Friday, January 22, 2016
Want to remove some uncertainty from your SRMs? Try labeling entire proteins!
Wow. You want to talk about quality control for your validations? This is how you remove uncertainty from your measurements. You produce an entire protein completely labeled with your protein of interest and you spike that whole protein into your normal protein extract.
What are the advantages? No concerns about tryptic digestion efficiency -- that is controlled for. No concern about chromatography shifts.
Disadvantages? You've got to construct an entire protein that is isotopically labeled!!! Fortunately, Miral Dizdar's lab at NIST has assembled thorough instructions on how they do it in this work from Prasad Reddy et al., (paywalled).
Is it tough? Sure it is. Heck, for some proteins it might not even be possible (there are some proteins that E.coli just may not be able to make) but if you want absolute certainty you are measuring the right things, there might not be a better way to do it!