Is it possible to use the Skyline program for the quantitative analysis of various modified forms of the same peptide in different samples with known relative ionization coefficient k? akulyyasov  2020-06-12 02:06
 

Dear Brendan,
I have a question for you regarding the use of the Skyline program for the quantitative analysis of various modified forms of the same peptide in different samples.
According to the results of the experiment, I got two samples. In one control tube, a small amount of the biotinylated form of BAP is present, and basically there is a propionylated form of BAP.
In another tube, the amount of biotinylated form of BAP is much higher than propionylated BAP.
Does the Skyline program provide for the possibility of calculating the level of biotinylation between samples taking into account the known value of the relative ionization coefficient k of two modified forms of peptides?

With best regards
Arman

This Biotin Acceptor Peptide (BAP) is a non-naturally occurring peptide and is absent in the NCBI and Uniprot database.
BAP is the result of a design in which the amino acid sequence is modified to reduce substrate activity compared to the known natural AviTag peptide.

Biotin Acceptor Peptide (BAP) - tryptic peptide for LC-MS/MS
BAP biotinylated - ILEAQK(Bio)IVR, m/z value of (M+2H)2+ (648.4)
BAP propionylated - ILEAQK(Pro)IVR, m/z value of (M+2H)2+ (563.2)

The relative ionization coefficient k between the biotinylated and propionylated BAP peptides was estimated as k = (HP - LP)/(LB - HB), where HP corresponds to the area of TIC of heavy propionylated, LP of light propionylated, LB of light biotinylated, and HB of heavy biotinylated BAP. K were calculated for two independent experiments, giving an average value 11.9 (Kulyyassov A, et al. PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo.//J Proteome Res. 2011 Oct 7;10(10):4416-27. doi: 10.1021/pr200189p).

I also uploaded one experiment in Panorama database

https://panoramaweb.org/Nat Center for Biotech Kazakhstan – Mass Spec Lab/BAPSx2_9hrbiotpulsonNidigest1_RA3_01_562/project-begin.view?
 
 
Nick Shulman responded:  2020-06-12 10:21
I am not sure that I understand your question.

This sounds like the sort of calculation that you would need to do in some other tool such as Microsoft Excel using data exported from Skyline.

If you would like to learn about how to export data from Skyline, you can take a look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

In the Document Grid, there is a column available on the Peptide Result called "Modified Area Proportion" which might be helpful in terms of telling you what percent of the observed peak area came from a particular form of the peptide.
-- Nick