Getting started, developing MRM's for the 1st time.

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Getting started, developing MRM's for the 1st time. neil dodsworth  2019-02-19
 

Hi All
I'm new to all this and don't have a proteomics background. I'm trying to rapidly develop MRM methods for some known proteins to start with, with a view to quantitating them in mixtures. I have some digests ready to go on. I don't need databases to search for unknowns at this point. I've pasted my protein sequence into the box, and created an in silico digest of my protein with all the tryptic peptides listed, with their resulting theoretical fragment ions, displayed below as per tutorials, home page etc.
However, ..... that's as far as I've got and attempts via Edit and Settings etc. in the toolbar to create Transition lists and MRM's have failed. I've tried editing some of the fields such as instrument type, m/z ranges etc but nothing happens. I don't generate an MS/MS spectrum or any MRM's. So what am I doing wrong and what steps do I need to take to create my methods?

many thanks

Neil.

 
 
Brian Pratt responded:  2019-02-19
Hi Neil,

You're very close! The Targets window is your transition list, you now just need to write it out in a format that your mass spec understands. The File > Export menu will help you output a properly formatted transition list based on the transitions you created by doing the digestion on your protein.

Skyline doesn't generate spectra, it reads spectra from mass spec runs to help refine your transition lists. But that's the next, possibly iterative, step.

I hope this makes sense?

Good luck with your research!

Brian Pratt
 
neil dodsworth responded:  2019-02-26
Hi Brian
many thanks for the quick response, I'm sure there'll be more questions to follow,
best wishes
Neil