Saturday, November 12, 2016
How does spectral counting do for intact proteoform quan?
Spectral counting isn't my favorite thing for bottom-up proteomic quan. It totally works for highly differentially expressed things -- and there is probably over 1,000 studies out there with clear evidence of this, but I'm an XIC kind of guy.
This new study from Lucia Geis-Asteggiante et al., does something I've certainly not seen before -- applying spectral counting to top-down proteoform quan.
They start with an exosome lysate and it looks like go for the proteoforms under about 30kDa. An Orbitrap Fusion Lumos, which is tuned up nicely and, unless I'm forgetting something, clearly states every parameter you'd need to reproduce this on an identical instrument.
They spike in some proteins of known concentrations and evaluate the performance of spectral counting in determining quan...and it works surprisingly well above 2x!
In a very minor criticism of the methodology, they do the MS1 and MS/MS at 120k resolution and I think this might be just a little excessive at the MS1 level. (I think they did it so they could use baseline isotopic resolution for further analysis) and probably leads to sampling less proteoforms than what we normally see with "low-high" approaches.
This is definitely a proof-of-principle paper, though, and they definitely prove it works -- which I'll admit surprised me! I'm definitely interested to know how this compares to XIC-based proteoform quan.
As an aside, they process this all in the ProsightPD nodes in PD 2.1. Sounds like another set of 2nd party nodes are ready for primetime!
EDIT: Forgot the link to the paper! Here!