Orbitrap DIA search using converted Spectronaut library

Orbitrap DIA search using converted Spectronaut library zhangfangfei  2021-08-02

Hi Skyline Support team,

I used a converted library from Spectronaut for the DIA analysis.,by importing the transition list, other steps referring the instruction here https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/DIA-QE-20_1.pdf. The mProphet capture is in the attached. It seems to me a failed search with greyed RT difference and dot product. We got a very poor CV by replicates, low cross-correlations by retention time and quantification comparing with other DIA search results. The results are much better on other datasets.

What do you think could be the problem ? My guess would be 1) the neural loss ions or other ions are setting not properly from the converted Spectronaut library 2) retention time alignment failed (there displayed the RT in the library ) 3) the size of library is too large (300K precursors) 4)Is the Skyline Batch relevant for the DIA analysis?

As the library is very large, any operations took a long time. I would like to know your suggestions in how to quickly probe into the problem in quantification.

Thanks for your support

Nick Shulman responded:  2021-08-02
Can you send us your Skyline document?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral library (.blib) and extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.

Otherwise, you can upload it here:

When a feature score such as "Library Intensity Dot Product" is shown in italics like in your screenshot, it means that the score could not be calculated for some but not all of the peptides in your document. If you highlight that score in the Feature Scores grid and switch to the "Feature Scores" tab in the Edit Peak Scoring dialog, there will be an "Unknown" bar displayed at the right end of the bar graph. If you hover the mouse below that bar, binoculars will appear. If you click those binoculars, Skyline will display a list of the peptides that are missing that score. It is usually a good idea to figure out why those particular peptides are missing the score, and either delete the peptides from the document, or correct the problem which is causing them not to have scores.

One reason that peptides might be missing the "Library Intensity Dot Product" score is that Skyline requires the peptide to have at least 3 MS transitions in order to calculate the dot product.

After you send us your Skyline document we will be able to give you better information about what is going wrong.
-- Nick