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:
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.