Table of Contents

guest
2018-12-19
Welcome to Skyline Support
   Floriana Capuano
   Problem in analysis after saving
   Y Biotin Phenol (APEX) spectral library
Sign Up for Support and Notification Email
Follow the Skyline Support Board
MS1 precursor peak picking based on best idotp value
QUASAR error
Error in Importing Files
Build spectral library from Byonic searching results
Missing SILAC Ratio
Xuemei
Reverse external calibration curve
Jos
Eva Hunyadi-Gulyas
Absence of Acquired Time information in report documents
Importing Peptides with dimethyl labelling from MaxQuant msms.txt
identifying small molecule binder sites
Skyline 4.2 installation

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.

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Floriana Capuano


Hi Skyline team,

I am using Skyline to analyze DDA samples by MS1 filtering. In order to increase the depth of our analysis we use HPLC to fractionate our samples prior to MS analysis. Fractions run separately on Thermo instruments (Orbi-Orbi) and we, then, perform data search using Peaks. We are also spiking our samples with labelled peptides that we are interested in quantifying.

I am having troubles applying the spectral library I built using the .pep files generated by Peaks. Whenever I select the library in the peptide settings of my skyline document data disappear. If I do not tick the library box then I can see my data. I am using Skyline (64-bit) 3.7.0.11317.

In my Peaks file there are no spectral matches for the labelled peptides as the modifications are not added during the search.

Any help would be very much appreciated.

Best wishes,

Floriana




Problem in analysis after saving


Dear Skyline Team,

I would like to ask for your help in the following matter: I am working with proteomics, using Skyline 64-bit 4.1.0.11796. When I analyzed my data (after peak picking) I saved my Skyline datashet and closed it. After opening, it seems that the analysis (specifically my peak picking is lost/changed-please see the attached ppt). I tried to analyze the datashet in a different computer and also the option save as, instead of simple save but the same thing happens. Also, I opened some older datashets and the files seem ok (the analysis is not messed-up).

Maybe you could provide some help?

Thanks a lot,

Georgia




Y Biotin Phenol (APEX) spectral library


I have done some APEX proximity labeling experiments in which I enrich for Y-biotin phenol sites using anti biotin.  I would now like to build a spectral library to perform targeted quantification of these sites.  I have searched the data with MaxQuant and tried importing the peptide search as usually done, but it is not recognizing the Y-biotin phenol modified peptides in my msms.txt file and these are being excluded from the spectral library.  I have added the Y-biotin phenol modification to the list of modification in the peptides settings.  Is there some text from the msms.txt and skyline settings that needs to match in a certain way for these peptides to be imported properly?  

Thanks!

-Danielle




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MS1 precursor peak picking based on best idotp value


 

Hi Skyline team,

There was this post from 2013 about picking MS1 peaks based on their isotope dot-product value.  Basically, I want to define an isotope distribution and if it doesn't meet the criteria, then Skyline won't allow it to be picked.  Here's the post from before that suggested that this was not something that i could do, but it was in 2013 and I know you guys are updating Skyline all the time.  Has this feature been implemented?  This is more for a PK/bioanalysis type of measurement rather than for proteomics at all, so the data acquisition and analysis needs are different, but I thought that I'd ask.  

https://skyline.ms/announcements/home/support/thread.view?entityId=0f887145-f665-1030-8e9b-5d0a05bcbc14&_docid=thread%3A0f887145-f665-1030-8e9b-5d0a05bcbc14

Best regards,

chris




QUASAR error


Hello all. I have performed a calibration curve and I am trying to use QUASAR in order to calculate LOD and LLOQ. I have filled all the fields for the QUASAR input file (as I have previously done for other experiments), but it is giving me the next error:

 

Attaching package: 'gplots'

The following object is masked from 'package:stats':

lowess

[1] Processing arguments ...
[1] >>> Processing part 1 of 1
[1] Initializing ...
Error in calculate(data.subset, concentrationFile, output.prefix = outputPrefix, :
Duplicate transitions found -- try including neutral loss
Calls: tryCatch -> tryCatchList -> parse.cmdline -> calculate
Execution halted
Finished!

 

I think it started happening since I last updated the Skyline daily software. Could that be the reason?

 

Thanks for your help.

 

Carmen




Error in Importing Files


Hi,

 

I am getting following error in all the Skylines software that I use. I am requesting suggestion for that.

Here is the error message.

At 3:31 PM:
To extract chromatograms from C:\Users\Protein.Shared\Desktop\HPV\171020_Caski_Heavy_only.raw full-scan settings must be enabled.


Inner exceptions:
Exception type: pwiz.Skyline.Model.Results.NoFullScanFilteringException
Error message: To extract chromatograms from C:\Users\Protein.Shared\Desktop\HPV\171020_Caski_Heavy_only.raw full-scan settings must be enabled.
To extract chromatograms from C:\Users\Protein.Shared\Desktop\HPV\171020_Caski_Heavy_only.raw full-scan settings must be enabled.
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 92
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1263
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 249




Build spectral library from Byonic searching results


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
 

 




Missing SILAC Ratio


Hello,

Great tool! I was hoping you could explain an issue I am having.

I have imported K602 and R604 SILAC labeled data and have confident library assignments for my target peptides (green circles). For K602 labeled peptides there is a total ratio provided after the dot product.  However, this information is not provided next to R604 labeled peptides. Peptide settings -> Modifications have been set up so that a 'HeavyR' Isotope label type is associated with the 'Label: 13C(6) 15N(4) (C-term R)' Isotope modification and accordingly for 'HeavyK' and 'nolabel' isotope label types.  Please let me know if there is anything else that I can provide.

Thanks!

Gary

p.s. Does total ratio refer to the light/heavy ratio?




Xuemei


Hi,

I export a transition list from a skyline file with retention time. The RT exported are different from I saw in the library. How do I change settings to get the experiemtal RT exported? Thank you.

Xuemei




Reverse external calibration curve


Hello!

I was wondering if Skyline supports external calibration curves in which you inject different concentrations of heavy peptide and you use the light peptide (at fixed concentration) to normalize.

Thanks!

Cristina




Jos


Dear Skyline team,

thanks for developing skyline and your consistent support. I use Skyline (version 4.1.0.18169, 64-bit) for analyzing a DIA data set of 15 .raw files (Thermo) by a library of 300 raw files (both samples and library ran with iRT's). The settings I used are pretty much the same as they are recommended in the two DIA-Webinars. For my analysis, I have adjusted the iRT and used shuffled decoys implemented in skyline, then I tried to improve the peak scoring by using mProphet.

However, in the outcome not all options are available (see the figure attached) that may can lead to an improvement of the peak scoring.

And secondly, I experienced while exporting protein list following group comparison that the proteinlists varies a lot between different treatments (from 10.500 down to 500). Is there any default filtering criteria (hidden) which reduces the number of exported protein lists within one analysis file?

Thank you for your comments and suggestions. I would be happy to help you out if this is necessary!

Best, Jos




Eva Hunyadi-Gulyas


Dear Brendan,

I would like to use Skyline(64-bit, 4.1.0.18169) for quantitative 
proteomic analysis. I have managed to create a spectral library 
already several times, but now I am unable. I use ProteinProspector 
for DDA database search and generated pep.xml files, but the Skyline 
said:  "ERROR: 180802_03.pep.xml(line452): The .pep.xml file is not 
from one of the recognized sources".
Could you suggest what should I change?

I attach the pep.xml file.

Thanks for your help in advance,

Best,
Eva Hunyadi-Gulyas




Absence of Acquired Time information in report documents


Dear Brendan,

 

We've been working on a QC analysis of several BSA DDA runs performed on an Orbitrap Fusion. When we export the MSStatsQC report document from Skyline, we find that it lacks the "Acquired Time" data. We tried exporting the report from a single run, and it still doesn't work. We've attached the Skyline documents. Could you take a look?

 

Thanks,

 

Lakshman




Importing Peptides with dimethyl labelling from MaxQuant msms.txt


Dear Skyline Team,

I would like to do DDA with MS1filtering in order to quantify a certain yeast peptide (TGAPNNGQYGADNGNPNGER) which is present in charge 2+ and 3+ with either dimethyl light or dimethyl heavy label in my dataset. I set the respective modifications in the Peptides settings tab and when I import my fasta file (which also contains some high abundant peptides to allow for matching between runs) I can see the expected m/z values present in my msms.txt (generated from MaxQuant Version 1.6.0.1). However when I next do the "import peptide search" all peptides vanish from the left side tab. When I go on I get the error message "The document does not contain any peptides". When I then try to add the Fasta file (again) I get the error message "The document must contain at least one precursor transition in order to proceed". After reading some of the earlier support threads I put the modifications.xml file from MaxQuant in the same folder (along with msms.txt and mqpar and Raw Files). I also tried the import replacing the Skyline names by the respective MaxQuant names, i.e. "DimethylLys0" instead of "Dimethyl (N-term)" in the peptide settings in Skyline. Still, Skyline somehow does not recognize the dimethyl modification from the msms.txt. When I try the import with default settings, Skyline suggests that there is Dimethl (K) my data and also an unkown K modification with the mass of 34.0 which corresponds to the heavy Dimethyl (K), but when I add them, I still get no transitions (but of course the respective N-terminal modifications are missing). When I try the import with just the Dimethyl (K) and Dimethyl (N-term) set as variable modifications (no heavy label), Skyline starts the import, however the peptide I am interested in is then only present as in the non-modified version. I tried all this with both the Skyline version 3.5.0.9319 and the Skyline version 4.1.0.111796. I would also like to add that the double dimethyl labelling settings I tried out now have worked before for another double dimethyl labelling dataset, which was also searched with the MaxQuant Version 1.6.0.1 but searched against a Trypanosoma database. I attach my msms.txt, mqpar and skys file in the hope that you can find out where the error is.

Ida

 

Dear Skyline Team,

I have found a workaround for my problem. I set both the light and the heavy dimethyl as structural, variable modifications. During the "Import peptide search" Skyline suggested that there is an unknown modification with the mass of 34.0 on each amino acid. I then added the heavy modifications on K and Nterm (giving them a different name) additionally as heavy modifications (mass shift of 6). The import started and I now get my peptide in the heavy and light version.

 

 

 




identifying small molecule binder sites


Hello

 

  We are analyzing samples of recombinant protein which were incubated with small molecules that attach covalently to cysteine. The objective is to identify the positions of modification and extract the chromatograms of the labeled peptides for characterization. Essentially I define the molecule's formula in MaxQuant (saved in the modifications.xml file) and define both the molecule and carbamidomethyl as variable modifications. The analysis by MaxQuant clearly identifies peptides containing the molecule as well as their sequence and the exact labeling site (including if there are two cysteines in the same peptide). My objective was to import the peptide search into skyline to build a spectra library and to extract the chromatograms of the carbamidomethyl and molecule-bound peptides. However, the peptides with the bound molecule are never added to the library - only carbamidomethyl peptides are. This is even though MaxQuant clearly identifies the modified peptides and they are defined in the same way. 

 

  Is there anything you can recommend to help in this problem?

Details: Windows server R2 enterprise 64 bit

             Skyline 64 bit 4.2.0.18305

             MaxQuant 1.6.0.16

  I attach the screenshot from skyline. Cysteine containing peptides are added to the library (carbamidomethyl containing) and some are not (molecule-labeled). 

 

 

  I also attach the screen shot from the MaxQuant output showing the identification of the molecule-labeled peptide. 

 

Thank you

 

 




Skyline 4.2 installation


I have this really basic issue with installing the Skyline 4.2 in my PC laptop that is running Windows 10 home edition. The 4.1.0 edition used to work fine but now that the data are generated in Skyline 4.2, I am unable to install it. The installation starts and then I get the error message that is something to do with the Microsoft.NET framework. I could not easily update the .NET framework also.
 
On the other hand, I was able to update to Skyline 4.2 easily in a Windows desktop running Windows 7. You might have heard from others who have similar issues. can you please let me know how to resolve this issue? I use Mac for everything and have a Windows laptop just  for running the Skyline.