Sunday, November 9, 2014
Max Planck heavy analysis of QE vs. QE HF
Do you have a Q Exactive? You need to read this paper in press at MCP out of Max Planck! It is an awesome analysis of the original Q Exactive vs a Q Exactive HF. If that wasn't awesome enough, it is also a full out analysis of how any Q Exactive works and how best to optimize them to squeeze every possible ID out of your complex shotgun analysis. One of the authors on this great paper is some guy named Makarov, so you can probably assume that these numbers are pretty accurate.
You should be reading this paper right now, but I'll pull out some bullet points I made on the plane this morning and tried to put into some semblance of order:
For shotgun analysis on both instruments, they found the ideal target for MS/MS to be 1e5. A great chart backs this up.
For the QE, they recommend the ideal isolation window is 2.2 Da
The QE HF should be set at 1.4 Da for highest efficiency
For a QE HF, the ideal gradient for maximum numbers of IDs on a 50cm column is 150 min. They were not able to increase their IDs with a longer gradient!
At a gradient of this length, with a 2.2 Da window, 8% of MS/MS spectra have a second peptide in them of high enough intensity that it can be sequenced. At a 1.4Da window, this drops to around 6%
What about the head-to-head?
Same gradient -- QE HF gets 48% more peptide IDs than the Q Exactive
Both instruments have a dynamic range of about 3 orders of magnitude, but the QE HF can reach some lower copy number proteins than the QE classic.
Both instruments get an efficiency on a HeLa digest of about 62% (of MS/MS have peptide spectral matches).
The cool new flatapole may reduce the ion beam by as much as 75% (way way more robust now!)
The overhead is much better on the QE HF. It may be as low as 6ms!!!
Again, you should read this awesome paper. QE or QE Plus or QE HF, there is awesome info in here that will give you more peptide and protein IDs!