Wednesday, February 28, 2018
Why do your alkylation and reduction separately? Do them together!
Okay -- I'm prepared. This is probably one of those things everyone has been doing that I just missed while I wasn't in the lab full time. Admittedly, I don't prep many samples, and I never really have. But I'm prepping a bunch of patient plasma samples this week with some different methods and I forgot how much it sucked for me. I forget what I'm doing all the time. I can't read my own handwriting and I get very...very...distracted....
What was I..umm....
Oh yeah! Until recently when everyone all of a sudden seemed very interested in PTMs on cysteines (WTFourier?) I blindly reduced and alkylated. Now that I'm thinking about it, I'm pretty concerned. There is this blog post
...and associated paper...
Okay...so that's bad news...serenity now... These are patient samples that I'm really really interested in (note to collaborators -- I'm 150% invested in every sample, not just these). How do they deal with it in the study I'm using as my template? Oh. They don't use IAA. I should have read ahead.
For reference, this is my model study for patient plasma samples. If someone brings you plasma, just do what they did. (I have to mix it up a little myself because I'm looking for something super weird.)
When you get to the reduction and alkylation steps you need to go back in time to this study.
Where you find this gem!
The figure at the very top of this post is 1b, demonstrating the efficiency of this method. BOOM! Reduction and alkylation in one step! No weird iodine things. No DTT cross-reactions with your alkylating step. Less places to get mixed up and forget where you left 80 samples. Everyone wins!
EDIT: 3/1/18. Definitely check out the comments people have time to make on this post!! This story is more complicated than this. I need to spend more time on this as well.