Sunday, November 25, 2018
What do you get when you run an HFat full X transient speed?
If you can't tell, that's a picture of the racing stripes I'm considering requesting a local auto-body place cut and install on my HF. Now that I'm looking at it, it seems a little dumb. It looks more like suspenders.
Okay -- so you should probably check this out on your own, but in case you aren't crazy and want to see how it turns out -- these are my results so far. (Oh. And this is an extension of this post a few weeks ago)
500ng of human cell digest at 60k MS1 15k MS/MS (normal HF speed) -- 45 minute gradient;
Okay -- pretty normal.
What if I change nothing, but drop the MS/MS to 8,000 resolution (and the fill time down to 11ms to make sure transient is the limiting factor)?
Loads more MS/MS scans.
Less of everything else. Let's look at why:
Even with the normal method, we're maxing out our fill time the majority (66.85%) of the time.
Cutting that number in more than half? That's totally dumb. Makes me sound as dumb as all these "peer reviewers" keep implying that I am, right?
"Hey Ben, why did you choose 11ms?" Because I'm dumb. Or because I wanted to verify that I really was increasing the number of MS/MS scans and boosting the number of ions sampled per cycle. And -- I'm increasing the number of MS/MS scans and the number of ions sampled per cycle.
And I wanted to see if I could get anything with 11ms of fill time. Almost 1900 protein groups in 45 minutes with a massive fill time limitation? That's AWESOME. Less than what I get with a normal method an nLC -- but, in case you've never visited this blog before, I HATE nanoLC. Necessary evil and all that, but I'll have a huge party when it's a dead technology we laugh about suffering through.
We've been evaluating options to nanoLC. And I've got a solution that shows some promise, but the peaks are waaaaaay too sharp. Like 4 seconds wide sharp. At the top of a 4 second peak, there is a TON of signal. But if your cycle time for only 20 MS/MS events is a full second? You've got high odds of missing that peak apex, and all that signal is gone.
(In case you're wondering 1ug of digest doesn't look much better)
So....more optimization is necessary...but for specific applications, maybe the HF deserves to get those flames!