Wednesday, October 31, 2018

Advanced Precursor Determination -- Bad for TMT? Part 2.

Honestly -- even after reading the second paper -- I'm not sure I get it....

CPTAC-3 has some heavy hitters again in this newest project and a few months back they demonstrated their results of some serious TMT 11-plex optimization.

Their results?

Don't use APD
Don't use MS3 based TMT.

I rambled about that here.

The second one I get. For global proteomics I also go to MS2-based TMT quan. I'd rather get 15 peptides per protein with more isolation interference than getting 9 peptides per protein with less interference. The first one -- cheeeeeeeese --- I can't wrap my head around....

A second study on this topic replicates and elaborates on these findings and is brand new here.

Okay -- honestly -- maybe I get it....and maybe it's just denial....cause I've got some TMT 10-plex data on the PC behind me that is some of the best I've ever seen and it came from this study from the Olsen lab.  

The author's report 16,700 TMT10-plex labeled phosphopeptides. I'm pretty sure I got around 10k when I reprocessed it myself (and I'm a picky jerk about PTMs) with offline fractionation and short short gradients (6 hours total run time or something ridiculously short). 

And maybe it's the offline fractionation that improves the coisolation? And maybe phosphopeptides are just simpler? Because on the HF-X -- at least at launch -- APD was always on....

Lots to think about -- later! 

1 comment:

  1. I do not understand the whole point behind APD (except getting more money from customers). In my understanding APD allows you to actually see which co-eluting precursors you isolate for MS/MS. Then, you are trying to get MS/MS for each of these precursors. But since their masses are very close all these MS/MS scans will be chimeric, at best they will just contain a different ratio of precursors. Therefore despite a huge increase in the number of acquired MS/MS scans you get only a minor increase in the number of ids. APD will not affect accuracy of TMT quantification since it does not separate co-eluting precursors.