Monday, September 7, 2020

Synergistic optimization of LC and MS parameters on Tribrid instruments!

Okay -- we all know how to set up the perfect DDA experiment on a Tribrid, right? I sure felt like I had it locked down, but I learned a lot from this incredibly thorough and inciteful new study. 

The goal was to start with one of the most important studies ever written for us proteomics nerds in the lab and see where a Tribrid stands in a similar analysis. How many features are there? How many can we get with this thing? What are we missing and why? 

There is a lot in here, but I'll just go over the highlights. 

1) One of the most interesting findings might be some really suboptimal results using the "top speed" methods. The authors find that even though they predict 50 OT or 60 IT scans should be occurring per cycle, they only obtain 18 OT or 12 IT scans per cycle. Ouch!  I need to check some old data later to see if I'm seeing anything close. 

2)  By analyzing the amount of time needed to get to 50,000 charges in MS/MS, they find the following:

97.4% of the peptides required >20ms of fill time to get there. Totally agree. 
22.4% of acquired MS/MS scans require >150ms to get to that same state (lower abundance)
And....they cite a study I should really already know that suggests that for the ultra-low abundance stuff, 2 seconds is required to get to that target. Two(2) seconds?  Whoa. 

3) Okay -- and this is super cool.
The authors look at the reproduciblity of label free quantification at different flow rates, gradient lengths, and loading. 
High load? Worse quan. Sure, it looks prettier to have tons of signal, but the quan falls off a cliff. What they find is that when the MS1 fill time drops to <1ms you lose some dynamic range in your experiment. 
This makes sense, right? Automatic Gain Control (AGC) is trying to keep the C-trap and Orbitrap from overloading by capping your fill time. Is it more accurate when it has 10ms to compensate or 0.1ms? I'm going to guess it gets better at the higher numbers.
They find the best quan on their 20cm 1.8um columns at <1ug and do not recommend going over 2ug regardless of the gradient length on the column. 

Is this the most exciting study of all time? Maybe not, but the findings here are definitely worth thinking about before you set up that next global study on your Tribrid. 

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