Monday, February 11, 2013
Is iTRAQ 8-plex ruining your experiments?
I have done a lot of iTRAQ. In my last job we used the iTRAQ 8-plex kits as our most common experiment. Imagine my surprise when I was sitting in a presentation where the weaknesses of the iTRAQ 8-plex kit appeared to be a given!
While I'm still waiting on further clarification and possibly a follow-up meeting with the technical experts to assembled this publication, I'm doing some digging. And what I've come up with is that in the last 9 months or so the iTRAQ 8-plex kit has become one of the most reagents in our field.
There have been some extremely critical papers recently, such as this one from Evans et al., where they show that iTRAQ constantly underestimates the ratio fold change. A discussion of the paper at Shared Proteomics suggests that this may have been an issue with the HCD fragmentation on the Orbitrap XL.
The guys on the forum also brought up this paper from Pottiez et al., that appears to completely refute these findings. They show that using a MALDI-TOF/TOF and studying known ratios experimentally that the iTRAQ 8-plex is considerably better than the 4-plex kits.
Further searching led me to this presentation from Matrix Science (MASCOT), presented at ASMS 2010 that also delves into the under-representation of iTRAQ ratios as described in this 2009 paper from Yen Ow et al., that has a pretty shocking illustration on the front (below).
When I moved into iTRAQ after years of label free and SILAC studies, I read a lot of iTRAQ papers. The fact is that it is such a popular technology that there are just a staggering number of them out there -- and it is impossible to read them all. This is how I interpret all this stuff I've been reading the last week: Every technology is going to have some controversy around it, but if people are still getting good results from that system, then we need to roll with it until something better comes around. I think the data out there that says which system is better for iTRAQ essentially neutralizes itself out. 4-plex and 8-plex are each going to have their distinct advantages and disadvantages. As for the ratio suppression issue in iTRAQ -- we aren't using this as a definitive filter. When we see a 2.3 fold up-regulation we don't really, literally, believe that protein is up-regulated 2.3 fold, or that it is an inferior measurement to another protein that is up 2.8-fold. We know that in quantitative proteomics we have a huge error bar on every measurement of a complex digest. If we are using iTRAQ to implicate pathways and keep take those ratios with a "grain of salt", as I think we all are doing, 8-plex iTRAQ is currently the fastest and best way to screen multiple samples at once.
Now if something better comes around, it comes around, but for now, we're going to keep running our 8-plex iTRAQ data.