Friday, April 13, 2018
Determination of Site-Specific Phosphorylation Ratios in a single PRM run!
I've been sending this great new JPR study to lots of people for weeks now and I forgot to post it here! (If you think this blog seems frantic -- you haven't received many emails from me....I need an embedded exclamation mark filter...)
Honestly, the picture does a great job of describing the workflow, but I love to type!
The phosphopeptides are going to have low relative ion signal on their own -- maybe too low to find in cell lysate even with a PRM, but a TiO2 enrichment will do a great job of enriching your single phoshorylation site. And -- okay -- hard to admit, but some proteins are too low to find with high confidence without a stupid antibody enrichment thing --particularly in body fluids, but do you have to run the instrument 3 times? These authors say --
Just combine it back together and schedule the single (per appropriate replicate, of course) LC-PRM assays. If your protein and phospho are high enough in abundance that you don't have to add in the extra variables of TiO2 and IP and can just build your own -- maybe using the amazing resources at PhosphoPedia -- even better, but if you don't have enough sensitivity, this method could save you valuable instrument time!