Welcome to Skyline 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.

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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Requests 
Showing: limited to 100 requests
Advanced peak picking models for small molecules
(3 responses) Mathias Kuhring 2017-12-13
Are there any plans for advanced peak picking models for small molecules? The machine learning approaches (such as mProphet) rely on decoys, which seem to be available for peptides only. Will there be other algorithms or at least more parameters (e.g. smoothing, selecting biggest peak instead of closest RT, ...) to improve peak detection and integration for small molecules?
In particular with low concentrations and intensities, I often observe that a) a small noise peak closer to expected RT is preferred to considerably bigger peaks (which are often detected and marked with the dashed lines, but not selected). And b), rather jagged peaks are identified as several peaks (which often contributes to problem a). I think, having some control of peak integration parameters might help here.

Best, Mathias
view request
Library versus DDA import for PRM
(2 responses) abrahampe 2018-02-21
Hello,

I generated DDA data on a QExactive Pro instrument that I then used to build a spectral library from Crux results. I have created a .ssl file as instructed here (https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec%20input%20and%20output%20file%20formats).

I imported the .ssl and created a library that appears to be correct.

Next, I then imported the results (.mzML) files that were used to generate the library. Interestingly, the peak picking (transition settings: MS/MS filtering, acquisition method=targeted, orbitrap, resolution 60k @ 200 m/z) does not seem to be working properly. I would expect the dot product scores to be a value near 1, given that the library was generated from the same data. Oddly, the peak picking retention times are, in many cases, nowhere near the ID time. For whatever reason, Skyline isn't identifying the transitions near the ID time. I have used the parameters outlined in the webinar document and I've looked through the MS/MS manually and the mass tolerances are well within the set parameters. I'm not quite sure what's happening. I've looked at previous posts regarding this topic and so I've made sure to check file names, for example.

Thoughts?

I am using Skyline 64bit version 4.1.0.11796.

--
Paul

 to obtain a refined list of precursors that will be used for a PRM approach.

I am using the Webinar-17 document as a reference.
view request
MS1 Full-Scan Filtering
(1 response) pap adam 2018-02-21
Dear Skyline Team,

 I am working with Skyline's MS1 Full-Scan filtering workflow to estimate the reproducibility of an O-glycopeptide enrichment protocol. In a nutshell: I have three technical replicates in which I try to verify the presence of O-glycopeptides at MS1 level using confident MS/MS identifications (both ETD and HCD).

My problems:
 1.) I use the custom .ssl format to generate the spectral library. In this library there are ETD and HCD identifications of O-glycopeptides (for one peptide ID there is only one ETD or HCD spectrum). My problem is with the HCD glycopeptide identifications. For ETD I can insert the modifying sugar structure into the peptide sequence, but for HCD it is not acceptable because the sugar, during collisional activation, flies off from the peptide therefore the site cannot be assigned. Is there any recommendation about how to indicate the neutral loss of the sugar in the peptide sequence?

 2.) I have 12 raw files in .mzML format (see raw_files.jpg). Skyline consideres for MS1 filter only the raw files that are mentioned in the .ssl file (see results_file_found.jpg and ssl_file.jpg). Why does that occur? I expected that Skyline will search the peptides, listed in the .ssl, in all the provided raw files at MS1 level.

I would really appreciate your help! Thank you!

-Adam
 ssl_file.jpg  raw_files.jpg  results_file_found.jpg 
view request
Labelling approach in DIA
(4 responses) William 2018-02-20
It is said that DIA usually not compatible with labelling approaches like SILAC. However, to get better quantitation, the labelling method could preferred in SRM or PRM. I wonder if I have known protein targets (and library from DDA), can I use Skyline to extract chromatograms for both light and heavy peptides (normalise to heavy) for quantitation? Is there coisolation and cofragment issues?
view request
QC with Skyline Alone
(2 responses) victor nesati 2018-02-19
I was reading a bit about Skyline QC capabilities and looks like current workflow consists of
AutoQC Loader, Skyline processing and pushing those results to Panorama for further visualizations.
I am all for it except that we have limited access to internet on our data acquisition computer and while
first two legs of this pipeline are easily implemented, pushing to Panorama would be a bit of an issue.
If I understand correctly, Panorama has a bit more options with regards to visualization, but in principle some basic info like Peptide Areas, RT, FWHM, etc could be easily tracked in Skyline itself. So in principle upload to Panorama could be skipped, correct ?
Cheers
Victor
view request
Transitions not found in results files
(4 responses) as3e15 2018-02-19
Dear Skyline team,

I am encountering a problem that i cant seem to find help for in your support section. I am doing an MRM experiment but it seems when i import my results files, some of the transitions have no data. I know this is not the case because some other runs (of exactly the same thing, in exactly the same conditions) have data for these transitions. to be more specific, it seems to be the precursors of every peptide which does not show up.

I am using a Waters Synapt G2-Si mass spectrometer.

I have attached a screenshot of what i mean.

I hope i have made this clear and given enough information for you to help.

Many thanks,

Andy
 No transition data.pptx 
view request
MS1 XIC QUAN
(15 responses) victor nesati 2018-02-13
HI, I have this interesting situation during MS1 XIC quantitation. I imported MSF file from Proteome Discoverer, along with mzML file, and for some reason Skyline finds Peptide which was not in PD output. This created a bit of an issue as I really do not need to quantitate something which was not found as a result of data base search. Is there any way to filter out those results based on confidence ( yellow/red colour ), oidp score , XIC intensity or some other metrics in Skyline prior to exporting in Excell ? Or alternatively create the library which contain only peptides found in DB search and thus circumventing this issue from the very beginning ? I think this issue was raised before
just wanted to try to tackle it from different perspective. Of cause I can manually remove it but that feels like missing a point, as the goal is to minimize human intervention and subjectivity.

Thanks for advice
Victor
view request
Quickly Delete Peptides with Missing mProphet Features
(1 response) phillip ihmor 2018-02-19
Hey Skyline team,

I'm running a DIA workflow and have >150k peptides in my spectral library. I am using Skyline 4.1.0.11717. I want to employ the mProphet reintegration to filter for well identified peptides. When setting up the reintegration model, several key metrics are grayed out as not 100% of the peptides have values in that metric. Now, I understand that the mProphet model cannot deal with missing values, so one has to exclude them. To loose 1k peptides in a panel of 150 is no issue. The thing is just that I only know one way to delete them: click on the binoculars of each grayed out feature score, double click on each peptide in the "Find Result" list, press delete and repeat until the list is finished.
I can do that for 20 peptides, but not for >1k peptides?! Is there any way to do that quicker?
Is edit -> refine -> advanced the right place for that? I can't find an option.

I believe this issue became a problem when switching to 4.1. Before that, the mProphet model seemed to be more robust/applicable to more peptides? But maybe I just have a wrong setting somewhere. The library intensity dot product for instance seems only to be calculated, when more than 2 transitions are available. I've found the new feature in transition settings -> library -> pick minimum product ions, but that is just for the selection of the library, not in the DIA data, right? There seem to be peptides that just miss some transitions due to random / noisy data, which then then screws up the whole mProphet model? That seems quite drastic. Am I missing a setting here?

Furthermore, it would be tremendously helpful if you could give me some rule of thumb on the expected number of identified peptides. With an unfractionated library of 40k peptides, I can identify about 12k peptides at a qvalue < 0.01. Is that expected? I found one statement by George Rosenberger for openSWATH, that he identifies 70-80% of all peptides of an unfractionated spectral library.
I know, there are a million reasons, why my number might be low, but some ballpark figure could help me to gauge my analysis. In most papers, I find the number of positively identified peptides, but not the size of the spectral library. That seems to be kind of crucial... In the Navarro paper, you get >30k peptides, but that is from a deep fractionated spectral library. With my 150k library and current limited features in the mProphet model, I get next to no peptide to pass the qvalue<0.01 threshold.
My DIA run was over a 90min gradient on a Q-Exactive.

Thanks for all the hard work. I can't imagine how much work it must entail to build something like skyline...

Best,
Phillip
 2018-02-19 2 transitions lead to missing dotp.png  2018-02-19 search for peptides with missing features.png 
view request
very slow performance of skyline with saved .sky file after opening
(1 response) kguehrs 2018-02-19
Hello Skyline team,

I use Skyline (64 bit) 4.1.11796 with a MS1 filtering approach. There are 5 Mascot .dat files and 5 Thermo .raw files (not named the same way but correctly assigned)that I copied to my local harddrive. My computer is not very highly equipped in terms of both processors and RAM and therefore I do not expect it to be superfast. Preparing the library took quite a while but after this was finished I was able to manually remove peptides or poor peaks, edit peak boundaries for re-integration and do refinement of the protein list without too much delay. I saved the project without any problem.
However, when I try to complete the editing after opening the saved .sky file another day the performance of Skyline is very slow compared with the same manipulations on the day of project creation. Switching between samples or retention time or peak area windows needs a very long time and I see in the Windows task manager that Skyline is using lots of processor capacity just for switching between replicate tiles what I cannot understand. Is there any difference in the way Skyline links the files (spectra and chromatograms) between a freshly created and an re-opened saved file?
If the problem is my computer I would be interested to learn the minimal and recommended hardware requirement for the different versions of Skyline. Perhaps, it would be a good idea to place this information on the download site of Skyline (either as text or as a link).

Did you ever hear before about the behaviour I described. Is there any suggestion how I can prevent the current situation?
view request
Skyline Roaming Profiles
(2 responses) Marco Schmidt 2018-02-14
I already mentioned the problem in another request, but to summarize the problem I created this new request.

https://skyline.ms/announcements/home/support/thread.view?rowId=24266

Skyline hast two possible install options:
1. clickone application
2. admin install

Togehter with windows roaming profiles there are some problems.
1. is installed (incl. config) in the local part of the profile, which is removed on logout.

2. is installed in %programfiles%, but the users config is still in the local part of the profile and deleted on logout.

External Tools:
1. are installed (incl. config) in the local part of the profile and deleted on logout.

2. as mentioned on the download page, the external tools can only be installed by an administrator and the config is installed in the profile of the administrator. This makes the installation useless, because if a user runs skyline, it does not know about the installed tools.

The user can not use the tools which are installed by an administrator!

Even if the user would have administrative rights and install the tools, the config is still in the local part of the users profile, which is deleted on logout.


Now I tried to add a script which uses skylinerunner to install the tools via "one-click" for the user. But unfortunately the skylinerunner does not recognize the admin-install-skyline and downloads/installs a fresh one (which is of course deleted on logout).

Seems that I have to copy the config file to the correct location in the users profile on login.

Of course, there are several other possibilities to solve this (e.g. a local skyline user on each host, with the login and password distributed to all users).
view request
Problem with 4.1.1.11798 cannot open existing projects or load transitions
(4 responses) eer55 2018-02-18
Hi there,

I just downloaded the new version of Skyline daily. (v4.1.1.11798)and ran into a few problems. I cannot open existing projects. i tried to load a transition list and that failed too. I'll attach a word doc with the error details.

Error loading .sky file: The file contains and error on line 45 at column 10 (details attached)

I am running windows 10.

Thanks very much,

Elden
 Sky_errors_.docx 
view request
Isolation list export for Fusion Lumos 3.0
(7 responses) mwfoster 2018-01-08
Hi all,

Is there any possibility that you could update the isolation list export format for the most recent version of Tune for the Fusion Lumos? The attached file was exported from the Lumos method editor.

Thanks,

Matt
 Mass List Table.csv 
view request
mProphet discrepancy between Skyline Versions
(1 response) doellingerj 2018-02-16
Dear Skyline Team,

thanks for the amazing Software!!!

I am currently working on DIA data generated on a QE instrument. When I analyze one run using Skyline Version 3.5.0.9321 I can detect 13058 Peptide Sequences with q < 0.01 using mProphet. When I open the same document, which was generated in Version 3.5.0.9321, in Skyline 4.1.0.11714 and reintregrate the data using mProphet I can only detect 10662 Peptides with q < 0.01. What is the reason for this large difference and how can I get back my "lost" peptides?

kind regards

Joe
view request
Custom Spectral Library
(10 responses) gwilson9 2018-02-13
Hello,

I am encountering an error building a custom spectral within Skyline. I get a series of errors after loading the .ssl file:

Error: unknown escape sequence
Error:
Error: reading file ForSpectralLibrary.ssl

The .ssl file is templated after the example found here: https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec%20input%20and%20output%20file%20formats

and spectra files are .ms2 files created from Thermo .raw files with MSConvert.

I have attahed the .ssl file and one of the .ms2 files (truncated for file upload). I would really appreciate your help trouble shooting, thanks!

Gary
 ForSpectralLibrary.ssl  20180207_InterlabPhosphoSample_StandardSample_Run1_30KMS2_.ms2 
view request
How can I add a disulfide bond between two peptides in the Skyline document?
(25 responses) danielacgranato 2015-10-17
Hello,

I am interested in measuring a peptide containing a disulfide bond. How can I do that using Skyline?

I added my peptide sequence and considered the cysteines to have a dehydro modification in order to get it with the correct m/z value but Skyline shows no precursors in the ion chromatogram. I have confirmed the presence of this peptide though by a software that predicts disulfide bond formation and also using XCalibur.

Thank you very much for your help. Best.
view request
PRM reproducibility question
(1 response) jung165 2018-02-13
Hello this is Jinwoo Jung who are working with PRM analysis.

I am about to start PRM analysis for my experiment and I have one quick question about its reproducibility

I have performed PRM analysis with Q-exactive device with triplicate run but it shows some peptides with over 30% coefficient variance between multiple analysis.

Retention time variance is about 1~2 minutes. I think this is not much acceptable values for variance.
I also analyzed same peptide with SRM, but it shows almost same retention time between multiplicate run.

I would like to ask those variances are acceptable. If not, is there any parameters that need to be modified for better result???

I also attached my result and method of PRM analysis

I really appreciate for your help.
 skyline question.pptx 
view request
Error opening a skyline file
(2 responses) jmurphy 2018-02-14
Hello, when I try to open a file it gives the following error: Failure opening file...The file contains an error on line 1 at column 1. Is this file corrupt and I have to redo the processing? I have attached the full error.

Thank you,
Jen
 skyline issue.docx 
view request
remaining skyline program files after upgrading ?
(1 response) kguehrs 2018-02-14
Hello Skyline team,

I just upgraded to 4.1 64-bit on a Windows7 machine. Performing a Windows search for the term "skyline" and browsing the file tree I noticed that there are remaining files from previous installations.

Might these file produce problems at some point? Is it possible to get a clean installation of the current version and remove the old files not reqired anymore. I do not know whether some of the old files are reqired to read or reprocess former analyses. Therefore it is difficult to manually remove the files left from previous versions.
view request
Can't open my skyline
(1 response) Sadr 2018-02-13
Hi,

I can't open my skyline file; can you help me on it?

Please find the error message in attachment, for full details.

Regards,

Sadr
 SKYLINE error.txt 
view request
iRT issue
(6 responses) chloe bardet6729 2018-01-18
Dear Skyline team,

I use the last Skyline version, but I also used the previous (3.7) and it doesn't work.
The MS I have is an Sciex5500QT

I would like to use an home made retention time calibrator containing 14 peptides and I have an issue to export correct calculated retention times.
When I use the 14 peptides to retention time calibration for 86 peptides from an LC gradient N°1 to an LC gradient N°2 (HPLC vs UPLC) using a basic linear regression, it works on Excel. However when I am using Skyline after reading the tutorial, I failed. Predicted retention times did not appeared in the "export transition list" and "calibrated retention time" that are noticed in the "export, report, peptide results" were not well calculated.

After the 14 peptides calculator built, I import results for the 86 peptides with gradient N°1 and I calculated iRT for the 86 peptides. This works, I had a great regression. Then when (in another skyline file) I import the results for the 14 peptides with gradient N°2 the RT predicted for the 14 peptides seems great, but I didn’t succed to export the RT predicted for the 86 peptide with the new gradient. When I wanted to export the transition list, Skyline asks me to choose the result file that I want to sort the retention times (or an average) never it talks about “predicted retention time” as you describe in your tutorial.

Thank you by advance for your help.
Best regards

Chloé Bardet
view request
problems locating skyline installation in windows server 2012 R2
(18 responses) tobias.kockmann 2015-11-17
Dear skyline support,

I have a running installation of skyline and skyline daily on windows server 2012 R2. But skyline is not displayed in the list of installed apps, nor can I find on my system when using the search tool. I suspect this has something to do with the roaming user profiles used. As soon as I right click on a skyline doc, skyline opens up, so it is def. there and functional. Is there a way to solve this problem (get a stable link to start skyline)?

Greetings,
Tobi
view request
q-value issue when attempting to build library from .pdresult
(1 response) monica lane 2018-02-09
Hi Skyline team,

I'm attempting build spectral library from Proteome Discoverer 2.2 search result (Mascot search node in workflow and consensus workflow nodes that calculate q-values at PSM, peptide and protein level). I'm using the .pdresult file but getting following message from Skyline (in both Skyline v 4.1.0.11714 and Skyline Daily v 4.1.1.11756):

System.IO.IOException: ERROR: This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives.
ERROR:
ERROR: reading file Heavy_peptide_pool_2X_013118_2.pdResult

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 125



Do you have a workaround or fix so that I don't have to set cut-off to zero?

Thank you!
view request
Adding (glycan) modifications as a list
(2 responses) Erik de Graaf 2018-02-06
Hi Skyline Team,


I have generated list of all glycans I could expect like this:
Name    Mol Formula
H5N2    C46O35H76N2
H6N2    C63O49H103N3
H7N2    C80O63H130N4
H8N2    C97O77H157N5
H9N2    C114O91H184N6
... ...


Is it possible to add a lot of modifications like this list to the Mod table, just by simply copy/pasting into a Skyline config file?

This would help the glycoproteomics field a lot.

Thank you,

Erik
 Glycan-mod-list.xlsx 
view request
targeted metabolomics with Skyline
(2 responses) arnaud garcia 2018-02-08
Dear Skyline developpers,

I work on a project of using this software to extract feature from Orbitrap raw data to obtain a standardized profile of metabolites. The goal is
- Upload raw data files
- Upload a database of molecules to screen for ( with exact masses/ formula and known retention time)
- Extract from the database the area of the masses that are found only in the time windows (and if there is nothing, then the area should be left blank).
- Get in an excel file (in the best scenario) the observed area, peak info, masses and retention time for each molecules screened.

After uploading the list (using the "Edit > insert > transition list") and the insertion of raw data, it occurs to me that I couldn't get the selective windows of retention time correctly even after "re-scoring" of the data and changing parameters. For many metabolites it was good because they were found within the right range. However (fig 1 in the file attached), many ions were returned by the software to be matching our molecules of interest while they were out of the suggested ranges.
Same, another problem was the mass accuracy of my list. I was working with an Orbitrap format and couldn't reach manually the mass accuracy settings ( from Settings > Transition Settngs > Full Scan). The accuracy at my selected settings were too high for my study so i had to switch my precursor mass analyzer to "centroided" in order to manipulate the mass accuracy, even if it is less than ideal.

So here i have two main questions :
- is it possible to integrate areas automatically for a set m/z windows and a set Retention time windows regardless of the kind of data used (orbitrap, Waters, Agilent..)?
- Is there a way to select easily the mass accuracy of the selection of masses when selecting MS1 Orbitrap files?

Retention time accuracy and mass accuracy are really important for selectivity in metabolomics and since the goal of Skyline is also to cover small molecules integration, those parameters are still confusing to me.

Thank you very much and have a lovely day,

Arnaud
 results.pptx 
view request
linking Human proteome to skyline
abasit 2018-02-08
Hi skyline,

I have acquired protein digest in SWATH mode in SCIEX 5600 tripple TOF. I would like to search the identifiable peptides in my scan. How I can link human proteome (FASTA) downloaded from uniprot to skyline as library of peptides or proteins?

Thanks

Abdul Basit
view request
Skyline 4.1 gives a different order for isotopologs than previous version
(1 response) john adamson 2018-02-07
Hi Guys,

We have set up a template for small molecule C13 incorporation into metabolites, using previous versions of Skyline.
When opened with Skyline 4.1 64 bit, the isotopolog order is altered, and this could cause problems with our end users and their manipulation of peak areas of the different species.(see attached PDF)

I might add that we depend heavily on skyline for our targeted metabolomics and proteomics and really appreciate the effort put into the program by your team. I can send relevant Skyline files if the need arises.
 JHA-Isotopolog Order Issue.pdf 
view request
Lipidomic Analysis
(1 response) elizabeth waldron 2018-02-08
I analysed labelled lipid standards using a DDA experiment on the Q-Exactive machine. I imported my data into Skyline and found that Skyline was only extracting data around a specific time frame. The outcome is that my MS1 data is being extracted beautifully, but my MS2 data looks like a scheduling experiment gone wrong. I have attached a screenshot of my Skyline document. Can anyone advise why Skyline is extracting MS2 data in a limited time frame and not over the whole chromatogram?
 Troubleshooting_OctanoicAcid.PNG 
view request
File from previous version does not open in Skyline 4.1
(7 responses) afmpinto 2018-02-07
After updating to version 4.1, files from the previous version do not open.
This is the error one of those files gave me.

Failure opening file
The file contains an error on line 233 at column 10.
unable to locate precursor calculator for isotope label type heavy and mods (null)

System.Reflection.TargetInvocationException: There is an error in XML document (233, 10). ---> System.InvalidOperationException: There is an error in XML document (233, 10). ---> System.IO.InvalidDataException: unable to locate precursor calculator for isotope label type heavy and mods (null)
   at pwiz.Skyline.Model.DocSettings.SrmSettings.GetPrecursorCalc(IsotopeLabelType labelType, ExplicitMods mods) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\DocSettings\SrmSettings.cs:line 249
   at pwiz.Skyline.Model.TransitionGroupDocNode.CalcPrecursorMZ(SrmSettings settings, ExplicitMods mods, IsotopeDistInfo& isotopeDist, TypedMass& mass) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:line 724
   at pwiz.Skyline.Model.TransitionGroupDocNode..ctor(TransitionGroup id, Annotations annotations, SrmSettings settings, ExplicitMods mods, SpectrumHeaderInfo libInfo, ExplicitTransitionGroupValues explicitValues, Results`1 results, TransitionDocNode[] children, Boolean autoManageChildren) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:line 78
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupXml(XmlReader reader, Peptide peptide, ExplicitMods mods, Double deltaMass) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1237
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupListXml(XmlReader reader, Peptide peptide, ExplicitMods mods, Double deltaMass) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1042
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideXml(XmlReader reader, PeptideGroup group, Boolean isCustomMolecule) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 873
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideListXml(XmlReader reader, PeptideGroup group) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 756
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 726
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupListXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 568
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 536
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1947
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass12b.<OpenFile>b__129(IProgressMonitor progressMonitor) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 258
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1837
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 176
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 262
view request
QuaSAR reshape2 and ggplot issues R 3.0.1 and QuaSAR-1_32
(2 responses) peter.scholl 2018-02-07
Folks,

I was initially learning to use QuaSAR via an old tutorial .zip file QuaSAR-1_0 while running Skyline 4.1.0.11714 and obtained well know error messages in the immediate window. After upgrading QuaSAR-1 to QuaSAR1-_32 some of the same error messages appeared.

We are running Win 7 SP1 64-bit and R-3.0.1. The last page of the attached .ppt displays the error message.

The skyline shared document has also been attached.

Am I making a mistake or is there something "going on" with the R packages reshape2 and ggplot?

I'd like to use QuaSAR but find myself currently "stuck" at this point.

Many thanks for any help you can provide to resolve this problem,

Pete
 QuaSAR_Tutorial_share.sky  02072018_QuaSAR_tutorial_errors_2.pptx 
view request
Custom transition list data
(4 responses) B.C.M. van der Helm 2018-02-06
Dear all,
I am analysing lipid data with the skyline software. One of the problems i have is that the lipids can be separeted in classes and (total-) fattyacid chain lenght (2 groups). and i was wondering of it is possible to ad a function in edit>insert>transitionlist where you can select the annotition for an specific transition, so that when you export the data in a report, you also be able to sellect those data from you transition import. The note function won't work because those are multiple parameters. I see the annotion function back in the notion. But how can i add my data to those anotions by the import of an transitionlist?

The transitionlist is imported from an .CSV and combiend with the MS/MS data and exported to R/Excel for data analysis and now i have a "loss" of data between import and export of the transitionlist. Adding the data later is an pain in the ass because there are more than 1500 transitions and a typical batch has more than 100 samples masseured in duplo. Also there could appear an error in the recombining of the data.

I was wondering if you have anny idea's to help me with those problems?
I work in both skyline 4.1.0.11714 and skyline daily 4.1.1.11756
Bas
view request
Import Peptide Error
(5 responses) rbao1357 2018-02-06
Hi guys,

I am a new user of Skyline (and to the MS field). I have been experiencing an error over the last 2 weeks when attempting to import peptides in the initial steps.

I have previously created technical replicate files under names (RB1, RB1b, RB1c etc), under the (.group), (wiff), (.wiff.scan) formats. These files are all stored in a single folder. When attempting to extract chromatograms, the peptide search is duplicating the first (RB1.wiff) file, rather than finding (RB1b etc) files. I have tested this with a number of different files, and the software is not locating the other files under different file names. I have also tested against (RB1) and (RB2) only, and it successfully builds the peptide spectrum.

This generates the below error.

An item with the same key has already been added.

Skyline version: 4.1.0.11714 (64-bit)
Installation ID: cf11b43b-5d9e-48e1-b3b6-ba6bcb35d288
Exception type: ArgumentException
Error message: An item with the same key has already been added.

--------------------

Stack trace:

   at System.ThrowHelper.ThrowArgumentException(ExceptionResource resource)
   at System.Collections.Generic.Dictionary`2.Insert(TKey key, TValue value, Boolean add)
   at pwiz.Skyline.Model.Results.MeasuredResults.set_Chromatograms(IList`1 value) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\MeasuredResults.cs:line 80
   at pwiz.Skyline.Model.Results.MeasuredResults..ctor(IList`1 chromatograms, Boolean disableJoining) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\MeasuredResults.cs:line 57
   at pwiz.Skyline.SkylineWindow.ImportResults(SrmDocument doc, List`1 namedResults, String optimize) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2601
   at pwiz.Skyline.SkylineWindow.ModifyDocumentInner(Func`2 act, Action onModifying, Action onModified) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Skyline.cs:line 671
   at pwiz.Skyline.SkylineWindow.ModifyDocument(String description, IUndoState undoState, Func`2 act, Action onModifying, Action onModified) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Skyline.cs:line 635
   at pwiz.Skyline.SkylineWindow.ModifyDocument(String description, Func`2 act) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Skyline.cs:line 629
   at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.WizardFinish() in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 606
   at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.NextPage() in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 358
   at System.Windows.Forms.Control.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnMouseUp(MouseEventArgs mevent)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ButtonBase.WndProc(Message& m)
   at System.Windows.Forms.Button.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)

Exception caught at:
   at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
   at System.Windows.Forms.Control.WndProcException(Exception e)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.SkylineWindow.ShowImportPeptideSearchDlg(Nullable`1 workflowType) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2866
   at pwiz.Skyline.SkylineWindow.importPeptideSearchMenuItem_Click(Object sender, EventArgs e) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2853
   at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
   at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
   at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ToolStrip.WndProc(Message& m)
   at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at pwiz.Skyline.Program.Main(String[] args) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Program.cs:line 311


I have attached screenshots of the way the Skyline is behaving in a pptx. Are you able to provide any insight?

Thanks,

Rebecca
 Skyline Error RBAO.pptx 
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How do I create skyline file (.sky file) using command line
(5 responses) Bhoomi Dhananjay Bhatt 2018-02-05
E:\Skyline-64_4_1_0_11714\SkylineRunner.exe --in="C:\Users\shiro\Documents\TargetedMSMS_2\Low Res\test1.sky" -import-fasta=E:\prm\9606_refseq_2017_11_29.fa --import-all=E:\core\ --exp-translist-instrument=Thermo --exp-file=E:\core\test1.tab

Here, test1.sky was created using skyline GUI prior to running this line of code.

Thanks,
Bhoomi
view request
Overlaying same peak across different samples
(2 responses) victor gonzalez 2018-02-05
Dear all,

I would like to know if there's a way to get a graph in which I could plot an overlay of the profile of all the peaks corresponding to the same transition, across all of the available result files. I have checked the tutorials and webinars, but I was unable to find an example in which such feature was employed.

Thank you so much in advance, and best regards,

Víctor.
view request
add multiple modifications on a sequence at the same time
(1 response) Jongmin Choi 2018-02-05
We have multiple modification such as phosphorylation on 1~2 sites and acetylation or oxidation together.

Actually, I have big phospho-peptide list, so I don't want to add modification one by one.

Is there any of short way to make multiple modification? Or if I can import a chart or file including sequence list with modification, it will be better.

Thanks,

Jongmin.
view request
Invalid cvParam error when loading mzID
(3 responses) solntsev 2018-02-02
System.IO.IOException: ERROR: Invalid cvParam accession "1002826"
ERROR:
ERROR: reading file s.mzid

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 125
view request
How to identify the peptides by using MS1 spectra?
(3 responses) zzhang9 2018-01-11
Hi,

How to identify the peptides by using MS1 spectra in Skyline? Similar as "match between runs" in MaxQuant, but I want to use the normalized retention time instead of the measured one. Thank you!

Best,

Zhenbin
view request
Opening 3.7 and earlier small molecule assays with 4.1 - error
(4 responses) Chris 2018-01-15
Dear Skyline support,

Our lab has encountered an issue in regards to some small molecule skyline assays from 3.7 and earlier. I believe it originates from our method of describing small molecules for import with Skyline in which we give the bare minimum for transition list import (molecule list name, precursor name, product name, precursor m/z, product m/z, precursor charge and product charge). Please find the error at the bottom of this text.

We already have a workaround (importing our transition list to feature the adduct form) so it's not essential for our work to continue but it does mean that we can't open some of our assays built in 3.7 and earlier. Curiously, some of the assays I've made for 3.7 do work in 4.1 so I'm still trying to work that bit out (likely I used additional transition list import columns). Reading into the error below, the calculated value is 0.577550289955 less than the declared mz value (which is my observed m/z) but the theoretical value of my small molecule is 819.2533 which is still 0.410850289955 greater than the calculated value. Is there something weird going on with adduct calculation for assay files from 3.7 and earlier?

This error has also occurred with a colleague's skyline assay with a different small molecule but funnily enough, the deviation between calculated and declared is different than what I observed? Sorry I can't be of much more help.

Please find one error below, I can provide the Skyline assay file in sharing format (.zip) via email if requested:
 System.Reflection.TargetInvocationException: There is an error in XML document (189, 9). ---> System.InvalidOperationException: There is an error in XML document (189, 9). ---> pwiz.Skyline.Util.AssumptionException: error reading mz values - declared mz value 819.42 does not match calculated value 818.842449710045
   at pwiz.Skyline.Util.Assume.Fail(String error) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1901
   at pwiz.Skyline.Model.Serialization.DocumentReader.ValidateSerializedVsCalculatedProductMz(Nullable`1 declaredProductMz, TransitionDocNode node) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1484
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionXml(XmlReader reader, TransitionGroup group, ExplicitMods mods, IsotopeDistInfo isotopeDist) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1476
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionListXml(XmlReader reader, TransitionGroupDocNode nodeGroup, ExplicitMods mods) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1365
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupXml(XmlReader reader, Peptide peptide, ExplicitMods mods, Double deltaMass) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1237
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupListXml(XmlReader reader, Peptide peptide, ExplicitMods mods, Double deltaMass) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1042
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideXml(XmlReader reader, PeptideGroup group, Boolean isCustomMolecule) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 873
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideListXml(XmlReader reader, PeptideGroup group) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 756
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 726
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupListXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 568
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 536
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1947
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass12b.<OpenFile>b__129(IProgressMonitor progressMonitor) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 258
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1837
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 176
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 262

Cheers,
Chris
view request
mProphet for DIA - Retention time difference when peptides not detected in some samples
(2 responses) Tomas Vaisar 2018-02-01
Hi,
I have a set of samples I ran DIA on where part of them are negative controls in which number of peptides is not detected. When I do the mProphet model the Retention time related parameters are grayed out and when I use Find to see why, many peptides show up with Retention time difference missing from peptide.
Is this because of peaks not detected in some samples? If so is there a way around it?

Thanks,

Tomas
view request
Cannot display multiple peptides across replicates using the
(1 response) fredrik edfors 2018-02-01
Hi,

Just found out that I cannot display multiple peptides at the same time in the same Replicate Comparison view since the last update. The y-axis automatically goes to 10^306 when normalizing to heavy. The non-normalised function does still work however. Cannot zoom in either.

Thanks in advance,

Fredrik
 Screen Shot 2018-02-01 at 12.02.13.png 
view request
Exporting a single MS1 area value per peptide
(2 responses) rcrobinson 2018-01-26
Hi Brendan,

I'm processing some peptide data in Skyline 3.6.0, and I would like to export the MS1 areas to Excel afterward. Currently when I export the data, I get a separate row for each peptide charge state, and the corresponding area value for each charge state. However, since I have around 3,000 peptide sequences, the Excel file becomes really large when each charge state gets its own row. Is there some way to export the data with only one row per peptide sequence? I am flexible in the type of data that gets exported; I just need to export some sort of abundance measurement for each peptide: it could be peak height or area for a single charge state (maybe the most abundant one), or some sort of total area/height. Would this be possible?

Thanks so much!

Randall
view request
Rollback installation
(8 responses) Joakim Bygdell 2018-01-26
Hi,
I need to get my hands on an installer for Skyline 3.7.0.11317

//Joakim
view request
Not abble to install the Skyline on new computer with Windows 10 Pro OS
(3 responses) Bennett Lab 2018-01-31
Hi Support Team,
I am unable to install Skyline 4.1 on out new lab computer having windows 10 Pro OS.
The issue is Microsoft .NET 3.5 Framework install. When clicked on the install windows, it shows error code 0x800F0950.
On our older computers with windows 7 OS, the program was downloaded and is running fine. his is windows 10 specific issue.

-Amit
 Capture.PNG 
view request
CE Optimization Observation
(2 responses) mburgess 2018-01-31
Hello-
I am using version 4.1.0.11714 of Skyline on a Windows 7-run PC operating a Waters Xevo TQ-S. I have utilized the CE Opt tutorial in the past and generated a series of methods with +/- 5 steps around a calculated collision energy value. The output methods from Skyline produce the proper number of transitions in the MassLynx MRM method and the data is collected normally. However, when I input the data from the runs into Skyline, I am only observing +/- 3 steps currently. Not sure if anyone else is seeing this. Am I missing a setting somewhere?
Many thanks in advance,
Mike B
view request
Install issue
(1 response) Brett Phinney 2018-01-30
Hey everyone, not sure if this is just my messed up win10 computer but I tried to install

https://skyline.ms/software/Skyline-release-64_4_1/setup.exe

And win10 would not let me install it even as the admin.

This seemed to fix it

https://superuser.com/questions/1252575/unable-to-install-clickonce-application-due-to-security-settings-windows-10


just an FYI if someone else has this issue

Cheers

Brett
view request
Skyline m/z tolerance window not work with Altis files
(4 responses) Lian 2018-01-26
Hi dear Skyline developer,

I have an issue with the m/z tolerance window when using skyline 3.7.1 to analyze Altis raw data files. Sometimes, Altis automatically changes the parent m/z by 0.001 when importing more than 10 transitions for the same compound (e.g. change parent m/z from 852.233 to 852.234). I am assuming Altis is relying on Skyline to still recognize it since the smallest isolation window of Q1 and Q3 is 0.2 but skyline does not. When the resulted raw data file is imported into skyline, skyline does not recognize the transitions with parent m/z changed by 0.001. This issue still exists when I changed the method mass tolerance m/z to 0.6 in transition settings/instrument in skyline.

I heard that this issue is resolved previously but probably the raw data from the new version TSQ-Altis is still not compatible. Could you please fix it?

Thanks

Emma
view request
webminar download
(1 response) Stephane Claverol 2018-01-30
Hi,
Webminar and tutorial download fails every time. Is there a ftp server where i could download the archives? Best regards
Stephane
view request
win32 error 5
(9 responses) Karin Wolters 2016-09-02
Hi Brendan,

we just installed Skyline on one of our PC's, but when our student tried to follow the steps in the tutorials (trying to load the rat-proteome raw-files) she repeatedly got the error-message ('win32 error 5" -> see attached screenshot).

I also tried to open a shared Skyline-file (with measurements loaded into it) that is running fine on another PC, but got the same error-message while loading it on this PC.

Not sure what we can do about this, hope you can help us out

Karin Wolters
 temp.pptx 
view request
cannot install skyline-daily
(13 responses) Shuxuan 2018-01-27
I got the following error(see attached), when I try to install skyline daily.
How do I install Newtonsoft.jason in the GAC?
Please help.
 微信图片_20180128134608.png 
view request
MIDAS prefix
(2 responses) Enzo 2018-01-29
Dear team:

For some reason, my library file generates a prefix "MIDAS_" automatically, not sure what's this for. Could you please help me sort it out?

Thanks for your help!

Cheers
Cheng
 Capture.PNG 
view request
Feature Request: A new column in .blib RefSpectra table (iRT method and value)
(2 responses) Chris 2018-01-27
Dear Skyline Support,

First of all, thanks for the great software and recent new features.

I'm currently preparing a small molecule .blib library (~200 precursors) and have been considering criteria to achieve correct automatic peak picking in samples containing unknowns using the spectral matching feature.

It looks like I can improve the success rate of correct automated peak picking with ion mobility CCS or retention time values. Unfortunately molecule retention time can be variable between samples/batches/columns/labs and is difficult to pick the right peak when peaks are quite close together. Maybe implementing an iRT field into Skyline would improve the retention time filtering option?

Although iRT is not currently applicable using the Skyline small molecule feature, it would be nice to be able to give more information in a spectral library to make it more widely applicable for other labs. I'm interested if this is in the pipeline or a solution already exists for the proteomics side of Skyline that I'm not familiar with?

Cheers,
Chris
view request
Reporting raw chromatograms for downstream processing
(3 responses) scurran 2018-01-29
Dear Skyline Support,

I like working with my traces in a downstream processing platform such as Python or Excel. I like the freedom to customize my graphs and etc. One *slow* way to do this is to manually copy the trace data for each transition (MRM) and paste into a .csv file. I would prefer an automated way to do this.

I would like to make a report that has my peptide name, the replicate, and the data for the trace (time and intensity). There is an option for this under Reports, but the output does not have the trace data (see attached image). Am I confused? Is there an easy way to do this?

Thank you,

Sam
 trace report.png 
view request
Can Skyline handle MS3 data for small molecule runs?
(2 responses) lparsons 2018-01-29
I'm not sure if this question has been asked recently - a search through the site here showed a few people asking about MS3 data over the past few years - so I thought I'd raise it today. I have data from a small molecule run where we would like to use the presence of a specific MS3 transition as evidence of a particular adduct of interest, and then subsequently look at the area or intensity of the MS1 parent that lead to the eventual MS3. This is a DDA run, I can produce a list of the MS3 ions (and their relevant MS2 parents) that we are interested in.

thank you
Lee
view request
Use iRT for a merged library
(2 responses) yangyunwww 2018-01-24
Dear all,

Does anyone know how to use CiRT/SiRT in Skyline to calibrate the RT variations between the selected spectrum in a merged library and the corresponding query spectrum in different runs? SpectraST converts the retention times of all identified peptides in all runs into iRT scale. Does Skyline use the same strategy, or Skyline only calibrate the RT variations between the library spectrum and the query spectrum? Can Skyline automatically extract RT values of CiRT/SiRT peptides in all runs from corresponding RAW data?

Thanks,
Antony
view request
Chromatogram information unavailable
(3 responses) VP13 2018-01-25
Dear Skyline team,
I have problem with chromatogram information unavailable. Every time I import wiff results, some of the analytes show chromatogram information unavailable. For some time I was creating new transition lists only with the analytes which were not processed and repeated inserting new transition lists and importing results until I obtained chromatograms for all the missing analytes. But this procedure is a little bit inconvenient and does not longer help anymore. Can you give me a piece of advice?
I am using version 4.1.0.1174, Windows 10 and working with MRM data.
Thank you in advance,
Veronika
 Skyline support.pptx 
view request
"No Valid Precursor m/z column found…"
(6 responses) sa660 2018-01-24
Hi,

My name is Shimon and I am a 3rd year BSC student with Professor Don Jones at the University of Leicester. I'm trying to import 3 sets of transitions from Mass Lynx Version 4.1 into Skyline. 2 of the transitions have successfully been imported but the third has not. The one that isn't being imported is highlighted in the Excel screenshot. When I try to import I get the error: "No Valid Precursor m/z column found…" shown in the 2nd screenshot. These transitions are from a Waters Xevo MS and both precursor and product ion mass are set to monisotropic. The peptides were produced using a Tryptic Digest

I have restored both the ion and method match tolerance m/z back to the default 0.055 but that doesn't seem to have made any difference, other parameters that I am using that I think might make a difference are shown below:
-    Enzyme = Trypsin
-    No of missed cleavages = 2
-    Structural modifications = Carbamidomethyl (C)
-    Max Variable mods = 3
-    Max neutral loses = 1
-    Isotope label type = Heavy


I hope you can help.

Thanks,

Shimon
 Screen Shot 2018-01-24 at 15.58.28.png  Screen Shot 2018-01-24 at 15.57.47.png 
view request
Can Skyline 4.1 export information in the library?
(2 responses) yangyunwww 2018-01-25
Can we export peptide sequence, RT, spectrum quality score etc. in Skyline libraries to a file like Excel? If yes, how to do? Cause I want to quickly compare this information among many libraries. I noticed in Q&A of Webinar 3 (2015), the answer was no. How about Skyline 4.1 or Skyline daily?

Thanks,
Antony
view request
Details about merging libraries in Skyline
(3 responses) yangyunwww 2018-01-18
Dear Skyline developers,
Many thanks to your creative work in offering a better and better Skyline!
I want to use a single merged library to quantify several targets in PRM data from many samples. I searched the resquests&responses about merging libraries in Skyline, but I still have some doubts as follows:
1.    Merging libraries from redundant libraries (.redundant.blib) and non- redundant libraries (.blib) both of which are built from .msf files (PD), or get a merged library directly from .msf files, what are the differences among the 3 merge libraries? According to my test, the merged libraries from .redundant.blib and .msf files are exactly the same. In addition, the 3 merged libraries contain the same identified peptides, but the size of the merged library from non- redundant libraries is smaller, which is due to redundant spectra are not included? generally, we can use the merged library directly from .msf files?
2.    Since “Blibfilter refines the redundant library to choose just one representative spectrum for each peptide”, how to view the redundant spectra? If we activate or deactivate “keep redundant library”, how does it affect skyline results, e.g., for PRM data? The “best” spectra for all the identified peptides are exactly the same in the 3 merged libraries mentioned above?
3.    During merging process, what are the detailed rules of selecting the “best” representative spectrum for each peptide, or give me a reference? Does it still use dotp value and the highest average similarity score during merging, the same as building a library? In my result, I found that Skyline picked a very noisy spectrum but with high intensity (see attached picture). Why does it not pick a much cleaner one with lower intensity? So intensity has priority in the picking process? Or is there an intensity threshold for Skyline to give priority to intensity? If yes, for all spectra with high enough intensity, Skyline will preferentially pick the one with less noise peaks and more targeted peaks?
4.    In addition, does Skyline consider both MS1 and MS2 spectra quality in picking the “best” spectrum during merging? If yes, I want to limit it to only MS2 spectra quality for PRM analysis, how should I do?
5.    Do the noise/interference peaks in the “best” spectrum have no influence in calculating dotp values in Skyline?
Thank you in advance!

Kind regards,
Antony
 Skyline picked the upper one.PNG 
view request
Removing Black Border
izzahshahid 2018-01-23
Thank you so much
view request
Integration problems?
(6 responses) alejandro.cohen 2018-01-18
Hi Skyline people!

I'm running some metabolomics samples acquired on a Qtrap5500 by MRM. I downloaded the date, and for some particular targets, Skyline does not seem to pick the appropriate peak! Even when I select the likely peak manually, and then Apply Peak to All, it still doesn't seem to find the right 'obvious' peaks for integration.

Attached the file. For example, look at Target number 2 on the list: 1-Methyladenosine. A very prominent peak for most positive samples is at 14.7min in most 'Pos' labeled samples, yet Skyline finds other minor peaks all over the XICs.

Could you see if I'm missing something?
Thanks
Alex
 Metabolomics_MRM_Pos_&_Neg_Q5500_Chi_b.sky.zip 
view request
1mz overlap DIA
(1 response) tannous 2018-01-23
Dear skyline team,
When generating isolation windows list in skyline for DIA experiments, if you check "optimize window placement", do you think there is still a need to do 1 mz overlap margin at either side of the windows?
thank you
Abla Tannous
view request
corrected p values more accurate than 5 decimal points
(2 responses) dkueltz 2018-01-22
Hi Nick, Brendan,
In the comparison/ MSstats results table corrected p values are displayed up to a 0.00001 accuracy - anything smaller is set to 0. This is not ideal for creating volcano plots from the exported data since all p values <0.00001 will cluster at -logp upper threshold of 5. Is there any way that p values can be exported for MSstats comparison results with greater accuracy (i.e. up to 15 digest past the decimal point or even more)? This would render construction of volcano plots from those data more accurate.
Thanks for any suggestions,
Dietmar
view request
Share minimum does no longer work with v 4.1
(2 responses) dkueltz 2018-01-21
Hi Nick, Brendan,
I am trying to share: minimal a skyline file but it does no longer work with version 4.1. I always get the same error (The given key was not present in the dictionary). This happens with both the full version and Skyline daily (both 4.1). The error is attached as a screenshot. I checked that the referenced paths to the irtDB, protdb, and blib files are all correct - so broken paths to required files cannot be the reason. The error mentions something about small molecules even though this skyline document does not contain small molecule data. Thanks for taking a look at this!
Dietmar
 skyline share min error 21Jan18.png 
view request
Removing that black outline in the image
(1 response) izzahshahid 2018-01-22
Hello All,

When I save results as metafile or image, it shows a black outline and does not get deselect from there.How can i do that?

Figure is attached and in sample one, RT lines are having outer black line.


Thank you

Izzah
 Skyline Help.pptx 
view request
Getting smoother MS1 data while alternating with PRM
(2 responses) monica lane 2018-01-19
There are situations where we'd like to monitor both the MS1 and MS2 peaks in PRM (i.e. we alternate full scan with PRM during acquisition). We have issue where MS1 peak shape is very choppy in the Skyline profile (vendor software shows smoother profile). How should we optimize MS1 settings in Skyline - or instrument acquisition - so that peak shape is more comparable to transition peaks?

I've uploaded example case collected replicates of PRM-FullMS-SIM analysis of same sample over time (15 PRTC's spiked into ecoli digest) on QEHF-X. PRM settings: Res@60K/1e6 AGC/119ms for 15 scheduled precursors (+-2min), FullMS settings: Res@60K/ 3e6 AGC/30ms,375-1600 m/z
 AutoQC_PRTC_stds_2017-12-14_11-02-50.sky.zip 
view request
Tutotial for skyline command line
(3 responses) momin amin 2018-01-17
Hello,

 Can someone suggest a turorial to learn how to run and write skyline scripts for command line batch processes.
view request
MSstats external tool problem
(1 response) reinders 2018-01-19
Hi,
I have a stragne problem with MSstats as an external tool.
I have comparison for a single analyte (small molecule) with stable isotope labeled standard (two conditions, 14 subjects, triplicate measurements). While a can use the "Group Comparisons" from the "View" menu, I get the following error message when trying to use Group Comparison from MSstats in the "Tools" menu:

>Error in aggregate.data.frame(mf[1L], mf[-1L], FUN = FUN, ...) :
> no rows to aggregate

The respective file is present and has data rows in it. What am I missing?
Many thanks!
Joerg
view request
Chromatogram information unavaliable
(6 responses) io 2018-01-19
Dear Skyline team,
Today I have problems to import wiff results, the chromatogram information is unavaliable.
I am using 4.1.0.11714 with windows 10.
Yesterday, I used the same Skyline file and I didn't have problems with the import
I have also checked the wiff files and theay are correct.
I attached a screenshot.
Thanks in advance
 190118.pptx 
view request
Peptides sorted alphabetically and report out
(1 response) kerry hassell 2018-01-18
Hello,

Is there anyway to sort the peptide alphabetically? Also, when exporting a report with transition settings, can there be an area for all the transitions instead of individually?

Thanks,

Kerry
view request
NETWORK DRIVE: Performance deterioration after upgrading to v4.1
(3 responses) gabe 2018-01-16
Hi, I'm noticing a significant slow-down in performance after updating to v4.1. The slow-down is happening when importing RAW files from a Thermo QE. I'm importing 96 files at a time, and it's slower when I use either 'single' or 'many' simultaneous imports. These are ~30 minute PRM runs with ~150 peptide transitions. It used to take ~5 minutes to load 96 raw files, now it's taking more-like 30'. Are other people noticing this? Can I provide any details to help troubleshoot?

Thanks

Gabe
view request
HPLC-QE lipids data (m/z 1300)
(9 responses) fuyanli 2018-01-09
Hi,

I am now working with archaeal membrane lipids (m/z 1302, 1300, 1298, 1296, 1292). These lipids were detected by QExactive with ESI and SIM. For each of these compounds, three adducts (Na, NH4 and H) were added.
I want to try to use Skyline to detect and integrate these compounds and calculate the peak area. But I am not sure if the peak algorithm used in Skyline is suitable for these large molecules. What algorithm method was used for identifying peaks in Skyline? Is there anyway to calculate the signal to noise ratio (S/N) in Skyline? What method is used in Skyline for S/N?

Thanks
view request
extracting fragment ion intensities from DDA
(2 responses) rliyana 2018-01-16
Is there a way to extract fragment ion intensities along with precursor ion intensities from low-resolution DDA data, just for few targeted peptides using skyline ??
view request
Skyline Small Molecule Targets Tutorial: Chromatogram Unavailable
(7 responses) louis riel 2018-01-13
Hello,
I am running through the small molecule tutorial, however after I import the raw files, the chromatograms are empty. Any help would be appreciated! I am using the Default Settings.
view request
Trouble Opening Small Molecule Files
(2 responses) ekropp 2018-01-15
Hi Skyline team,

I am having trouble opening saved files with small molecule data in them. I have a template that I use with my transitions (all transitions were checked for errors prior to import) and can import results/analyze data without issues. However, if I close the file and try to reopen the file I keep getting an error message and am unable to open the file.

The error message states: this file contains an error on line 1232 (line number often changes) at column9. The precursor and product ion polarity do not agree. When I check my transitions prior to saving, I can't find any mismatched precursor to product polarities. Nor can I open the file after this error. I have tried creating multiple files and testing different data sets and keep getting the same error when I try to open a saved file. I have to create a new file, each time I want to analyze a data set.

Is there a way to open the file to check for errors after this message or is there a way to figure out if there is an issue with one of my transitions in my data file? I am attaching the blank file, corrupted file, and snapshot of the error message.

Thanks,
Erin
 180109 DF6 Titration.sky  180109 DF6 Titration.sky.view  180109 DF6 Titration.skyd  Metabolite List 2.sky  Metabolite List 2.sky.view  Error Message.PNG 
view request
Missing OK button in the "transition list" window
(1 response) yunh 7701 2018-01-14
With the update for Skyline 4.1, OK button in the transition list window disappeared (refere to attached image) for users in Japan and China.
Until fixed, pressing the Enter key should work for OK and the Esc key for Cancel.
 skyline_erroir.PNG 
view request
Problems when Skyline-Files are used by serveral people or on different computers
Sebastian Malchow 2018-01-15
Hi Sykline Team,

thanks for the great work and the recent update!

Im trying to setup method/assay development in a somehow regulated environment.
This includes that different people work on different computers on the same Skyline file.

Unfortunately some details of the file are always stored somewhere else than the in the file itself.
A colleauge prepared a file for me, including a certain set of spectral libraries.
She put the files and libraries into certain folder we defined beforehand.
When I open the file its loaded as expected, but when I check where Skyline looks for the library I noticed that the folder is different from the speified folder. I used the same library-file from a different folder before and Skyline suddenly uses the old path to the library. This is problematic, because I expect that Skyline would use the file and path which was specified before by my colleauge. This way we try to make sure that we always have all libraries in the right place over the next ~10 years.

Its not hepling to use the File -> Share dialouge. Skyline is still using a different path to a certain library than the one to the unzipped folder...

Another possibility would be to store all libraries in one spot, but I worry that this will be problematic when we work with several people on Skyline files using the same files as libraries.

I hope you can help with this issue or can give an advice how to circumvent it.

Regards
Sebastian
view request
removing proteins
(3 responses) et2md 2018-01-12
Hey Skyline team,
The same way you can refine to keep proteins/peptides based on Names, Accessions, Preferred names, is there a way to remove proteins?
Thank you so much for the amazing work. Highly appreciate
Emmanuel
view request
MS1 Full-scan Filtering
(19 responses) gailwei 2018-01-10
I have Mascot DAT file and tried to see MS1 integration with Skyline. When I imported peptide search *.DAT, it always said the results file was missing in "Extract Chromatogram". I forced to "retry after import failure", it could continue, but a lot of info are missing. Any suggestions are truly appreciated.
view request
Defining Standard and Unknown for small molecule
(1 response) abasit 2018-01-12
Hi Skyline,

I would like to quantify unknown from my samples. I have a calibration curve of the standard. How I can do define standard and unknown from my samples in skyline, and how to use 1/x or 1/x2 (linear) weighting factor for quantification.

Thanks

Abdul Basit
view request
Error while updating Skyline 4.1
(1 response) Sadr 2018-01-12
Pleas can you help me with this:


System.Deployment.Application.DeploymentDownloadException: Downloading https://skyline.gs.washington.edu/software/Skyline-release-64_4_1/Application Files/Skyline_4_1_0_11714/Skyline.exe did not succeed. ---> System.Net.WebException: The operation has timed out.
   at System.Net.ConnectStream.Read(Byte[] buffer, Int32 offset, Int32 size)
   at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
   --- End of inner exception 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.DeploymentManager.SynchronizeCore(Boolean blocking)
   at System.Deployment.Application.DeploymentManager.SynchronizeAsyncWorker()

Regards,

Sadr
view request
Error while installing Skyline 4.1
(1 response) trinidad martincampos 2018-01-12
Hello and thanks for the wonderful work again!!

I downloaded the new version after receiving the email but I cannot install it. Here you are the error on a Windows 7 64bit machine:

PLATFORM VERSION INFO
    Windows             : 6.1.7601.65536 (Win32NT)
    Common Language Runtime     : 4.0.30319.42000
    System.Deployment.dll         : 4.7.2053.0 built by: NET47REL1
    clr.dll             : 4.7.2053.0 built by: NET47REL1
    dfdll.dll             : 4.7.2053.0 built by: NET47REL1
    dfshim.dll             : 4.0.41209.0 (Main.041209-0000)

SOURCES
    Deployment url            : https://skyline.gs.washington.edu/software/Skyline-release-64_4_1/Skyline.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-release-64_4_1/Skyline.application resulted in exception. Following failure messages were detected:
        + Downloading https://skyline.gs.washington.edu/software/Skyline-release-64_4_1/Skyline.application did not succeed.
        + The request was aborted: Could not create SSL/TLS secure channel.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
    No transaction error was detected.

WARNINGS
    There were no warnings during this operation.

OPERATION PROGRESS STATUS
    * [1/12/2018 11:45:23 AM] : Activation of https://skyline.gs.washington.edu/software/Skyline-release-64_4_1/Skyline.application has started.

ERROR DETAILS
    Following errors were detected during this operation.
    * [1/12/2018 11:45:28 AM] System.Deployment.Application.DeploymentDownloadException (Unknown subtype)
        - Downloading https://skyline.gs.washington.edu/software/Skyline-release-64_4_1/Skyline.application 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.DownloadManifestAsRawFile(Uri& sourceUri, String targetPath, IDownloadNotification notification, DownloadOptions options, ServerInformation& serverInformation)
            at System.Deployment.Application.DownloadManager.DownloadDeploymentManifestDirectBypass(SubscriptionStore subStore, Uri& sourceUri, TempFile& tempFile, SubscriptionState& subState, IDownloadNotification notification, DownloadOptions options, ServerInformation& serverInformation)
            at System.Deployment.Application.DownloadManager.DownloadDeploymentManifestBypass(SubscriptionStore subStore, Uri& sourceUri, TempFile& tempFile, SubscriptionState& subState, IDownloadNotification notification, DownloadOptions options)
            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 request was aborted: Could not create SSL/TLS secure channel.
        - 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.




Thanks a lot!!!!
Trini
view request
Some small issue with Skyline 4.1
(2 responses) schen19 2018-01-12
Hi Skyline team,

Thanks for the new update! I really like some of the new features such as the ones on Document Grid. I used to do the data rearrangement in MS Excel but now I can do it within Skyline. However, I noticed that when I group the Peak Area view by annotations, clicking on the peak area bar of certain group no longer brings up the corresponding chromatogram. This is not a big issue but it would be convenient to have the function back.

Also, the detail slides attached to the new update announcement don't seem to be publicly accessible either.

Thanks!
Shimin
view request
light intensity channel doubling
(3 responses) erik.soderblom 2018-01-10
I observed an odd issue within Skyline where if I change which peak I want to integrate, it actually changes the light channel intensity (increases it by quite a bit). I've tried to capture this on the attached slides. I was hoping it was just a visualization thing, but I exported the numerical data and confirmed the actual intensity value is changing. It doesn't appear to be happening on all peptides.
Thanks in advance for looking into this.

Skyline-Daily: 64b. 4.0.9.11707
 light_channel_intesity.pptx 
view request
filter peptide list for modifications at specific amino acids
(11 responses) kenhsu 2018-01-08
Hello,

We are interested identifying peptides with particular modifications on amino acids from lists that contain both modified and unmodified peptides.

What is the best strategy?

Thank you.
view request
Build spectral library from Byonic searching results
(21 responses) Qiongyu 2017-12-15
Hi,
I used the mizd files exported from Byonic to build a spectral library. My file name is:

***.raw_20171214_Byonic.mzid

However, after I added those files, an error came out.
The error says:

Cound not find spectrum file
***.raw_20171214_Byonic[MGF|.mzXML|.mzML] in current directory.

My Skyline is version 3.7.0.10940.

I am kind of confused now. I thought that among mzid, mgf, mzXML, and mzML files, mzid file is the only type of file that can be used to build a spectral library. But why is the file shown in the error has those other extensions?
How can I fix this problem?
Best,
Qiongyu
view request
iRT chromatogram unavailable and split peaks
Midy 2018-01-04
Hi Skyline Group,

I can see all iRT peptides with other software but in Skyline only 3 out of 11 peptides. For other peptides it shows chromatogram unavailable with 64 window DIA isolation scheme, margin 0.5 Da. Sometimes it shows split peaks. Could you help me with this? Thanks.

Best regards,
Midy
view request
SWATH Ion Library with controlled Protein FDR%
(1 response) andrea armirotti 2018-01-04
Hello guys
Thanks for the great work you're doing with Skyline.
I'm wondering if I can use it to add quality to SWATH ion libraries. I mean the following: I want to 1) Acquire fractionated DDA data on a given proteome 2) Use a NON Protein Pilot software to search for IDs. I want to use a software that gives active control of protein FDR by searching Vs a decoy db (Mascot? MaxQuant?) 3) Retrieve the list of proteins at 1% FDR and import it back into Skyline 4) Export the transition list back into a .txt file that the SWATH microapp in Peakview can read.
I recently acquired a set of DDA data that I searched with MASCOT. I then imported it in Skyline as .dat and I now have my Skyline library with my peptide and transition settings. How do I convert it back into a txt? I understand I can't do it directly fron Skyline.
I tried the BiblioSpec workflow: BlibBuild to create the library from the .dat, then BlibFilter to remove PSM redundancy, then what? Is this the right path to obtain a .txt list of transitions usable with Peakview?
Many many thanks for your support
Andrea
view request
iRT alignment issue when importing SWATH runs
(1 response) laura declerck 2018-01-04
Hi,

Some time ago Skyline made it possible for me to build a library out of multiple searches on the same raw data files (Issue #501, "Improve BlibBuild handling of multiple searches on the same raw data files").
I needed this feature, because to annotate the iRT peptides in my data I needed different search parameters (Enzyme=Trypsine) than when I annotate histone peptides (Enzyme= ArgC, because all Lysine residues are blocked with a PTM or a chemical derivatisation by propionylation).

In the Skyline peptide and transition settings I chose the parameters that fit my histone peptides. This results in the fact that I am not able to retain iRT as a target, because I use 1) ArgC as an enzyme and 2) Propionylation (N-term) as a fixed modification.

When i want to import my SWATH results, iRT needs to be in my target list to be able to perform iRT alignment. However, to able to have iRT in my target list I need to change my settings in Skyline to 1) Trypsin as an enzyme and 2) no fixed modification on the N-termini. When I do this I lose a lot of my histone precursors and transitions, because they do not fit the settings anymore.

Is there a way that I can perform iRT alignment without loosing my targetlist? Would it for example be possible to use iRT alignment in the background, without putting it in the target list?

You can find the .sky file on this dropboxlink: https://www.dropbox.com/sh/pdyssp7hhdsndit/AACSrbExC0cT1fk1088gHYe2a?dl=0

Thanks a lot in advance for the help.

Kind regards,
Laura
view request
iRT Retention Time Issue
(7 responses) johnny perez 2017-12-29
I am having some trouble adding peptides to my iRT database. I am using an apomyoglobin digest as a home-made custom iRT standard. I only detect about 7 peptides, which I understand is not ideal, we are currently looking into buying a commercial digest. Now, I did 4 unscheduled injections with the peptide STDs spiked in, I calibrated the calculator using the detected peptide STDs and when I go to add the targeted peptides to the iRT database, skyline gives me an insufficient correlation error.

Any assistance would be greatly appreciated.
 iRT Skyline Error.pptx 
view request
Modifying adduct type textbox for small molecules crashes Skyline
(2 responses) Chris 2017-12-20
Dear Skyline support,

I'm using Windows 7 64-bit and Skyline daily (4.0.9.11664).

I've come across a crash that closes Skyline unexpectedly, likely due to my unintention methods. When I modify the adduct of an already-specified small molecule transition using the text box, e.g. removing the - from [M-2H] and replacing with +, Skyline instantly crashes (stack overflow, please find full log below).

When using the intended method (using the arrow on the side of the text box), it doesn't crash so I think I've performed adduct modification in an unexpected way.

I've attached the Skyline assay that caused the crash and please find the error log below:

Problem signature:
  Problem Event Name:    CLR20r3
  Problem Signature 01:    Skyline-daily.exe
  Problem Signature 02:    1.0.0.0
  Problem Signature 03:    5a3856df
  Problem Signature 04:    System.Windows.Forms
  Problem Signature 05:    4.7.2102.0
  Problem Signature 06:    59371148
  Problem Signature 07:    0
  Problem Signature 08:    ffffffff
  Problem Signature 09:    System.StackOverflowException
  OS Version:    6.1.7601.2.1.0.256.1
  Locale ID:    3081
  Additional Information 1:    1c98
  Additional Information 2:    1c98c898ba4042187feba63d99e9d7e6
  Additional Information 3:    ba93
  Additional Information 4:    ba935b4e6adfbd97b792a80604cf6fb1

Cheers,
Chris
 Adduct_crash.sky.zip 
view request
PRM with skyline
(1 response) jung165 2017-12-21
First of all, I would like to appreciate for Skyline team's support

What I would like to ask is the parameters for full-scan for PRM analysis with Q-exactive (orbitrap)
I could find the tutorial for low-resolution (LTQ), but is it possible to imply those parameters for Q-exactive??

I tried to choose orbitrap on precursor mass analyzer(in full-scan tap) and it showed me to choose resolving power.
Should I have to set resolving power with same as that of Thermo Xcalibur Instrunment Setup??
I attached picture of those.

I am kind of confusing with those parameters for Q-exactive.
Is there any tutorial with Q-exactive (high resoution) experiment??
or can you give me any information about parameters for Q-exactive analysis

thank you very much for your help
 skyline_question.pptx 
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Unusual behaviour in determination of peptide uniqueness
(4 responses) Sebastian Malchow 2017-12-20
Hi.

I often add peptides to Skyline (64-bit 3.7.0.11317 unplugged) which I consider unique for a protein on a proteome level.
For me this means that no other protein produces the same peptide when digested with the protease of choice (in my case trypsin KR|P, see 1.PNG).

Upon peptide import via Edit -> Insert -> Peptides Im getting the error message that one of the imported peptides is not unique. Manually checking shows that Skyline associates the peptide to the protein PRAG1_Human (see 2.PNG), which is not correct, because for this protein the peptide is not a tryptic peptide.

I think thats the reason why Im getting the message that this peptide is not unique.

Is this a bug or I understand the "Enforce peptide uniqueness " setting wrong?

Best regards
Sebastian
 2.PNG  1.PNG 
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DIA isolation window vary with time
(3 responses) qi tang 2017-12-18
Hi Skyline Group,

During a LC run, with time increasing, precursor range changed. So, for better fragmentation, I wonder if DIA isolation window can vary with time?

Thanks!

Qi
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Global vs Surrogate standards
(1 response) sstoychev 2017-12-19
Dear Skyline team, could you provide some information on the differences between Global/ Surrogate/ QC standards when it comes to sample normalization. How do these work i.e. are the peak areas of peptides set as Global standards averaged or summed prior normalization.

Also, please have a look at the attached screenshot. Why is the "standard selection" window grayed out hence not allowing for selection?

Regards,

Stoyan
 Skyline_stds_screenshot.jpg 
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Possible file-locking issue with Daily?
(3 responses) lparsons 2017-12-18
I've been using a Skyline-daily build to look at small molecule data in a SWATH-type experiment. However I find that if I try to re-import data (Edit -> Manage Results -> Re-import) that Skyline throws an error ("Failed importing results file (...) [read_file_header()] Unable to open file (...) [invalid permission or file locked]")and tells me it cannot read the file that has already been read in. After that the file is no longer loaded in skyline (no data displayed and the file name no longer present).

If I then go to the command prompt (without closing Skyline) and try to rename the file, windows tells me that I cannot do that, returning "Device or resource busy". If I close Skyline I can then do that, but not before.

In non-Daily releases of Skyline I don't recall having this problem, I could re-import files without this happening. I am trying to re-import files after changing settings (transition settings in particular though occasionally trying other settings as well). This is a system running Server 2012R2, where I have admin access.

thank you
Lee
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Target and confirming transitions
(2 responses) Mathias Kuhring 2017-12-13
Is it possible to assign different roles for transitions of the same small molecule? For instance, using one fragment as target for quantification while using another one just for confirmation. The first question in the "Question and Answers" of Webinar 16 goes in a similar direction. However, it only indicates a differentiation between quantitative and qualitative transitions, but doesn't specify additional usage of the qualitative transitions.

Best, Mathias
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Fail to import SWATH results: Sequence contains no elements
(1 response) laura declerck 2017-12-18
Dear Skyline team,

Yet, another issue appeared.

I fail to insert my SWATH results (.wiff files):


At 5:00 PM:
Failed importing results file 'C:\Skyline\Histones_Micro\data\171205_LDC_Hist_SWATH_60W_4000_B.wiff'.
Sequence contains no elements


Inner exceptions:
Exception type: System.Reflection.TargetInvocationException
Error message: Sequence contains no elements
Sequence contains no elements
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Util\Util.cs:line 1832
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Collectors.ReleaseChromatogram(Int32 chromatogramIndex, ChromGroups chromGroups, TimeIntensities& timeIntensities) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1143
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 555
   at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 76
   at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 213
   at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 142
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 455
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 272
---------------------------------------------------------------
Exception type: System.InvalidOperationException
Error message: Sequence contains no elements
Sequence contains no elements
   at System.Linq.Enumerable.Average(IEnumerable`1 source)
   at pwiz.Skyline.Model.Results.OverlapDeconvSolverHandler.BuildDeconvBlock() in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\OverlapDemultiplexer.cs:line 142
   at pwiz.Skyline.Model.Results.AbstractDeconvSolverHandler.Solve(ILsSolver solver) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\AbstractDemultiplexer.cs:line 524
   at pwiz.Skyline.Model.Results.AbstractDemultiplexer.GetDeconvolvedSpectra(Int32 index, MsDataSpectrum originalSpectrum) in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\AbstractDemultiplexer.cs:line 388
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 318
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 215
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.<SetRequestOrder>b__8() in c:\Users\nicksh\svn\skyline_4_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 540



First I thought it had something to do with the fact that my library is built out of multiple .dat files searches with different parameters to be able to extract iRT.
But, when I do not use iRT calculation, this problem still appears.

Would you be able to help me?
I put the .Sky file and the .wiff file in a dropbox link (https://www.dropbox.com/sh/t1l7uuygdy8n31x/AACJc5l1CO8JRVhf7ixPZvkCa?dl=0).


Kind rergards,
Laura
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Evaluate SWATH product ions without known parents
(22 responses) lparsons 2017-12-11
I would like to use Skyline to draw chromatograms of product ions that were acquired in a SWATH-type experiment (where the exact mass of the parent is not known). I have a list of product ions I am interested in however if I use Edit -> Insert -> Transition List I cannot bring in the product ions without providing information on the parents.
I have selected "Small molecules", rather than "Peptides" for type on the Transition List import tool.
Is there a different way to use Skyline to draw chromatograms of specific product ions without concern for a parent ion? I tried making a list where I had the suspected parent ion m/z as well as the observed product m/z (with charges of both set to 1 for this case) but when I imported that and then imported the data file skyline showed no chromatographic data (presumably because the product ions have no parents associated with them?).

thank you
Lee
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Combined analysis
(2 responses) AnnaKwasnik 2017-10-19
Hi, can anyone advice? I am combining different methods into one method in Skyline and the problem that I am facing is match of the data to the spectra libraries. Each method was developed using different spectra library and while the methods work well for the specific library they do not when I combine them into one Skyline file, the dotp for some of the peptides is lower in combined file. It seems like top library have the spectra for that peptide but its transitions were optimised on different library. Does anyone know if I could overcome this problem and selectively select the libraries for this combine Skyline file or do I have to keep and analyse methods separately?

Thank you for any help,

Anna
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Filter to remove C terminal Arginine peptides
(4 responses) tcj11 2017-12-13
Hi, I was wondering how to
1)generate a filter to remove peptides with C terminal Arginine. Our heavy isotope control tissue only has lysines labeled, so I'd like to automatically remove all the R peptides, leaving only the K to more readily design SRMs.

Any ideas?

2) remove peptides with missed cleavages:
I tried K.*K|K*.K.*K and it didn't seem to work.

3) I also tried filtering to remove ragged ends, but this often seemed to remove some additional peptides:

ex A. - removed both 1. K.KVVFSTGSYPR.L (missed 1)
2. K.VVFSTGSYPR.L
other examples of peptides removed with ragged end filter
1. K.TWVLTPK.L
2. K.ADAHWITTPNR.M
3. R.EEIVYQWK.R
4. R.SSVEVGDTR.S

Thanks, Tija
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Build spectral library from Byonic searching results
(1 response) Qiongyu 2017-12-14
I use Byonic to search RAW data generated from Orbitrap. After that, how can I generate a spectral library directly using Byonic results?
Thanks!
Qiongyu
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