Friday, April 10, 2015
Another hurdle in top down proteomics surpassed by the Kelleher lab
We miss so much data when we digest our proteins. The long term goal is for us all to get to running all of our proteomics experiments top down. Problem is, its really really hard. There are hurdles all over the place. Proteins degrade, proteins are hard to separate, proteins ionize poorly, proteins fragment unpredictably and the data processing can be a nightmare -- to name a few.
One by one, the Kelleher lab has led the way in chipping away at these hurdles. Is it still hard to do top down proteomics? You bet your sweet peppy!
One of the ones I mentioned above is separating proteins. Thanks to insolution isoelectric focusing techniques like the GelFree System, the separation of proteins is a whole lot easier looking than it has been in the past. Problem is...that system (and things like it) use SDS to get good separation. And SDS is not mass spec compatible. You've got to get rid of it.
Until now, the only way of getting rid of the SDS is to crash the proteins out of solution with a precipitation technique. Have you ever done this? It sucks. You precipitate the protein and it is never the same again. You lose a lot of it because 1) maybe it didn't crash out efficiently (its really hard to tell) or 2) it won't go back into solution again or 3) you just turned all the proteins into a big gross mess. It sucks. You tell me that you are going to have to precipitate your protein with acetone or something and I'm going to assume what the mass spec is giving you isn't all that relevant to the biological system you had a minute ago. Okay, maybe it isn't that bad...but sometimes it really seems like that. Did I mention precipitating proteins sucks?
But now? How about an online separation technique where you just wash out the stupid SDS!?!?! It exists. It looks a lot like an online FASP system from the figure above. The proteins can't go through but the nasty detergents are gone. According to the paper it takes like 5 minutes. And then you can go right from a clever separation of your proteins to identifying what you have.
You can check it out in this paper from Ki Hun Kim et. al., here.
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another nice approach to remove detergents (including SDS) is Single‐Pot Solid‐Phase‐enhanced Sample Preparation (SP3), recently published in MSB: