank you,
Xiaofang

 PD.JPG  MaxQuant.JPG 
view request
Error parsing spectrum while building a library
(6 responses) pamrollahi 2020-05-18

Hello,
I am trying to build a new library using a ".msf" file exported from PD and get this error (attached snapshot).
I was wondering if anyone here has faced such an issue and can kindly help me solve it.
Thanks!
Pouya

 Capture.PNG 
view request
MSP Spectral Library import for small molecules failed
(2 responses) stolltho 2020-05-17
Hi there.

Wanted to import .msp spectral libraries from:
https://mona.fiehnlab.ucdavis.edu/downloads
https://minedatabase.mcs.anl.gov/#/download
http://prime.psc.riken.jp/compms/msdial/main.html#MSP

.msp import works for HMDB, MoNA and ReSpect but NOT for GNPS, Fiehn HILIC and LipidBlast (throws an error message, see example attached) from https://mona.fiehnlab.ucdavis.edu/downloads

Spectral library import from
https://minedatabase.mcs.anl.gov/#/download
http://prime.psc.riken.jp/compms/msdial/main.html#MSP
does not work at all, i.e. shows zero molecules after import

win10
skyline-daily 20.1.1.134

cheers,
Thomas
 lipidblast error.png 
view request
Importing Data
(7 responses) dawn dufield 2020-05-07

Hi I have 4 questions

  1. Is there a way to import acquired data that you don't have a skyline file for and have skyline automatically create transitions for those that are in the data file? vs creating it first in skyline and then importing. This would be much quickly for some small molecule data

  2. How does skyline handle Sciex Qtrap IDA data? If you acquire an IDA data file say for the top 4. Is there an easy way to review that data in skyline?

  3. When I'm integrating a specific peak and try to "apply to all" many times it does not still pick the same peak. How can I force this so I don't have to select each window in every file. (This is especially true when data is not great or peaks are noisy or low)

  4. What is the "best conditions" for transitions settings in your opinion for finding the best peptide to follow. Should you really cast a very wide window and use all transitions to last ion?

thanks
Dawn

view request
How does skyline store and handle the settings files
(1 response) hogan 2020-05-06

Hello Skyline Team,

Firstly I hope you and your families are well!

I have come across some strange behavior with trying to update and save the settings for a skyline document to be used elsewhere. How does skyline handle the .skys files. The only way I have ever seen a .skys file is by using the "Share.." command in the settings drop-down to generate one. Where do all the other .skys files live and how does skyline interact with them? Are they specific to a local installation of skyline? I can provide very detailed and specific examples if necessary.

Thanks!

Kyle

view request
Panorama error with Skyline daily document
(3 responses) Chris Ashwood 2020-05-14

Hi all,

I hope you are well. I have been encountering a reproducible error lately, working on a Skyline document that uses the new log iRT feature. There are other niche features used including a spectral library of 500 molecules. The document itself is sensitive and sizeable (95 MB) but I can share it privately. Please find the Panorama warnings/errors below:

14 May 2020 20:23:29,961 WARN : The version of this Skyline document is 20.11. This is newer than the highest supported version 4.
14 May 2020 20:24:06,450 INFO : 98% Done

14 May 2020 20:24:06,677 INFO : Done parsing Skyline document.

14 May 2020 20:24:10,809 INFO : Failed to complete task 'org.labkey.targetedms.pipeline.TargetedMSImportTask'

14 May 2020 20:24:10,812 ERROR: SqlExecutor.execute(); uncategorized SQLException for SQL []; SQL state [25P02]; error code [0]; ERROR: current transaction is aborted, commands ignored until end of transaction block; nested exception is org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block

Caused by: org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block

Caused by: org.postgresql.util.PSQLException: ERROR: duplicate key value violates unique constraint "uix_auditlogentry_document"

Detail: Key (documentguid, entryhash)=(577e6540-bd2e-4a40-85dd-c009a80ba168, VeCVOeCev0LQhxtTovC3nshjfOY=) already exists.

14 May 2020 20:24:10,853 ERROR: SqlExecutor.execute(); uncategorized SQLException for SQL []; SQL state [25P02]; error code [0]; ERROR: current transaction is aborted, commands ignored until end of transaction block; nested exception is org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block

org.springframework.jdbc.UncategorizedSQLException: SqlExecutor.execute(); uncategorized SQLException for SQL []; SQL state [25P02]; error code [0]; ERROR: current transaction is aborted, commands ignored until end of transaction block; nested exception is org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block

Caused by: org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block

Caused by: org.postgresql.util.PSQLException: ERROR: duplicate key value violates unique constraint "uix_auditlogentry_document"

Detail: Key (documentguid, entryhash)=(577e6540-bd2e-4a40-85dd-c009a80ba168, VeCVOeCev0LQhxtTovC3nshjfOY=) already exists.

Cheers,
Chris

view request
DIAPASEF Scoring Issues
(9 responses) Max McCabe 2020-05-12
I have been attempting to use Skyline for processing DIAPASEF data from analysis of neat plasma on the timsTOF Pro. The issue is that although everything during setup and upload appears to be proceeding successfully, I am unable to achieve separation between target and decoy distributions when generating a scoring model. Additionally, the data have a lot of interference in most of the ID chromatograms. I am unsure if the issues are due to library quality, Skyline import settings, or improper integration of ion mobility.

 I've set up the Skyline document as described in the Large-Scale DIA Webinar with a couple of alterations to accommodate ion mobility data where I have been able to find the settings. I'm using an ion mobility resolving power of 40 following recommendations in an earlier support thread and have tried both centroided (assuming that this process is internally supported) and TOF analyzer settings when uploading raw .d data with the same results. The DIA scheme is imported directly from the data and appears to be correct.

I am using a spectral library generated using MSFragger with a 0.99 score cutoff and have checked to confirm that the library contains ion mobility values as well as RT.

I've played around with the setting and am at a loss for what the issue could be because the data has been successfully searched using other pipelines. I have zipped the .sky and associated files into an uploaded folder titled "Skyline Support Upload".

Thanks in advance for the help!
view request
RT peptides
(12 responses) smanda 2020-04-14

Hi Brendan,

We have 50 peptides (including some from Biognosys) which we use as RT peptides in all samples. I was wondering if there is a way I can use all of them for my RT alignment in Skyline? I see currently it takes 25 or so and says 20 required. Am I missing something.

Regards,
Srikanth

view request
Only coeluting product ions as quantitative (extended)
(8 responses) Tobi 2018-12-17

Dear Skyline team,

I noticed that setting the "Quantitative" checkbox also works with copy and paste, but no matter if done by copy and paste or by manual clicking, it seems impossible to get the same checkboxes as in the Coeluting column because of constant recalculations overwriting manually set checkboxes (in PRM)

For just a single file one could think of exporting and reimporting a transition list filtered for coeluting product ions, but for a larger experiment, this is not feasible. Is it possible to somehow fix the quantitative column to make it directly susceptible to manual editing (in general just setting "not coeluting" to "not quantitative")? Is there a smarter way to do that?

I know about unchecking integrate all, but it would be much better to have full control over the quantitative state of a transition compared to just removing the target from the target list which impacts all datasets.

The attached file shows what would be the desired output, its just not fully implementable for a whole experiment.

Thank you for all thoughts and ideas on this topic.

Best,
tobi

 181116_PRM_quant_coeluting.sky.zip 
view request
Using Prosit for heavy ions
(7 responses) denisoleynik3007 2020-05-08

Does Prosit work for a prediction of MS/MS spectra of heavy ions (in particular heavy y-ions)?

view request
About Synapt G2-Si (Waters). Data-independent acquisition
(3 responses) eriosc 2020-05-05

Hello!

I would like to know a few things about Skyline.

I have used commercial software most of the time; however I would like to know if Skyline supports * .raw files from Waters coming from a Synapt G2-Si mass spectrometer. I ask this because I am interested in doing Label-free (DIA) using HDMSE and UDMSE mode. Is it possible to do this?

Additionally, I would like to know if with Skyline it is possible to identify crosslinked peptides (XL-MS) using the same way HDMSE and UDMSE (DIA). If possible, it would be great because I have some ideas to develop using this methodology.

Thanks and regards

view request
issue when importing peptide search from a MaxQuant msms.txt file
(5 responses) marie locard-paulet21061 2020-05-01

Hello, I am trying to import the results of a DDA search from MaxQuant (1.6.14) for generating a library in Skyline (for DIA data analysis).
I work with Skyline (64-bit) 20.1.0.76 (0d62150d0). My samples do not contain iRTs, and kept all default parameters for the "import peptide search".

Here is the error message that I get (copied and pasted from Skyline):

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

Command-line: C:\Users\malopa\AppData\Local\Apps\2.0\5ZNXQLMA.P6A\TRMG87OZ.R8T\skyl..tion_e4141a2a22107248_0014.0001_72292abfbc2fefb5\BlibBuild -s -A -H -o -c 0.95 -i MQDDALib_MC1_Carba -S "C:\Users\malopa\AppData\Local\Temp\2\tmp3F62.tmp" "C:\data\Skyline\MQDDALib_MC1_Carba\MQDDALib_MC1_Carba.redundant.blib"
Working directory: C:\data\MQ\HeLA_21min_Library\MQDDA_MC1_Carba\combined\txt
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\malopa\AppData\Local\Apps\2.0\5ZNXQLMA.P6A\TRMG87OZ.R8T\skyl..tion_e4141a2a22107248_0014.0001_72292abfbc2fefb5\BlibBuild -s -A -H -o -c 0.95 -i MQDDALib_MC1_Carba -S "C:\Users\malopa\AppData\Local\Temp\2\tmp3F62.tmp" "C:\data\Skyline\MQDDALib_MC1_Carba\MQDDALib_MC1_Carba.redundant.blib"
Working directory: C:\data\MQ\HeLA_21min_Library\MQDDA_MC1_Carba\combined\txt
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 160
---------------------------


I attach to this post the msm.txt files and corresponding mqpar.xm.
please let me know if you need more information.

view request
Setting up Skyline in a Virtual Environment
(2 responses) halve021 2020-05-01
The University of Minnesota is working toward a virtual delivery pilot in the research space. Can you tell me if the license for Skyline allows the software to run in a Citrix or XenApp environment?

If this is not possible, can they be delivered via Remote Desktop Services?

Thank you for your time,
...Justin Halverson
IT - University of Minnesota
view request
Refinement Suggestion: Min peptides per protein EXCEPT X
alejandro.cohen 2020-05-01

Hi Skyline people

Suggestion for refinement in Skyline\Advanced menu:

Add an option to remove Min peptides per protein with another tick box reading 'EXCEPT for proteins smaller than': X (Da). The value of the intact protein could be estimated from the FASTA file, maybe?

Some smaller proteins (<5-10KDa) give very few good (sometimes only one!) unique tryptic peptides, yet very good quality and signal. You dont want to discard them just because you didn't get enough number of peptides.

Your thoughts?

Alex

view request
Manual integration of SST is not transferred to AutoQC/LabKey
(3 responses) katrin freiburghaus 2020-04-27

Dear Skyline team

I am using Skyline with AutoQC Loader to get my SST uploaded to the Panorama data management system, where I can track the instrument performance (Xevo TQ-S triple quadrupole, Waters). All is working nicely, excpet when the Skyline algorithm picks a wrong peak in my data. I can adjust it manually in the Skyline file, but it is not updated in Panorama.

Could you tell me what I need to do so that my manual adjustments are adapted in Panorama?

Thank you very much and kind regards,
Katrin

view request
General question about peptide quantitation
(2 responses) caho 2020-04-29

Hi there,

May I please ask, with regards to peptide quantitation, is the sum of the peak areas of all fragment ions taken (so the total peak area of the precursor, precursor [m+1], precursor [m+2] fragment ions), or is generally the fragment ion with the greatest peak area used? May I also please ask how is the peak rank determined for each peptide/fragment ion?

Thank you so much for your help!
Cally

view request
Precursor metabolite data from SWATH
(3 responses) lefelit 2020-04-09

Hi

I have metabolite data from TOF MS acquisition on a SCIEX triple TOF instrument and I am trying to extract the ion chromatogram information for more than 200 precursors. I am not interested at this stage for fragment data. I built a method with all the molecules of interest with the accurate masses and when i applied it to a test mixture of pure standards the software does not find almost 60% of them. I set the mass window to 0.2 and the RT window to 1min and still does not find them. What could be the source of the problem? How could I also customise the threshold setting to different value per precursor?

Many thanks

view request
Installing Slyline error
(4 responses) alejandro.cohen 2020-04-28

Hi Skyline people,

I moved to home office, and I'm trying to install Skyline (regular or daily) on my laptop running Window10 , and in both cases I get the following error:

An error occurred trying to download 'https://skyline.gs.washington.edu/software/Skyline-release-64_20_1/Skyline.application'.
See the setup log file located at 'C:\Users\Agus\AppData\Local\Temp\VSDF497.tmp\install.log' for more information.

BTW, I can't seem to find that Temp folder in the C: drive anywhere...

Any ideas?

Thanks

Alejandro

view request
MSP Spectral Libraries for Small Molecules
(6 responses) Christina lucas 2020-04-22

Hi there,

i am trying to Import an MSP library to Skyline.

First attempt was an Test Library created in ThermoFishers mzVault exported as MSP (attached). Which was not working, no element was shown.
Second attempt, as Thermo might have some Export issues on that was to download the HMDB (or any other file in MSP) from https://mona.fiehnlab.ucdavis.edu/downloads

Still I get no content in my library.

So far I could not find anything in Forum thats giving me a hint how to open them correctly.
I am using daily 20.1.1.83 on a Win 10.

Best,

Christina

 Acyl_Test.msp 
view request
Prosit .msp library import is missing iRT values
(4 responses) anatoly.urisman 2020-04-25

Hi Skyline Team,
I was wondering if the iRT values in Prosit generated .msp libraries could be parsed upon import into Skyline. I use a simple conversion script as a work-around, but this adds an extra step. I suppose another solution would be to change Prosit output to Skyline-readable.
Thanks!
Anatoly

Here is an example of Prosit .msp format...

Name: TIFGGSDSTNQGPSNGER/2
MW: 912.41136795554
Comment: Parent=912.41136795554 Collision_energy=28.0 Mods=0 ModString= TIFGGSDSTNQGPSNGER///2 iRT=32.720001220703125
Num peaks: 46
175.118952167 0.10104765714257913 "y1/0.0ppm"

view request
iRT-Pierce combo library import converts all unmodified peptides to K+8, R+10
(1 response) anatoly.urisman 2020-04-14

Hi Skyline team,
I really like the feature where you can combine multiple blib libraries in Import Peptide Search wizard to create a new combo library. This allows me to use very different search results for iRT peptides (I use Peirce iRT mix, which is K+8 and R+10 modified) and for target peptides (which are usually unmodified) to generate blib files.
However, when I use this combo library to add target peptides, unmodified peptides are incorrectly showing as K+8 and R+10 modified (even though the reference sequences and spectra are correctly showing as unmodified in the library browser). Decoys are also modified as a result. As a work-around I can remove the modifications using Refine --> Advanced --> Remove Label Type (heavy), but I have a feeling that this was not the intended behavior during target import, so just wanted to bring it to your attention.
I've uploaded a file that illustrates this problem (DIA_2020-03-14_90min_v2.sky.zip).
Thank you for looking into this!
Anatoly

view request
noenzyme setting
(1 response) egulyas 2020-04-27

Dear Brendan,

I would like to do peptidomics study. The identified peptides have no specificity on any side. How can I set it in the "digestion" ? (both side unspecific or no enzyme)
Thanks,

Eva

view request
A question about MSstats
(4 responses) 1214800593 2020-04-26

Hi
I want to do a DIA project, but when I use the MSstats,it report error like attachment.
please help me!
THANKS!
Steven Liu

 MSstats.png 
view request
pepxml file generate
(3 responses) pucci biagio 2020-04-23

Hi,
i'm working with bruker tof and I'm use mascot as engine software...I'm not able to generate pepxml file to use for MS1 DDA analysis..
help me!!
regard

view request
Skyline is CFR part 11 compliant software
(1 response) Stephane CHARMONT 2020-04-23

Hi,

I'm currently working on pharma environment and specially at quality Level 2. But we are doing business with external service provider for clinical trials analysis where their lab need to be GxP qualified.
I would like to know if Skyline software is CFR part 11 compliant software and at the same time I would like to know if skyline incorporate an Audit trail system as usual qualified software from MS supplier or vendor?

Thanks a lot

Regards

Steph

view request
Peak selection for MRM
(1 response) bart van puyvelde 2020-04-24

Hi Brendan and Skyline team,

I am currently doing some data analysis for an MRM experiment and I got a question concerning the peak selection.

I would like to force Skyline to always select the peak within a 0.1minute RT window or even smaller.
I tried to do this by changing the Full-Scan settings, but apparently it does not work.

Any suggestions to get this working? I can't share the Skyline file through the support page because it contains patient data.

Best,

Bart

view request
Peak with high mass error not found
(2 responses) katrin freiburghaus 2020-04-23

Dear Skyline team

I encountered the following problem: Our Q-Exactive Orbitrap was not well calibrated and produced data with high mass errors (up to 70 ppm deviation) for all small molecules. I imported the SST files into Skyline and it did not integrate any peaks (see attachement "Skyline_Kynurenic_acid") using the attached transition settings. But when I integrated the exact same file using TraceFinder software, I saw the peaks (attached TraceFinder screenshot for kynurenic acid, mass error -53 ppm).

I adapted the "method match tolerance m/z" in the Transition Settings to the allowed 0.6 m/z, but still no integration of my peaks.

Could you let me know what I am doing wrong and how I can get my results in spite of the high mass error?

Thank you very much for your help and best wishes
Katrin

 Skyline_Kynurenic_acid.PNG  TraceFinder_Kynurenic_acid.PNG  Skyline_Transition_Settings.PNG  Skyline_Transition_Settings_2.PNG 
view request
Retention time Prediction
(1 response) scv01958 2020-04-23

Hi,

I had a question regarding the retention time prediction tool of Skyline. I understand that the tool is capable of using imported data to calibrate a calculator and build up an iRT database. This iRT database is then very useful in validating retention times recorded in future trials. However, I am confused on the "prediction" capabilities of this tool. Let's say I have a calibrated calculator already made consisting of an iRT database of related peptides/molecules. I have a new peptide/molecule that is related, but slightly different in some way, the difference is a change in structure or charge. Will this calculator be able to predict the retention time of my new compound based on the database I have already built?

Thanks.

view request
Unknown modifications
(3 responses) Wilfred Tang 2014-12-06
In building a library (Settings menu, select Peptide Settings, go to Library tab, click on Build button), it appears that Skyline does not like "unknown" modifications (modifications not mappable to Unimod(?) based on delta mass). See attached screen capture. I am using Skyline-daily (64-bit) 2.6.1.6899.

Why does Skyline need to know the modification name? Shouldn't the delta mass alone be sufficient information?

Is there a way to use "unknown" modifications in Skyline? We commonly deal with data having lots of glycan modifications - there are a wide variety of glycan modifications, and most are not in Unimod.

Thanks,
Wilfred
 Screenshot 2014-12-06 22.26.51.png 
view request
Pressure output?
(9 responses) alejandro.cohen 2020-02-20

Hi Skyline people,

I work with an Agilent 1290 coupled to a Qtrap5500 running MRM metabolomics which I analyze using Skyline (.dam methods, .wiff datafiles). Analyst is recording the column pressure output signals, however viewing these using Analyst is cumbersome and time intensive when working with big sample batches. We have encountered erratic behaviour of our chromatography, accompanied by unusual pressure profiles for samples, which tend to correct themselves after a few runs.

As part of our QC, it would be great if skyline would be able to add (overlap) the pressure traces on each chromatogram. Is this possible? I know each vendor 'hides' the pressure traces in different output and channels... but the information is there :)

I know this request has been done in the past... here another kind 'reminder'

Cheers

Alex

Alejandro Cohen, Ph.D.

Scientific Director
Biological Mass Spectrometry Core Facility. Room N-105
Life Sciences Research Institute
https://medicine.dal.ca/research/biological-mass-spectrometry.html

view request
FAIMS unit on Thermo Lumos
(8 responses) erik.soderblom 2019-11-20

Hi Skyline Team! I wanted to "bump" a thread entitled “Optimizing FAIMS on new Thermo instruments” from Martijn van Duijn back in September. We are now in the same boat (acquired a shiny new FAIMS device for our Lumos!) and at this point we just want to incorporate the data (say, 3 different CVs across 7 different peptides) into Skyline from our normal system suitability PRM acquisitions. I see where compensation voltage is a selectable unit under the ion mobility settings, but it doesn't look like Skyline is able to extract data for a single ("best") CV in the chromatogram view. Is this possible with current Skyline Daily (19.1.1.309) yet?
Thx,
Erik

view request
Exporting Isolation Lists
(5 responses) cjeline1 2016-01-29
Dear all, we are experiencing a similar problem as that documented by Josua Troesch on the 27th. Running skyline version 3.5.0.9319, exporting a Thermo fusion isolation list from a skyline file fails to render the generation of a .csv file. Do you have any suggestions to resolve this issue?

Thanks,

Pandey Laboratory
Johns Hopkins School of Medicine
view request
Set a Fixed Mass Tolerance in Skyline
(1 response) lancia darville 2020-04-20

We are processing small molecule data using Skyline and would like to know if there is a way to set a mass tolerance in Skyline? We have been observing a either a spilt or a broad peak for a targeted molecule when processing the data in Skyline which aligns with our stable isotope standard, however the mass error is ~10 ppm. If the same data in processed in Thermo’s XCalibur with a mass tolerance set to 5 ppm, the interfering peak is removed and we only see our target with a mass error between 0.5 and 1 ppm in the samples. We would like to be able to use Skyline to process all the data, however this will require us to set a specific mass tolerance. Please advise if a specific mass tolerance can be set in Skyline and if so how?

view request
Removing precursors for DIA data
(4 responses) epcasavant 2020-04-17

Hi,

I am trying to run my data through MS Stats and ran into the error that said I needed to remove the precursors. I saw your answer here (https://skyline.ms/announcements/home/support/thread.view?rowId=37139) and followed the following steps you outlined:

  1. View > Document Grid
  2. Choose "Transitions" from the Views dropdown at the top of the Document Grid
  3. Right click on the "Fragment Ion" column and choose "Filter"
  4. Set the filter type to "Starts With" and type "precursor" into the textbox.

However, how do I delete the precursors? When I select them all int he document grid, it looks like it literally deletes all of my data.

Thank you,
Ellen

view request
AutoQCConfiguration
(2 responses) m kuppusamy 2020-04-16

We have two Bruker Impact II Q TOFs and would like to implement the skyline QC pipeline. I could not find the option for Bruker in the instrument type while configuring the AutoQC. Would it be possible to include Bruker data types as well in the future updates of AutoQC? Agilent too has .d format folder but the analysis file itself is in a different format (.baf for Bruker), would AutoQC be able to recognize .d folders irrespective of vendors?

view request
Waters HDMSE Raw Data Visualization
(2 responses) brad williams 2020-04-13

I have been testing the latest Skyline builds (20.1) and Skyline Daily (20.1.1.83) and they both do not allow the raw data to be visualized for MS1 precursors. The raw data plot shows up blank and the MS/MS information is there. Has this been reported before? I have attached a screen capture to show an example. The datafile type is HDMSE.

 MS1 Spectrum Viewer_041320.pptx 
view request
How to analyze and quantify multiplexed peptides (Q Exactive PRM) with Skyline?
(3 responses) gillespiekevinp 2020-04-09

I'm having a lot of trouble with analyzing my RAW files generated by a Q Exactive HF.

I am only trying to analyze one protein. In my "Inclusion List" I constructed with the Thermo software, for each peptide, I have Multiplexed multiple precursor charges. But I have the heavy internal standard peptides and light peptides Multiplexed separately.

Using Xcalibur Qual Browser, I check for the precursor ions of these peptides (Full Scan) or the product y and b ions (DIA), and my peaks are present and reasonably intense.

However, I can't figure out the correct Transition Settings in Skyline to reflect any of this Multiplexed PRM data when I import it. And I can't figure out how I would generate and export the ratios of either Light/Heavy precursor ions or Light/Heavy MS2s from Skyline.

Any tips? I can attach files as needed to clarify. I've gone through tons of tutorials by this point, and any additional help would be appreciated.

view request
Phosphorylation Variable modification
(5 responses) dawn dufield 2020-04-13

I am looking for some phosphorylated peptides and was wondering how your variable modification worked? I added 2 sites of phosphorylation as variable in a specific peptide and only see it predicting the double phosphorylation as a potential MRM. I was hoping it would give me none, 1 (either on 1 site or other) and both.

Any help would be appreciated

Thanks
Dawn

view request
Tryptic peptide selection
(3 responses) djlj1 2020-04-13

Dear all

apologies if this is elsewhere, but i haven't been able to find it. Is there a set of criteria for selecting suitable proteo- and quanto-typic peptides in Skyline??

view request
How to find a suitable library for a certain protein (with enzyme digestion)
(2 responses) HENG 2020-04-10

Hello.

Recently I am analyzing a protein called leptin (Protein Name: P41159) with AspN digestion using Skyline. But even download some MS/MS spectral library form PeptideAltas and NIST. I cannot find a suitable library that match the transition of my peptides.

So whether there is a pathway that I can find certain library can match the transition of my peptides, when I input my target proteins and digestion enzymes in the searching field. Or other suggestion maybe you can give to me.

Thank you very much.

Heng

view request
Purple bands on chromatograms
(2 responses) samyukta sah 2020-04-12

Hello,

I have been getting some purple bands across my chromatograms and it looks like it's happening when I manually select the peak boundaries. I was wondering if I could know why exactly that's happening and if they could be removed? I have attached a picture of it. Also I was wondering if there is a way of displaying the full names of my analytes on the chromatogram?

Thank you

 Standard 1.emf 
view request
Data of MSX-DIA demultiplex error using MSconvert
(7 responses) lxy0901 2020-03-31

Dear Skyline team,

I ran a DIA MSX experiment on QExactive HF-X set up using instructions by 5 m/z window, DIA, MSX 4.Data was collected in the profile mode. The method was set according to the template. I want do the demultiplex of RAW files by the Mscovert software, but this software displayed some error information. as shown:

Starting...
Opening file "J:\lxy\LXY-data\DIA\MSX\20191218\HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.raw" for read...
Calculating SHA1 checksum...
Processing...
Writing "J:\lxy\LXY-data\DIA\MSX\20191218\HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.mgf"...
Failed - System.Exception: SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.
在 pwiz.CLI.msdata.MSDataFile.write(MSData msd, String filename, WriteConfig config, IterationListenerRegistry iterationListenerRegistry)
在 MSConvertGUI.MainLogic.processFile(String filename, Config config, ReaderList readers, Map2 usedOutputFilenames) 在 MSConvertGUI.MainLogic.Go(Config config, Map2 usedOutputFilenames)

I want to know if there is a problem with my method settings?
Or can the data produced by the HF-X not be processed by this software?

My computer system is Window 7 service pack 1, 64 bit. The skyline software version is 64 bit 4.1.0.11717. The MScovert Version is 3.0.19161-f1b0b59b1.

The raw data and skyline files are already upload , "HFX_LXY_IgG_DIA_Methods_1_MSX_dia_4_5Da_20191218_F1_R1_20191219191652.rar"
And the problem and the setting is shown in the attachments.

Thanks.

Jessica Li

 Mscovert error.png  Mscovert setting.png  setting1.png  setting2.png  setting3.png 
view request
DIA data upload with 15N-APOA1 iRT peptides and Chromatogram Library (elib)
(5 responses) Tomas Vaisar 2020-04-09

Brendan et al,

This may be covered somewhere, but I could not find it in any tutorial or here on the Support pages.
I have 15N-APOA1 iRT peptides in my samples and I enumerated proteins/peptides (database search) in my samples using the Chromatogram Library approach (6 DIA runs of the pooled sample with overlayed 4 m/z windows segmented over 400-1000 m/z). I ran the data through EncyclopeDIA (Walnut search part) and created a chromatogram library (elib). Now I want to get this into the Skyline and create iRT calculator using the ChromLibrary data so that I can upload the data from all study samples using the iRT predictor.
Where this fails is that while I can create the iRT calculator for the 15N-APOA1 peptides alone, I cannot find a way to add the remaining peptides to the iRT calculator. The 6 DIA "Chrom Library" files load fine into the Skyline document (using Add one replicate), but whenever I try to update the calculator it fails because the 15NAPOA1 peptides are not present in all Chromatogram Library mzML files. (I am not uploading precursor ions only b,y fragment ions).

What is the way to get this working? I am sure there is a way people do it.

Thanks,

Tomas

view request
qick question feature description
(4 responses) Tobi 2020-04-08

Hey everyone,

can you please tell me what exactly the Library intensity dot-product from the mProphet scoring is? During recent webinar it was explained that it is the dotp between data and library. This opens the question on how can it be part of target decoy scoring if there are normally no dotp values for the decoys. It is quite important to know as this is the attribute with often the highest weight.

With best wishes,
tobi

view request
MSStats volcano plot labels
(2 responses) cabarnescabarnes 2020-04-09

Hi all,

This might be somewhere and I just didn't find it, but can you help me find out how to control the labels on the MSStats volcano plots creation in the Skyline implementation? Is it possible to use only the gene names or something else instead of like it is in the attached plot?

I hope this isn't a multiply duplicated question that I just didn't find.

Best,

chris

 VolcanoPlot.pdf 
view request
caTools MSstats not being downloaded during installation
(5 responses) lake n paul 2020-04-04
Just a heads up. A critical package for Mstats is not being downloaded, therefore MSstats is not working correctly.


Warning: dependency 'caTools' is not available
trying URL 'http://cran.us.r-project.org/bin/windows/contrib/3.5/gplots_3.0.3.zip'
Content type 'application/zip' length 592572 bytes (578 KB)
==================================================
view request
How do I limit the RT window when use 'Apply Peak to All' function
(4 responses) qzhang31 2019-10-17

Hi there,
I am running MS1 filtering for a bunch of peptides, I have a standard with high concentration and every peptide's peak is properly picked. Then I am loading some data with much lower concentration of those peptides and I find that skyline will pick wrong peaks at a very different RT.

How do I tell skyline to just look at 1 min RT window within the peak in file A for all the other files?

Thanks!

view request
Feature request: color dotP labels based-on value
gabe 2020-04-06

We analyze datasets with many samples (typically 96) and rely critically on the dotP values displayed above each Peak-Area bar. Depending on the monitor and resolution, sometimes the dotP text is so small that it's very difficult to read. It would be helpful if these values could optionally be colored based on the value -- something like >0.9 green, between 0.7 and 0.9 orange, below 0.7 red. Ideally these would be user-configurable.

Thanks

view request
exporting transition lists
(1 response) dawn dufield 2020-04-05

When i try to export a transition list I get the following message. Is there a way to set this up properly

The settings for this document do not match the instrument type SCIEX

view request
Precursors per sample injection thinks it is transitions per sample injection
philip remes 2020-04-04

Hi Brendan,

This is a repost of issue 723. Sorry I didn't know to first post to Support and not Issues.

I've attached the skyline file. To reproduce what I saw,

  1. File->Export->Isolation List
  2. Instrument Type -> Thermo Fusion. Interestingly, if I use the first Instrument in the list, Agilent QTOF, and enter 216 in max precursors, the number of methods is correctly set to 9
  3. For Fusion, if 216 is set to max precursors, the number of methods is 138.

Thanks
Philip

 200404_export_list_debug.sky.zip 
view request
small molecules: LOD, LOQ and S/N
(3 responses) ayson 2020-04-02

Hello! Good afternoon! I have run standard curve on some small molecules in an Agilent QQQ. I changed my LOD and LOQ settings from the molecule settings. My questions are the following:

  1. I am using quadratic fit and not linear because most of the molecules I ran followed this pattern. But when I got my LOD's, I am getting negative values. I believe it is because of the nature of quadratic fit. Can we have absolute values for our LOD's?

  2. I am also noticing that for some of my molecules, the LOQ's are so much lower than the LOD's. Usually, LOD's are lower than LOQ's. Can you explain why I am getting this?

settings:
20% for max LOQ bias
15% for max LOQ CV
LOD as blank + 3*SD

  1. how can we export signal to noise ratio?

Thank you!!!

view request
quick question feature description
(2 responses) Tobi 2020-04-02

Dear all,

https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/DIA-2_6.pdf

can you please help me with a quick explanation? In the link above, it is said "DIA precursor window for exclusion tells Skyline not to include transitions that fall in the DIA isolation window".

In the current Skyline daily, the pop-up tool-tip says you can change between symmetric and setting-based exclusion windows, and contrary to the above it is phrased as if now some kind of exclusion is applied in any case.

What is the current function of checking/unchecking this feature? The Tutorial and text next to checkbox indicate a On/Off function while the pop-up tool-tip indicates On-variant A / On-variant B.

Looking forward to get a short help on this.

With best wishes,
tobi

view request
failed to install MSstats from the Tools store
(4 responses) chenxiaotong 2020-04-01

Dear Skyline Team,
At the very beginning, I can successfully install the MSstats tool in my computer, but it couldn't work and an error was reported in the immediate window as shown in the attach file below. And then, I tried to replace the R 3.5.0 with R 3.6.1 which has the "caTools" package that can be installed. But the skyline kept reminding me to install the R, then I installed and uninstalled MSstats and R again and again. And suddenly, I couldn't install the MSstats anymore and an new error was reported in the immediate window as shown in the attach file below.
Best Regards,
Xiaotong Chen

 MSstats Error.docx 
view request
final library match issues
(2 responses) Tobi 2020-04-01

Dear Skyline Team,

please find attached files to explain issues to finalize fixing the library match window and related Prosit mirror dotp. Summed up, the dotp seems to falsely include removed y1,y2 etc. leading to reduced dotp-value while target list and document grid get it right.

With best wishes,
tobi

 Library Match issues.sky.zip  Library Match Issues.xlsx 
view request
Waters Xevo auto comments out transitions more than five
mattkarasu 2020-03-30

We have a waters Xevo XS and have been struggling to find a setting that will allow us to use more than 5 transitions per peptide. At the moment we have been doing the work around of simply deleting all the text in the comment boxes during the editing phase. But now we are getting into transition lists of more than 9000, so are making 10 to 30 methods of 400 transitions each.
Q. I read in an earlier Issue in Support that it was called Issue 304 and is unresolved? IS there a way to get around it via template editing or such?

view request
small molecules: Error with calibration curve: all of the external standards are missing one or more peaks
(7 responses) ayson 2020-03-27

Hello! What are some of the criteria of having calibration curve? Currently, I only have standards and also blanks but no quality control samples. I have already labeled my standards as standards and blanks as blanks and I have also put in the concentration. But whenever I am opening calibration curve this is what I get: Error: All of the external standards are missing one or more peaks

Is it because some of my standard samples especially the lower region do not have any peaks detected? Thank you!

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Error reading iRT standards transition list
(2 responses) HENG 2020-03-27
Hello. I am learning your Skyline Importing Assay Libraries documents. And when I proceed to the following steps (see my attachments) and click OK button. The skyline showed this Error reading

---------------------------
Skyline
---------------------------
Error reading iRT standards transition list: Product m/z value 171.11 in peptide TPVISGGPYEYR has no matching product ion.
---------------------------
OK More Info
---------------------------
System.IO.InvalidDataException: Product m/z value 171.11 in peptide TPVISGGPYEYR has no matching product ion.
   at pwiz.Skyline.FileUI.CreateIrtCalculatorDlg.OkDialog() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\FileUI\CreateIrtCalculatorDlg.cs:line 194
---------------------------

I am sure I was following every steps of the document. So how can I solve this problem?

Thanks.

Best,
Heng
 微信截图_20200327164534.jpg 
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Small molecule - custom report - cannot get light and heavy on same row
(4 responses) MartijnvF 2020-03-27

Dear all,

I am working on getting my custom report to show both the areas of the light and heavy molecule on the same row in the report. However, I only get the areas on different rows, see attachment.

Cheers,
Martijn

 custom_report.tif 
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Existing Quantitative Experiment Tutorial_Peptide Setting problems
(2 responses) doloph 2020-03-27

Dear Ladies and Gentlemen,

I am currently trying to get familiar with your excellent software and am therefore working through the provided Tutorials. However, while I was working on the Existing Quantitative Experiment I am facing some problems. The user interface looks quite different when setting up the data and the program does not allow to pick the expected "Labels:13C(6)15N(4) from the drop down menu. Additionally, when typing in the parameters manually the, software gives back several error codes. Does someone know what I have to change?

Kind regards,
Dominik

 Screenshot (350).png  Screenshot (351).png  Screenshot (352).png  Screenshot (354).png  Screenshot (355).png 
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Small molecule: Skyline cannot properly analyze Agilent QQQ with waste diverter in the beginning of the run
(4 responses) ayson 2020-03-26

We have acquired HILIC data in Agilent QQQ with diverter valve to waste from 0-0.2 mins. From 0.2 to 5 mins, mass spec was turned on. When I analyze the data in Skyline, I am only seeing the chromatogram from 0-0.2 mins but never 0.2 to 5 mins. The data looks fine in Masshunter Quant but not in skyline. I have sent you more detailed report in the attached word document.

It is vital that skyline can process these types of data because more small molecules will be diverter their initial flow to waste to protect mass spec instrument. Thank you so much!!!

Best regards,
Marites

 032620_skyline_not properly analyzing Agilent QQQ data with waste diverter on.docx 
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Small molecule - calibration curve error: Matrix must be positive definite
(2 responses) MartijnvF 2020-03-26

Dear all,

I am trying to use Skyline to see if it will be suitable in our clinical laboratory for quantification and monitoring of MS performance. I am getting an error I cannot find anything previous help on. The calibration curves are not showing:

Error: Matrix must be positive definite.

Cheers for any help, much appreciated.

Martijn

 CAMMETA.sky.zip 
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backwards compatibility skyline v20 vs skyline v19
(1 response) kguehrs 2020-03-25

Hallo Skyline team,
I generated some dta with the newest version of skyline. I wanted to reload them on an other computer running Skyline 19.1.0.193 from an external disk. However, skyline refused to open the data because they are generated with a newer version. I do not have administrator rights for this computer and it is therefore somewhat complicated the upgrade skyline in short time in these days. Moreover, there is the question of the general backward compatibility of skyline files.

Wish you all the best and hope that we all can keep going.

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Increase in m/z of fragment ions when exporting methods for collision energy optimisation
(2 responses) sarah lennon 2020-03-24

Dear Skyline team,

I have just noticed that when exporting a Waters method for collision energy optimisation from Skyline Daily 20.1.1.32, the m/z of the fragment ion is increased slightly (+0.01 Da, +0.1 Da from first to last) with increased collision energy. Is there any reason for that ?

Thank you very much for your help,
Kind regards,

Sarah

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Prosit in skyline
(11 responses) Tobi 2019-12-05

Dear all,

thank you very much for integrating Prosit into Skyline. I noticed that the dotp in the new library match mirror view does not treat y ions from deferentially labeled peptides as equivalent. Is it possible to extract the Comparison dotp via results grid for all peptides at once?

Best,
tobi

 Prosit in Skyline.png 
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Peak quality score for small molecules
(4 responses) bwidner 2020-03-17

Hello Skyline Team!

I am working with small molecule data, and I would like to put a number on chromatographic quality for each peak. I would like this score to only be for MS1 peaks (or MS2, if I decide that's what I want to look at) and to include things like peak shape (i.e. gaussian), signal to noise, agreement with predicted retention time, etc. I have not so far found this in Skyline.

I looked over the Advanced Peak Picking Models tutorial since it includes scoring, but I do not think this is exactly what I am looking for. It seems more complicated than what I need; perhaps I am overlooking something?

I am new to MS and get a little lost in the terminology, especially when it is peptide terminology, as I have never worked on peptides. My apologies if I am overlooking something obvious, and thanks for your assistance!

Brittany

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LOD and LOQ calculation for Small Molecules
(1 response) h ravuri 2020-03-19

Hello Skyline Team

I am currently working on small molecules. I am trying to work out the LOD and LOQ on the molecule. I followed all the steps in the small molecule tutorial document. But could not figure out where I can get the steps or information on required samples sets to calculate the same.

Can you please provide any information on this?

Thanks

Halley

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Question on Limits
(6 responses) Tobi 2019-08-06

Dear Skyline team,

we have an outstanding project and want to work with DIA and Spectral libraries on a atrget list generated from huge fasta files in the GB range. Are there still any (fixed) limits for the number of peptides and transitions in the target list or the number of peptides in a spectral library that skyline can hendle? Older posts here yield different answers.

Best regards,
tobi

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Skyline for MAC OS
(1 response) ana normando 2020-03-17

Is there an available version of Skyline for MAC OS?

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Skyline-daily and Skyline 20.1 crashed in loading pepxml from MSFragger
(17 responses) fcyu 2020-01-30

In loading pepXML or ineract.pep.xml from MSFragger/FragPipe using the "Import DDA Peptide Search", skyline crashed with the following error. Looks like it can find the mzML file which is in the same folder, but crashed somehow. You may find the mzML, pepXML, interact.pep.xml, and fasta file in dev2.zip from file sharing. Could you please help to take a look?

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

Command-line: C:\Users\yufe\AppData\Local\Apps\2.0\KG99EWD8.1KH\L9HYCN5B.KLC\skyl..tion_e4141a2a22107248_0014.0000_e9276d4fd631b09e\BlibBuild -s -A -H -o -c 0.95 -i test -S "C:\Users\yufe\AppData\Local\Temp\tmp6570.tmp" "E:\dev\msfragger\dev2\test.redundant.blib"
Working directory: E:\dev\msfragger\dev2
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\yufe\AppData\Local\Apps\2.0\KG99EWD8.1KH\L9HYCN5B.KLC\skyl..tion_e4141a2a22107248_0014.0000_e9276d4fd631b09e\BlibBuild -s -A -H -o -c 0.95 -i test -S "C:\Users\yufe\AppData\Local\Temp\tmp6570.tmp" "E:\dev\msfragger\dev2\test.redundant.blib"
Working directory: E:\dev\msfragger\dev2
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 160
---------------------------

Thanks,

Fengchao

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Issue with the skyline cmd.exe with the latest release version of Skyline
(1 response) shobhabr20 2020-03-16

Hi Brendan,

An error related to command line runner is observed. I downloaded the Release version of Skyline 20.1.76. But the error description indicates that there is a dependency on Skyline daily.

Please find attached the screenshot for more details.

Thanks,
Shobha

 Error.png 
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Points Per Peak
(3 responses) sa825 2020-03-11

Good morning,

I am conducting PRM on the Thermo Q Exacitive and I wanted to ask if there was a setting in Skyline that allowed you to select the number of points per peak for the peptides of interest?

Thanks,

Shimon

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export a method with each transition CE optimized
(3 responses) tongzhang 2020-03-12

In a project to optimize CEs for each transition, I created methods files with the option of optimizing collision energy selected. I ended up with 50 injections for 391 transitions on a TSQ. I followed a Skyline tutorial (link below) to upload the raw data, and would like to export a method with the best CEs for each transition. I enabled the "use optimization values when present" option, but can not get the optimized CE exported. One thing I noticed is that a schedule method was used in the tutorial, but I still want to export multiple methods because my peptides elute in a pretty narrow window.
I also attached the skyline files if that helps.
Thanks very much!

https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/OptimizeCE-1_4.pdf

 optimizing_CE_each_transition.sky.zip 
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Import Results no longer works for bruker.d DIA data in version 20.1 (works fine in version 19.1)
(6 responses) dkueltz 2020-03-04

Hi Brendan,

We recently noticed that the import of bruker.d folders that contain DIA raw data from Bruker UHRQTOF instruments does no longer work in Skyline v20.1. It took us a while to track this problem to the version of Sklyline. We tried all other possibilities (spectral library. assay list, raw DIA files) and finally were able to determine that the results import works without any problem in v 19.1 but not at all in v 20.1. It would be great if you could look into this. Let me know if you don't have bruler.d raw data to test this with.

Thanks,
Dietmar

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adding modifications to peptide sequence
(6 responses) kbagramyan 2019-12-17

Hi Brendan,

I am glad that I found the question that I wanted to ask you too. We recently performed an MRM analysis using 6495 LC-MS Triple Quadrupole (Agilent). We optimized the MRM transitions for the quadruply charged precursor ions of the light and the heavy peptides. Here, are our transition settings used in the method: 545.3 (precursor, +4) and the transitions are 509.0 (b, 4+); 640.0 (b, 3+); 678.3 (b, 3+). The Skyline recognized the two of transitions such as 508.5155 and 639.67. The third transition of 678.3 in our method was 677.6849 in Skyline transitions giving 0.6151 Da difference. Unfortunately, this was the most intense peak but wasn't accepted by Skyline because I couldn't extend the method match tolerance above 0.6.
The data look very good and I was wondering if there is a way to extend the method match tolerance, so I can include that transition for accurate quantification?
I hope I was clear and was able to describe the problem.
Thank you in advance,

Karine

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Unable to export method to Masslynx 4.1 SCN950
(10 responses) Tran Tran 2020-01-16

Skyline ( 64-bit) 19.1.0.193
Masslynx V4.2 SCN895/ Waters Xevo TQD
Windows 7 professional 64 bit

Skyline is installed at the same computer as the instrument . I was trying to export CE optimization using the File/ Export /Method/Multiple methods but received an error ( screen shot attached). However, I was able to export into the .csv format using File/Export/Transition List

Waters Tech Support was contacted but they don't have a solution at the time. Masslynx security update isn't installed either.
Wonder if anyone else observed this issue and was able to resolve it.
Thank you,
Tran

 SkylineError.JPG 
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Generation of spectral library using Prosit
(8 responses) benoit fatou 2020-03-03

Hello Skyline Team,

I am trying to use Prosit to generate spectral libraries and I realized that Prosit does not recognize "propionamide" as a fixed modification on the cysteine residues even when I set this modification on Skyline.
Is there a way to modify the modifications into Prosit?

Thanks for your help,
Best,
Benoit

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Error generating spectral library from FragPipe in Skyline v20.1.0.31
(12 responses) Chinmaya k 2020-02-13

Hello,

I have not been able to generate a spectral library in Skyline from FragPipe/MSFragger output. Please find the attached error file. The same error has also been reported recently as well. (https://skyline.ms/announcements/home/support/thread.view?rowId=43828)

I tried to generate a spectral library separately using BlibBuid.exe and it was also ended up with the same error. Please find the attached screenshot as well.

Is there any other way to rectify this and generate a Skyline compatible spectral library?

--
Chinmaya

 FragPipe_SpecLib_BlibBuild.exe_Error.PNG  FragPipe_SpecLib_Skyline_Error.PNG 
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Peaks image export
(5 responses) davidz 2020-03-09

Hi there,

Is there a way to export all the peak images to a PDF file?
Thanks,

David

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Setting to limit the tolerance to be 5 ppm in peak finding
(2 responses) davidz 2020-03-06

Hi there,

Which setting would be the one to set so the ppm tolerance is 5 ppm or a given ppm value during peak finding?
Thanks,

David

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Showing Trendlines in Skyline
romeally 2020-03-06

I have a project where I am looking at change over 5 time points of a reaction. I ran 3 replicates of each and wish to show the changes over the 5 time points with variance. What is the best way to represent this in Skyline? I am calling each time point a replicate, but the document grid only allows a comparison of two conditions. Any suggestions?
Thanks! Bob

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Skyline not recognizing FIAMS CV rough tune data
(4 responses) cmwats2 2020-03-03

Hi,

I used Skyline to export SRM transition list for compensation voltage rough tune. I acquired the data on an Altis instrument. I imported the data selecting optimizing compensation voltage -> rough tune. I attached an image of the imported data. However, when I try to make an optimization library containing the peptides and best CVs, skyline says there are no new optimizations. I can successfully create the optimization library for collision energies. What am I missing for Skyline to select the optimal FAIMS CV for my peptides? I cannot make a medium or fine tune method because I do not have the rough tune data.

Thanks in advance,
Caroline

 SkylineCVissue.PNG 
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fail to import peptide search into skyline due to the retention time problem.
(3 responses) chengkang 2020-03-04

Hi,
I was trying to import my maxquant peptide search into the DDA in skyline, however, after I selected the .raw file of the search, an error message popped out:"The document specific spectral library does not have valid retention times. Please check your peptide search pipeline or contact Skyline support to ensure retention times appear in your spectral libraries." Can anyone help me out?

Thanks,
Chengkang

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Calibration curve of Sodiated Adduct not appearing
(5 responses) alejandro.cohen 2020-03-04

Hi Skyline people!

I'm running a MS1 filtering (Molecule Interface) of three catecholamines on a Vanquish QExactive system. Two of our targets (Epinephrine and Phenylephrine) work fine, however, I cant get Norepinephrine to show the Calibration Curve with all other parameter being identical. Interestingly, I only see the sodiated adduct on this target... wondering if this could be the issue?

Attached the corresponding file.

Thanks!!

Alejandro

 QExactive_Catecholamines_Positive.sky.zip 
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Separating HCD and EThcD spectra
(9 responses) jonasbecker 2020-02-26

Dear Skyline Team,

I ran a PRM experiment which used different fragmentation modes (HCD and EThcD) on the same peptides in the same run, i.e. HCD and EThcD spectra were recorded alternately. When loading these data into Skyline, it seems for some spectra that the MS2 chromatogram is "oscillating" (see attached picture1 & picture2). In contrast, this does not occure for precoursers fragmented only with HCD (see attached picture3).

Is there any filtering/data processing option available to separate the HCD and EThcD spectra so that Skyline can interpolate the peak areas correctly?

Thanks in advance and best regards,
Jonas

P.S.: If you could provide a private link, I could share you the skyline project.

 picture1.png  picture2.png  picture3.png 
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