Tuesday, April 3, 2018

A pull-down top-down KRAS isoform assay!!

KRAS is a little protein that is a big deal. As one of the most commonly mutated genes in cancer -- and one of the very worst -- loads of people (including a lot of friends of mine) are working on assays to figure out things like:

How much is there?
What mutant variants are there?
What is the ratio of normal to mutant?

With a goal being -- rapid -- sensitive -- and hopefully clinically adaptable!

Most of these assays are digestion shotgun based. Should/could we flip the paradigm and do TOOOOP  DOOOOWN PROOOOOTEEEEEOOOOMIIIICS!?!? Picture any author of this great new paper yelling that out the window of a car as they drive by you. It's much more fun that way.

There are some very good reasons for looking at these proteoforms from the intact protein level. Seriously. Again, it is a small protein. We're probably looking for a single amino acid substitution in a small protein. When we digest that protein and use standard global approaches identification is complicated by a couple of things. The wild type form is often still present AND there can be large differences in abundance between the WT and variant(s) forms.

From the intact protein level we can get 1) the intact mass shift (super important here) and verification from the MS/MS mass shifts. This is 2 points of evidence of the variant -- compared to one single peptide.

Having spent some time looking at this in global data I can tell you that the data processing shortcuts we use in virtually all proteomics software are NOT friendly to these proteins. Protein grouping and
strict parsimony. Remember those? These are trying to make your data report as simple and accurate as possible by making assumptions. Have you ever fed 2 virtually identical FASTA isoforms in and looked at data where both protein/peptide isoforms are present?

You'll get a surprising read-out; if the variant peptide is identified with confidence in run #1 it will appear that only the variant form is detected.

In run #2 if that peptide scores, for example, medium confidence (below your filter cutoff -- remember these are looow abundance proteins) the next run scores that only the WT is present. Protein grouping, strict parsimony, and the use of razor peptides confound your results.

P.S. Quick reminder: If you are still using Proteome Discoverer 1.4 or earlier -- please keep in mind that if you have equivalent information for 2 possible FASTA entries -- PD will always give you the smallest entry (because it is the highest % protein coverage -- this is why you see so many more shortened isoforms of proteins in global data than people using other software. In PD 2.0 or later, the longest peptide gets the nod.

You can avoid all this with top-down!

This team uses a pan-RAS IP (pulls down KRAS, NRAS, HRAS....

....um...thought I'd pull some lyrics from this album to extend the joke....nope. That parental advisory sticker is there for very good reason....yikes....moving on!)

On top of the peptide sequence variation, the terminus of KRAS can be modified in a number of ways --non-mass spec friendly ways.

Now I assume I've convinced you that we'd be better of studying KRAS isoforms/proteoforms with top-down rather than bottom up. But -- will this work in real samples?

This team optimizes that assay for cell lines in culture -- and then gets characterized material from CPTAC -- and shows it works there! I'm a little unclear on the amount of material it took from the fixed CPTAC tissue to characterize it, but I'm assuming we're looking at the normal amount -- what will fit on a slide and show they can determine -- on a precision medicine lab budget friendly Q Exactive, the KRAS isoforms present. As the title suggests -- this wasn't entirely their aim, I guess, they also reveal more basic info about KRAS mutant biology, but that is beyond me. I'm just psyched to have a simple straight-forward KRAS top down isoform characterization assay all ready to go!

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