MALDI mass spectrometry is beautiful and can have really impressive spatial resolution these days, but a single spectrum can only look at so many things at once. Even if you had an amazing ion capacity and dynamic range, once you divide that by thousands of tryptic peptides (and matrix) ions that are around you're not going to see much past the absolute highest intensity stuff.
I think the very best we ever saw from a single MALDI shot in Namandje's lab was 100 peptides(?) and I think reasonable FDR wouldn't have been so kind. That was also a very large sampling size with FTMS readout (high resolution and high capacity but low dynamic range - sort of averages out).
What if you could simplify your proteomic matrix so there was just less peptides hanging around? We've seen some interesting stuff recently for single cell loads where bigger peptides are better. Sounds like MALDI is something that could also benefit.
What about collagenases proteomics?
What?
Yes, collagenase. The stuff you use to rapidly extract DNA from tissue for quick genotype tests? Yup!
Unlike our friend trypsin that cuts at K and R and makes nice medium sized peptides, this protein is a lot pickier. It cuts at G-P-X domains - and while I'm very unclear on whether this would be helpful outside of regions where there is lots and lots of collagen - this study focused on the tricky proteomics of Extra Cellular Matrix ,or ECM (which appears to be lots and lots and lots of collagen).
Cool - so how on earth do you analyze peptides produced from this weird enzyme off of a MALDI spectrum? You can make a ridiculous number of guesses - or - you can do LCMS and use a lot of standard proteomic tools to understand the peptides and move backwards!
This study was a lot of work, btw.... LCMS is used to understand the peptide sequences including where and how they charge and their ion mobility - then the LCMS is used to inform the peptide picking from the MALDI.
End result? They analyze some patient FFPE tissues at 20um resolution and come back with hundreds of peptides identified by MALDI matching. When compared to trypsin collagenase helps them identify nearly 2x the peptides in the ECM and digesting directly off of tissue slices for LCMS is way more relevant than in solution digestion. There is a lot of biology here that they seem excited about that is outside of my wheelhouse, but there is some neat stuff here because the collagenase peptides often +1 charge in ESI and MALDI so they're straight-forward matches.
Ultimately, sometimes MALDI papers seems like pretty pictures and not a whole lot else, but this is not one of those. This looks like a really innovative way to get completely new insights from those FFPE blocks.
No comments:
Post a Comment