I've been doing MS for quite a while. Nanospray was definitely around when I started, but it wasn't the way that most labs did things. I toyed around with it a little in grad school, hated the existing technology at the time, and didn't do it again until my second Postdoc. I still kind of hated it. Microflow electrospray has so many advantages: the pumps are more precise, they are easier to set up, troubleshoot, they degas themselves, the clog less often, etc.,
Nanospray, however, has the distinct advantage of a 1 to 2 order of magnitude increase in sensitivity so we have to use it, right?
What if I said that I'm starting to see labs that are rejecting the use of nanospray and are going back to micro flow electrospray for proteomics and are generating very nice results? Here is the argument: When the first nanoflow experiments were being routinely done, say 10 years ago, this was a tremendous jump forward for these relatively insensitive instruments. The increase in analyzer sensitivity on current instruments is far more than 2 orders of magnitude. Is it 3 or 4 or 5? In any case, from this line of thinking, the advantages of microflow may outweigh the disadvantages of sensitivity for some experiments.
The image above is from one such experiment. In this experiment, a 3 uL injection of a BSA standard digest at a concentration of 2 pmol/uL was ran at 200uL/min on a 15cm C-18 column. It is difficult to see from the screenshot, but the gradient was (in total) 34 minutes. The base peak intensity is roughly 2 E 8.
The punch line? This digest was ran on an Orbitrap XL that could have had further optimization for this experiment. Yes, the injection is pretty high by most standards, but imagine this same injection on a fully optimized Orbitrap Elite or Q Exactive with a little slower gradient. We would easily be seeing a base peak of 1 E 9 due to the relative increase insensitivity and I expect we could drop this injection by 1,000 fold and still fully resolve this protein. Hypothetically, this doesn't sound like we'd be that far off from a normal experiment now, does it?
Just a thought, but I'm excited to see what will come of these labs that are switching over to higher flow rates.
You've just described one of the central themes of discussions in our lab when it comes to proteomics. We don't do anything at true microflow rates but we do have a standard capillary set up with ESI which we run at 9 uL/min. We run this when we have lots of sample - typically we're injecting 6-8 ug on column (300 um x 15 cm, 1.8 um particle size), 2.5 hour gradient on an Orbi-Velos. This works very nicely, but when sample is limited we're forced to go with the nano approach. We've just started using the new easyspray nanosource (75um x 25cm, 2um particle) and gives very similar results to our normal ESI/cap setup, all be it with <10% of the sample input of cap. For us the biggest headache has been switching back and fourth and having two separate HPLCs for cap and nano. Given the choice I'd do everything with ESI/cap, but that's not always possible.
ReplyDeleteThanks for the insight. That is great. More people seem to be doing this all the time. Hopefully we get back to a point in proteomics where the LC is the easy part! With nano, we definitely aren't there yet.
DeleteThis comment has been removed by the author.
ReplyDeletePretty good post. I just stumbled upon your blog and wanted to say that I have really enjoyed reading your blog posts.
ReplyDeleteproteomics services
Thanks for the feedback! BTW, I'm a fan of your company's approach to things. You're definitely going beyond what most core services are providing.
Delete