Saturday, October 21, 2017

Advancing top down proteomics past 30kDa!!

Okay...before you roll your eyes and assume this is going to feature the custom modified quadrupole Orbitrap with the 7 ion funnels that requires vacuum levels that can only be achieved on the International Space Station -- I don't think it does.  This appears to be a normal high field quadrupole-Orbitrap....that can scan at a 3,000 resolution and do 25 microscans.....

...I know! Not out of the box, but this is stuff that is just operational software. However, I swear there is stuff for everyone doing top-down in this new paper!

Realistically right now when someone says "I did comprehensive top down proteomics" it means "I got some great coverage of the proteins from this cell that were in the 15kDa range and I even got a few that were almost 32kDa." This is where the technology is -- but this range is creeping up all the time,

This paper is great because they got over 400 proteoforms that were in the 30kDa - 60kDa range and they did so by breaking one of the rules of top-down proteomics. They used NARROW isolation widths -- 3 Da!

(If you haven't done much intact protein fragmentation work, this probably doesn't sound too weird, totally is!)

This is a zoomed in MS1 spectra (140kDa QE Classic) of a 23kDa protein. There is a lot of stuff going on there -- however, the important part here is the signal -- it's only E5 in the MS1. That may be enough precursor for a peptide, but larger ions have a couple of problems. They are harder to isolate. They degrade more rapidly during trapping. They are harder to transfer from one place to another efficiently (for example, from the HCD cell to the C-Trap to the Orbitrap -- they're all places where you are going to lose more ions).

Another big problem is the ion spread. On a peptide with 12 amino acids, there are basically only 12 fragment ions possible, right? On something at 23kDa that fragmentation spread can be spread over 200+ different MS/MS ions (though there are obviously energetic biases).  So we cheat. We open up the precursor isolation window to get in as many ions from this one (or more, if multiplexing) protein charge state to squeeze in as much signal as we can and we use different scoring systems like the ones in ProsightPC that deal with co-isolation interference using strategies different than peptide engines. (Totally worth discussing some other time!) You will typically see isolation windows of 15Da or more.

Bigger proteins, however, mean more charges are accepted and the isotopic envelope is squeezed tighter together.

Easy example -- mAB -- if you capture a 15 Da window around this charge state you would really be isolating at least 5 different proteoforms!  This is going to make the job of sequencing the protein(s) much much harder, even for top-down algorithms that are expecting it.

Wow...that was a lot of words....I missed blogging!! Okay -- so it's a big deal in this paper that they narrow the isolation width. Apparently the segmented quad and the high field Orbitrap can get enough signal here that we can actually ID these proteins now! It's one of those things where, we've always done it this way, but the new technology advanced our capabilities more than we thought!

Worth noting, to get to the total number of proteoforms reported in the abstract they combine methods, including Autopilot, which you probably don't have. And they did use 3,700 resolution for the MS1 on the bigger proteins. However, that's not too far off from what some quadrupole-Orbitrap systems are capable of doing now when running unmodified versions of the vendor's software!

I'm glad there isn't a word count on this...but the moral of the story is that you may be able to get good results on fragmentation of intact proteins on some instruments with isolation widths that will enable single proteoform isolation, even on larger proteins!

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