Thursday, September 5, 2019

Determination of Proteolytic Proteoforms with HUNTER!


I really truly try to read at least one paper in its entirety each day. It's a rule that I started when I worked for the great Michal Fried and I thought it was the only way I'd ever be able to have a chance of having any context for how to help apply what I know how to do to the brilliant medical stuff she does. A really bad day for me is when I don't get to even a single one.

A great day is when I start something like this brilliant new MCP study and I am learning from the very first sentence!


What?

Wait. So....did you know this? Should I blame this on all-to-frequent head impacts?  Look, I know about caspases. I know that they're amazing things to ignore when we're doing proteomics (that your quantitative difference might actually be that one set of cells has decided to go into it's own death cycle and is degrading it's own proteins) but is there truly something that we should just be considering in all systems that is as broad-stroke as N-terminal degradation?    My ignorance in biology aside -- that we could be deriving important context from how one side of a protein is systematically degraded -- how on earth would you quantitatively measure something like this?

TAILS would be my first thought. This technique is covered in these past posts (1, 2, 3)

HUNTER is detailed here and seems like TAILS went all Super Saiyan....wait...[Google]


....okay...of course that is a thing. YouTube video here....

I'd like to point out here that TAILS is a tough experiment from the sample prep side. HUNTER looks crazy hard. As someone who IS NOT good at sample prep, this looks like something I'd only try once I found someone really talented at doing it (or programming a sample handling robot) to do it.

Fortunately......


The authors walk you through how to do it manually as well -- but here are step by step instructions (sorry) on how to set up a robot for an impossible sample prep design!

As further proof of 1) this technique totally works and 2) it can be applied to various biological systems and 3) it produces useful biological data from all of them -- they apply this method to a variety of human systems and to plants.

By selectively labeling and enriching for N-terminal peptides, they demonstrate the recovery, identification and quantification of >1,000 N-terminals even when they start with micrograms of material.....

The LCMS work is demonstrated on both a Q Exactive HF and a Bruker Impact II, showing that this technique with all of it's power and apparent biological significance can be applied in any proteomics lab. Do I fully get why you'd want to do it from a biology level? Nope! But I know an awful lot of biological models out there where the -omics hasn't solved the phenotype...and here is a fully mature technique provided in excruciating detail that might be the way to the answer.

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