Thursday, November 3, 2016
Study peptide methylation without antibody enrichment!
Keyun Wang et al., describe a super interesting methodology for getting to protein methylation sites in this new paper in JPR.
It takes advantage of two things:
1) That trypsin is going to pass right over a methylated lysine or arginine (the latter I wasn't sure about till I read this!) and
2) The methylation at these sites does not alter the overall charge of the peptide (this biologist didn't know this at all!)
SO....(ellipsis in case the next steps are obvious to any chemists reading this! Eureka?) they develop a chemical approach to exploit this and separate the bigger methylated peptides from the smaller non-methylated ones.
Best parts? (I like lists today!)
1) No derivatization!
2) Simple one-stage separation and it is seriously easy enough looking that I could definitely probably pull it off
3) It looks like it works great.
Further details: It appears to be basically high pH SCX (see what I did there?) and they get over 800 unique methylated peptides yanked out of a cancer cell line. I'm rushing through this one this morning, but they do the SCX on a tip (yeah!) and they use a SILAC-based approach to get some quan out of it.
Now, a critic might say that with some of the modern anti-methylation antibody approaches people have scored higher numbers of unique methylation events, but with this technique they don't see a bias toward particular peptide sequences!
Shoutout to PastelBio for 15 good papers I could add to my reading queue this morning!