problem with DDA search tutorial

support
problem with DDA search tutorial zahra mashhadi  2022-03-02
 

Hello,

I’m very interested in using skyline but also new to it. As I went to follow the steps in DDA tutorial, I got an error that it can’t find spectra for the new library (screen shot’s been attached here). No idea how to fix it. I really appreciate your help.

Thank you very much,
Zahra

 
 
kmp95 responded:  2022-03-02
Hello,
I wanted to add to this feed as I am having the same issue. There are a lot of other comments about this in the past, but I have been using skyline for months now and just updated and have just started to get this issue as well. I have included my error message below. I am not sure what the appropriate fix is, but for the time being I am planning to switch back to an earlier version of skyline.



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

Command-line: C:\Users\kmp95.WIN\AppData\Local\Apps\2.0\9NP975Q4.O6Q\842M5JVW.3XK\skyl..tion_e4141a2a22107248_0015.0002_38ac599f018d3163\BlibBuild -s -A -H -o -c 0.99 -i ABC_CoronaFormation_Samples_1G -S "C:\Users\kmp95.WIN\AppData\Local\Temp\tmpCCE4.tmp" "C:\Users\kmp95.WIN\Box\kits_project_5661-01_52186\Skyline Analysis\skyline\2022_analysis\ABC_CoronaFormation_Samples_1G.redundant.blib"
Working directory: C:\Users\kmp95.WIN\Box\kits_project_5661-01_52186\Skyline Analysis\skyline\samples_2
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\kmp95.WIN\AppData\Local\Apps\2.0\9NP975Q4.O6Q\842M5JVW.3XK\skyl..tion_e4141a2a22107248_0015.0002_38ac599f018d3163\BlibBuild -s -A -H -o -c 0.99 -i ABC_CoronaFormation_Samples_1G -S "C:\Users\kmp95.WIN\AppData\Local\Temp\tmpCCE4.tmp" "C:\Users\kmp95.WIN\Box\kits_project_5661-01_52186\Skyline Analysis\skyline\2022_analysis\ABC_CoronaFormation_Samples_1G.redundant.blib"
Working directory: C:\Users\kmp95.WIN\Box\kits_project_5661-01_52186\Skyline Analysis\skyline\samples_2
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 149
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
---------------------------
 
Nick Shulman responded:  2022-03-02
That error "No spectra were found for the new library" can happen if the DDA peptide search found no peptides. I once saw this happen when I used too low of a number for the mass tolerance.

If you actually expected some peptides to be found, then it sounds like Skyline is doing something wrong.
Can you send us your Skyline document, the raw files that you are searching, the FASTA file. It would also be helpful if you could send us screenshots of the pages as you step through the Import Peptide Search wizard so that we will know which settings you used.

In terms of sending the Skyline document, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

Files which are less than 50MB can be attached to this support request.
You can upload larger files here:
https://skyline.ms/files.url

-- Nick
 
kmp95 responded:  2022-03-02
Hi Nick,

I uploaded the files labeled "03022022_Poulsen_SkylineUpdate". Thanks for helping!

I used the following parameters:

Skyline Methods
-cut off score -> 0.99
-DDA Raw search
-irt standard peptides-> none
-workflow ->DDA with ms1 Filtering
Modifications
-Oxidation (M) - variable
-Carbamidomethyl (C) -fixed

Full Scan Settings
-precursor charge ->2
-Isotope -> Count
-Peaks -> 3
-Mass Analyzer -> FT-ICR, Resolving power 120,000; 200m/z
-retention time filter - only within 5 minutes
FASTA
-Trypsin, max missed=1

Search Settings
MSAmanda
-Ms1= 5 da
- Ms2= .8 ppm
-frag ions= b,y
-MS2 analyzer - default
-max mods - 3
 
Nick Shulman responded:  2022-03-02
Those mass tolerances that you are using on the MSAmanda search settings are strange.
I would recommend that you use "10ppm" for both the MS1 and MS2 mass tolerances.

5 Daltons is much too wide, and .8ppm is much too narrow.

When I use 10ppm for the MS1 and MS2 mass tolerances the search manages to find 411 peptides in "65136_c200LS.raw".
-- Nick