Wednesday, July 22, 2015
Proteomics in negative mode!
Wow! What is happening in this picture? Something flat out crazy and awesome.
The paper is in press at MCP here from Nicholas Riley et al., out of some guy named Josh Coon's lab.
What is it? A hacked LTQ Orbitrap with a new collision source. They call it the "Multipurpose Dissociation Cell" and it massively improves the signal, speed, and utility of ETD fragmentation. It improves it to the point that it makes doing proteomics completely in negative mode something that everyone has thought about something that is actually a possibility.
Just last week I spent some time explaining why we don't do negative proteomics for PTMs: poor ionization, fragmentation sucks, there aren't good tools for translating the data, etc., Amazing how this field evolves!
To get around poor ionization, this study switched the buffers around. High pH reverse phase!
To get good fragmentation, they use their awesome new MDC source to perform Acitive Ion Negative ETD (AI-NETD)
To do the data processing, they studied the AI-NETD fragmentation spectra and determined the new charge-reduction loss masses and processed the data to remove these components. Then they used a modified version of OMSSA that was set to read a(dot) and x fragment ions.
I had to pull the Wikipedia peptide fragmentation chart to figure out where things are coming apart. Wow, right?
This isn't the first run at doing negative proteomics. It isn't even the first run at doing negative ETD fragmentation. This is, however, the first time that we've seen this approach produce results on the same scale as we get from positive fragmentation studies.
In one run they broke the 1,000 proteins ID'ed barrier on yeast.
Using multiple enzymes they come close to 100% coverage of the yeast proteome. How's that for on the right scale!?!
But that isn't really where the power of this approach is needed. We can get whole proteome shotgun coverage. We've kind of got that one in the bag. This opens up a whole new capacity for ions that prefer negative charges. Like many post translational modifications do. Heck, this might even open us up for more high throughput analyses of completely different biomolecules like oligonucleotides.
I want to extend a personal thank you to the authors here. I was trying to come up with reasons to get out of bed early this morning and just when "staying employed" didn't seem like it was enough to tip the scales, I found this in MCP. Now I'm up, motivated and about to head out the door to see what else all you brilliant people are up to out there!