Sunday, July 22, 2018

CPTAC 3 -- First paper already! How the TMT protocol is set up!!

CPTAC3 (might not be the official designation, but that's what I call it) kicked off last year. What I knew about it -- full standardization. All labs have the same LC, same mass spec, use the same columns and chromatography, and they moved up from iTRAQ 4-plex to TMT 10 or 11.

Now we can all know the entire workflow!!

Interesting points? Despite having Orbitrap Lumoses, CPTAC is sticking to MS2 based TMT quan with a super interesting side note ---  they recommend running with Advanced Precursor Determination (APD) off. And suggest that the Q Exactive HF-X shouldn't be used for TMT quan because the APD can not be turned off...?

Interesting, right? But it kinda makes sense, if you are running at a lower resolution at MS1 (60,000 for CPTAC3) maybe you don't resolve as much of the lower abundance peptides that have higher coisolation interference. If APD is helping you to find more lower abundance peptides in the noisier ranges maybe those aren't going to be peptides with good ratios anyway.  I dig the logic. I also dig the 0.7 Da MS/MS isolation window. 

The study also suggests a really high degree of variation in HCD collision energies in their lab's Lumos devices. The actual settings table uses HCD CE of 34, but they emphasize CE optimization.

What kind of results are they looking at? About 10,000 proteins from xenografts with around 7,700 of them coming from human and the rest from mouse cell/protein invasion. They also show some really sharp correlation numbers suggesting their MS2 TMT quan is working just fine for them!

All the RAW and processed data is, of course, available at the CPTAC portal here.  EDIT: 7/23/18 -- I tried to download these files, but they don't appear to be posted yet. 

1 comment:

  1. Why not bump the intensity threshold instead of turning APD off?