Table of Contents

Welcome to Skyline Support
   Floriana Capuano
   Problem in analysis after saving
   Y Biotin Phenol (APEX) spectral library
   DIA import method error message: columns don't match

Welcome to Skyline Support

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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)

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,


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 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,


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?  



DIA import method error message: columns don't match

I am using a Thermo Exploris 480 running on Xcalibur 4.2.47.  I want to import a staggered DIA method from Skyline-Daily.  In method editor, after selecting DIA, import the .csv file, an error message columns don't match- The columns(s) [305.888916] from the data file are missing in the table.  Do you want to proceed without importing them?  Is there a way aroung this error?