MRM assay development

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MRM assay development ad6628  2022-10-12
 

Hi,

I have attended the introductory training on October 10 and 11 but I am not sure how to apply the training to my immediate problem. My boss wants me to establish MRM conditions for a QExecutive mass spectrometer to check on a synthetic peptide. I will attach the slides I have. The peptide has modifications as oxidation, acetylation and has an amide attached to the n-terminal. It can be analyzed as heavy versus light. I was wondering which tutorial I should follow to create the assay. Any advice would be greatly appreciated.

Thank you
Rita

 
 
Nick Shulman responded:  2022-10-12
I think the two tutorials that you should look at are Targeted Method Editing:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_edit
and Targeted Method Refinement:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_refine

-- Nick
 
Brendan MacLean responded:  2022-10-12

Hi Rita,
I have just uploaded "2019-08 Brixen Summerschool files.zip" to the Skyline support board file folder:

https://skyline.ms/files.url

This contains a more basic PRM tutorial than the one we used during the course. It still uses a spectral library and a protein sequence. Though, you can substitute Edit > Insert > Peptides where it uses Edit > Insert > Proteins for your peptide of interest. We used Edit > Insert > Peptides multiple times during Skyline-Online: Session 1. For the Absotlute Quant tutorial, we used it to add just a single peptide with no protein sequence information, as you may want to do here.

Since, your PowerPoint slides mention wanting to do this for a calibration curve, you will want to review the Absolute Quantification tutorial we covered during the session. It is actually very similar to what you seem to want to do, since it is just one peptide with a heavy standard reference. It is, however, an experiment performed with SRM on a triple-quadrupole.

So, I hope the extra simplified PRM tutorial for an Orbitrap will be helpful. If after reviewing the new tutorial, the PRM Orbitrap tutorial from Session 1, and the Absolute Quantification tutorial, you still feel stuck, post back to this thread and we will arrange for some kind of Zoom review to get you unstuck.

Good luck. Thanks for posting to the support board and for your time and attention during Session 1.

--Brendan

 
ad6628 responded:  2022-10-12

Thank you very much! I will try it.
Rita