Sunday, May 5, 2019

HDX vs FPOP -- They're both better than NMR, but which one is the bestest?

Structural mass spec is advancing like crazy. Crosslinking keeps getting better -- with new reagents, better separation and improved MS/MS techniques and -- of course better software (1, 2, 3 )leading the way. (Here is an awesome recent review on all things proteomics structural.)

Crosslinking gives us a lot of power by letting us know what amino acid residues are close or reeaally close together (is this one commercially available yet?)

There are weaknesses here, though. Things can be close together without being within the space necessary for us to chemically lock them together. Conformations of relatively "low stoichiometry" (hey -- if phosphoproteomics can use this terminology as if it's okay for 15 years, let's keep bending it, it's almost a meme now!) are going to be impossible to see AND we learn nothing about modified residues or the outside of the protein structures.

Two techniques are improving all the time that can give you a lot more of this information -- and they go head-to-head in this study I left myself a note to read in September and just found.

Chances are you know more about these techniques than I do -- but I'm learning 'cause I think they're only going to become more important all the time!  HDX requires some arduous sample prep up front or the purchase of an add on system for your mass spec that does all the work for you. Deuterium can't get to the inside of your protein as effectively, so anything that gets labeled is on the outside. Boom! I know what the inside and outside of this protein and protein-protein interaction is like and tons of smart software exists that helps interpret the data. Workbench is a good example!

FPOP uses hydrogen peroxide and

...lasers to modify the outside of proteins, protein complexes, and -- holy shit -- have you seen this?!? -- even works inside whole cells!!  Study 1, study 2. The downside is that the modifications on the outside of the protein may be unpredictable. Better data processing, better resolution and accuracy data have helped make this easier, but it's still tough at this point.

Both of these techniques are better than NMR, obviously, because I don't have an NMR and the whole concept seems really old fashioned, and helium is not getting cheaper -- if you are a new lab starting out you may find that it's hard to get Helium at all (we had to go through 3 vendors and lie and say we're doing medical research (!!kidding about the last part!!) -- to even get tanks!)

 In the study that I originally started talking about (comparing HDX of the same protein to FPOP) HDX comes out on top. The comparison might not be the most fair, though.

1) HDX analysis was performed on a QTOF system
2) FPOP analysis was performed on a QE Plus system

I know someone who has an HDX QE Plus (Hi!) and I wonder if she'd get the same results with the same material? I think the higher resolution and higher sensitivity of system #2 is kind of seriously essential here, maybe particularly because the QE Plus for the FPOP used 300ms(!!!) of fill time for MS/MS, indicating that even with a pure protein collecting a lot of ions is critical component, which the TOF utilized in this study can't do (only one TOF kinda sorta can, right?!? I'm only putting question marks here because I've discovered there was some really interesting hybrid TOF technologies that were developed in the past that we all seem to have pretty much forgotten about). looks like FPOP wins! I like this result because HDX sample prep seems too finicky for me to ever get it right!

1 comment:

  1. Nice overview! With the refrigator systems of modern NMR instruments the He consumption is significantly reduced (same for MRMS). Not being active in this field I guess the techniques are highly complementary concerning structural biology with MS useful for both NMR and cryo-EM analysis.