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.

5 comments:

  1. Interesting post, but I would suggest critically reading the paper you link above in MCP regarding off-site modifications with iodine containing reagents. There is a lot of interesting information contained within it regarding other reduction/alkylation combos, and you may not conclude that TCEP/CAA is the best choice after reading it. There are also other things to consider, such as the composition of your lysis solution, which if you are using deoxycholate, TCEP will likely not be the best choice for you (TCEP will precipitate deoxycholate). In addition, one aspect that is very clear in the above paper is that the actual method that you use for your processing can make a much larger difference than the reagents used. The observations of that work were strongly apparent in the in-gel data, but far less so in the in-solution (FASP-treated) samples (for reasons discussed in that work). This is also just the results of one lab processing in their hands...I wonder what the result would be looking for these unexpected modifications in a much larger data set (perhaps in the MSFragger paper, or the wide search window Gygi lab paper)?

    I'm not trying to advocate for the use of IAA (or any other reagents), but rather to suggest testing and tailoring your workflow to the results you obtain in your own hands.

    Cheers,
    Chris

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  2. I've been using iodoacetamide/TCEP at the same time for several years. I'll have to revisit the idea of using chloroacetamide.

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  3. Extensive and quite recent exploration of IAM versus CA here : https://pubs.acs.org/doi/10.1021/acs.jproteome.7b00022

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  4. I'll be impressed if this is now the only method the Mann group and their many academic descendants use... is it?

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  5. I'm curious with that as well Rob. After coming across this several weeks ago, my first two attempts utilizing the CAA/TCEP combo resulted in only 75% labeling efficiency. As Chris mentioned, further testing is always needed to see what can be achieved in your hands.

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