Sunday, March 27, 2016
QE Plus versus Orbitrap Fusion for quantitation!
What I'm talking about is this new paper from JC Williamson et al., in this month's Proteomics (paywalled...you can read it for $6, I guess).
The instruments compared are the Q Exactive Plus, which, in case you are confused about the large Exactive family these days, this is the Q Exactive with the advanced quadrupoles and front end optics. Better for proteomics than the QE classic and can be upgraded in various ways (280k resolution, "protein mode" and can be upgraded to an HF if you need more speed later).
The challenger here is the Orbitrap Fusion. This is a generation 1 Fusion. It starts out like a Q Exactive Plus with slightly different quadrupoles and the High Field (D20) Orbitrap but also gets a high speed ion trap, ETD and 2 powerful computers onboard that can make a load of decisions on the fly.
In this study they take some E. coli and heatshock it so they have a well characterized, though highly complicated, biological response.
For comparing the instruments they take some of the peptides extracted and use those for label free proteomics, and then they iTRAQ the rest. To further add interest to this paper they use both the MS2 for iTRAQ and the SPS MS3 method on the Fusion that everyone keep going on about.
How'd they do?
Actually it is really interesting. They do a lot of run optimization and come up with ideal settings for the two instruments that are a little different than what we've seen previously in the literature. They find the best isolation on the QE Plus to be 2.0 Da, rather than the 1.4 that came out of Max Planck. They also find the highest IDs on the QE Plus when using 5e4 for the MS/MS target. Of course, every sample load and LC condition changes things. But this group did a very thorough job of controlling their peptide load (reduced and did amino acid quan?) so it might be worth taking note.
In terms of MS/MS scans obtained per unit time, no surprises here. The Fusion ran laps around the QE Plus, both when using the ion trap for MS/MS and the Orbitrap. It is a much faster instrument. In terms of peptide IDs, the differences aren't as large. Still more IDs from the Fusion but at its highest speed, the Fusion came up with 13% more peptide IDs per minute than the QE Plus. Remember, this is a bacteria, the QE Plus might realistically identify every protein with a nice long gradient. The differences might be bigger with a more complex proteome.
Interesting observations here? Whether you are doing MS/MS iTRAQ or label free quan or MS3 SPS iTRAQ, it all works. You get the heat shock proteins that you are looking for (P.S. they use the Precursor ion area detection node in PD 1.4). The method with the best dynamic range? Label free. The worst dynamic range? QE Plus iTRAQ. Significantly better dynamic range than MS/MS? The SPS method.
Statistically, however, when looking at a simple genome (this guy only makes 4k proteins -- you can sequence it with a NanoPore USB drive) and looking at the data statistically? You end up with the same data with MS2 or MS3. Its when we get more complicated proteomes that these techniques and extra speeds where the instruments are going to look different, but for a "simple" organism the QE might do the job. And really, I guess we all know that. If you fractionate a sample and increase the run time, an Orbi XL will eventually get the same data as the fastest instruments out there. Its just a measure of how much time and work per sample.
Still, an interesting analysis and a thoughtful, independent look at comparing instruments and techniques.