Friday, October 26, 2012

Is the Top10 experiment obsolete?

We all love the Top10 experiment.  It's catchy, it was one of the original tried-and-true Orbitrap experiments, and since evolution has granted us 10 digits on our paired appendages, it makes the math for us pretty easy.
But is it handicapping our experiments?
The answer is complex and requires quite a bit of arithmetic.  I'm currently sitting in a bar at an airport waiting on a delayed flight, so I did some of the math, but my motivation appears to be dropping for some reason.
Here is some math I just did on a napkin (and then drew in Excel)  for the Q Exactive instrument:

Right?  Moving from a Top 10 to a Top20 vastly increases the number of spectra that you can get.  Yes, the QE is fast, but this is HCD scans read in the Orbi.  When operating a hybrid instrument and acquiring the scans in the IT, this speed can be comparable, depending on the resolution used.

Real life experiment:  Orbitrap Elite, complex sample, 5 hour gradient, same processing workflow:
Method                      Unique Protein IDs                      Total Proteins               Unique Peptides
Elite, Top 10 2034 2626 8536
Elite Top 25 2346 2942 10476

What is interesting here is that after a Top25, we saw diminishing returns.  This does make sense.  A Top30 method was no better than a Top25, and a Top40 was even worse.  Remember that it takes time to do each and every scan, and that by the time you get to scan 31, your peak may be gone.  The next 10 MS/MS fills may be focused on ions that have already passed in your chromatography gradient.  This is very dependent on the chromatography conditions, instrument speed, and sample load, but good to keep in mind!

Additionally, it would be nice to reproduce this experiment on other instruments.  I forget the resolutions used on the Elite here, but they were pretty high.  I'd love to take a look at a similar experiment ran on the XL and/or Velos at lower resolution.

1 comment:

  1. Hi Ben, as for your number of MS1 & MS2 scans in the figure, I got similar numbers to yours, but not exactly the same. Since I don't know much about these topics, it would be good to learn your way of thinking.

    This is how I did it. I calculated the number of MS1 & MS2 scans based on the cycle times.

    For Top10 method, the cycle time is (256 + 10*64) = 896 ms. So in 30s run, I think we'll have 30*1000/896 = 33 cycles, or 33 MS1 + 330 MS2

    For Top20 method, the cycle time is (256 + 20*64) = 1536 ms. So in 30s run, we would have 30*1000/1536 = 19.5 cycles, or 19 MS1 + 380 MS2 scans