Hi Bryan,

Yes, I guess that would work, as the approach you describe has been used very often for the analysis of a multitude of non-covalent protein complexes, some larger than 1 MDa. Spraying from ammonium acetate (e.g. 100 mM, pH 6.8), will result in ion signals as your protein complex likely contains residues such as lysine, arginine and histidine, so you should be able to detect this using the Xevo-G2.

If you have sufficient material, you could consider spraying it from a syringe or a glass needle, otherwise injecting it onto a SEC column running in ammonium acetate could also do the trick, although in the latter case less scans can be accumulated of course.

Good luck!

BTW. What do you think is the limit of the mass range on your Q-TOF?

Thanks for your response. I'm slightly more confident in doing this now than I was yesterday!

The Xevo has little white bottles where the NaCsI calibrant is sprayed through the instrument's syringes. I can fill up a bottle with my protein complex, but what concentration do you think is sufficient? I have 75 mg of material, so that's not really an issue. 

I think the quadrupole goes up to 4000 in a 'resolution mode but I'm sure the TOF is much higher. I imagine that many ions would simply be filtered out. Is that correct logic? 

Hi Bryan,

I'd just spray it through the inlet that you also use to infuse the solvent coming from the LC-MS using a syringe, to prevent contamination of your calibration reservoir and tubings.

Just start by preparing a solution of your protein complex of interest at 1 mg/mL in 100 mM ammonium acetate,pH 6.8 and see what kind of spectrum you get. If I do this for an antibody (150 kDa), the most intense charge state is the 28+at approximately m/z 5400.

Good luck!

Thanks Eef! It worked quite well! 

Bryan,

Just run the quadrupole on the Xevo as transmission only and don't worry about using it as a mass filter if you are just looking to ID the complex intact. Spraying in ammonium acetate in higher concentrations should be fine to keep the complex intact. I would also suggest running your source "colder" if you have the ability to adjust the capillary temperature. If you are still having trouble keeping the complex intact, I would suggest using a heavier damping gas such as Ar. Good luck!

Thanks Logan, I'm going to do another run this weekend, so I will see if I can run without the quad. 

I'll follow up Monday!

Hi Bryan,

don't forget that you can also change the detection parameters on the detector itself for those higher masses (more 'pull' is needed for the detector, so higher voltages). All the other comments above are useful, and like Eef, I would avoid the calibrant bottles: they aren't really meant for that type of sample at all and you will only add plasticizers and other things you don't ant to the source. Make sure the spray is even too - that is even more critical at low flows here and if you are using a syringe pump, make sure it isn't pulsing.

for deconvolution of the data I've sent you a separate note, because that won't be the same conditions either: with default maxent conditions you'll almost certainly create harmonics with that mass range.

St John

Thanks St John! I had some decent results but there is significant glycosylation microheterogeneity to contend with which seems to be rather difficult to resolve. I might need to tune better. I have the cone voltage set at 100 V and the capillary voltage at 1.5 kV. I had to drop the source down to 40 C which took about a day as well.

I am working off this publication which is for a Synapt but it's given me a good starting point for optimizing conditions for keeping the complex in tact -

The syringe I am using also isn't remarkably clean, it has some sort of detergent in it. 

Bear in mind that Michal's lab has a modified instrument too - the collision cells are very different to the model you have - try to ensure that the CID values are as low as possible and make sure that the bypass mode really is on! In the source you have, you can also 'cool' the ions by not having as much vacuum, which will prevent some (but not all) of the gas phase dissociation