Friday, March 13, 2015
Lets do intact proteins on the Fusion!
DISCLAIMER: Like anything/everything on this blog, this isn't official vendor stuff. I have successfully generated some intact protein data and a little bit of top-down using the Fusions I have ran into and people have asked me a couple times recently how I've done it. I'm sharing what I did.
With that out of the way! Intact protein masses are relatively easy to generate on the Fusion. The front end is very QE - like and you can generate intact protein masses that look as good as a QE without you having to work all that hard.
That being said, this is the most advanced mass spectrometer ever made. You can put in more effort into adjusting your parameters and get better data than the QE. It depends what you want. Do you need to confirm that your E.coli expressed protein didn't suffer a read-through? Inject 200ng on column and you'll get a mass that will confirm it. Do you need to know the position of a 1 Da shift in that protein? Then you're going to need to put in a lot more work. I can't tell you how to do that second one...yet!
The first one, though:
As with any Orbitrap intact run, I like to start with the lowest resolution first. On the Fusion, that's 15k resolution. You do have the option here, like on the hybrids, of starting with an ion trap scan first. Your choice.
I'm going to like these settings as a starting point for an intact mAB run. Another disclaimer: Good LC is going to help you a lot here. For this assay I generally use 200uL/min on a C4 column and divert the first 2-3 minutes to waste, particularly if this is a stored commercial antibody. There are lots of buffer components that are used to keep the mAB safe. Probably a good idea if that weird stuff doesn't go in the mass spec.
Starting points for mAB:
1e5 AGC target
50ms max fill time
MS1 scans from 1500-4000
S-lens RF at 60%
In source fragmentation energy (SID) at or around 40
325C ion transfer tube temp
And appropriate gas settings for the best solvation at your flow rate. Honestly, I LOVE that flow rate optimization button in the tune settings. You can likely tune it better yourself by putting in some extra work, but it gives you a darned nice starting point. (If you haven't seen it, you put in your flow rate and it sets what it considers the ideal gas pressures and HESI temperatures for best solvation.) Its really cool, particularly for those of us who mostly do nano!
Run these settings, tweak them a little (particularly the SID, often higher for bigger proteins) and run a nice clean intact protein. Average some scans and you're going to get something nice out of it.
Still doesn't look good?
Go into the scan headers and see what is going on. Are you hitting the maximum injection time? If so, move it to 100ms and see if that cleans it up. If that doesn't appear to be it, increase the SID. There is an application note out there on Rituximab (you can find it at Planet Orbitrap) that uses an SID of 80 on a QE with HESI. This is often the most important factor I end up moving around.
In general, you want higher SID for bigger proteins. This is my typical rule:
Light chain SID: 0-15
Heavy chain SID: 20-50
Intact mAB: 40-80
Why the big range? I'm not sure. If I had to guess, I'd say that the solvation of the protein is an additive effect. The goal is to get this big protein all into gas phase. I think the temperature/ESI voltage, SID, S-lens RF and other things all contribute to this effect. You can probably use more voltage and less SID to some degree. I'm not sure.
Once you've tweaked those to the best settings, take a look at the LC peak. Can you make your gradient steeper and sharpen your peaks so that you get more signal?
What does the isotopic envelope look like? Is it cleanly in the range from 2,000-4,000? Change your mass range to that. Get as much signal in there as you can.
Do you see things that don't belong and can't be deconvoluted into your protein of interest? Lower some energies! Lower the ESI voltage, crank back on the SID, lower the S-lens RF.
Intact protein measurements by mass spec is still a kind of art. There aren't a great list of settings that will work for every protein. Heck, I may sometimes re-optimize if I'm moving from one intact mAB to another. Secondary structure, # of basic amino acids exposes, number and types of PTMs, and on and on are all at work here.
Again, in case this wasn't clear: These are just starting points and are not vendor doctrine (as always, please don't sue me!). After I get emailed a question a couple times I figure I can save everybody some time by putting some stuff up here in the hopes that someone out there might find it useful. Correctly tuning the instrument for proteins and fully optimizing it for the higher m/z range is going to get you better data than this, but sometimes you just need to confirm your protein identity or number of PTMs and this is how I would get you there!