I have been called upon from time to time to do some intact proteins. Honestly, I believe I achieved the point where I finally failed enough at it (ask my postdoc advisor) that I'm reasonably good at it. However, I've ALWAYS had some questions about what is going on with the proteins as I kind of haphazardly change settings till I get a good signal. Especially when I'm tinkering with source parameters trying to, for example, get a low abundance mAB PTM to resolve a little better!
This brand new paper at NIST answers so many of the things I've wondered about in my head that I honestly can't come up with anything except -- how don't I know these authors?!? We're a pretty small community here in Maryland...but I'll sort this out later.
In this work they take several intact proteins of varying sizes-- all interesting (BONUS!), starting with CRP and up to the NIST mAB. They utilize three instruments, an Orbitrap Elite and 2 Q-TOFs. Systematically they go through a bunch of different parameters, including:
Source temperatures
Solvation energies (in source CIDs)
AGC targets
Resolution
They even go through the (always fun for those of us with multiple knee surgeries) manual optimization of the HCD gas pressures with the little knob hidden deep inside the later LTQ Orbtrap systems.
Etc.,
What they come up with is the best study I've ever seen on optimizing an LTQ Orbitrap system for obtaining reproducible intact protein measurements (particularly focused on the low abundance PTMs). Of course, these observations can be directly converted to the other systems -- since a lot of what they look at will be the same for the quadrupole-Orbitraps as well.
I've already sent links to this paper to a number of different people I know who will find this very useful.
Interesting note -- they calculate the intact protein theoretical masses using this cool little program from NIST that I'm embarrassed I didn't know (or forgot) about!
No comments:
Post a Comment