Tuesday, December 11, 2018

Nonhuman proteins in human milk?!?!??

(Image is from @Confused_Cow, a very strange Twitter account I am now definitely following)

What I actually want to put here is this confusing new JPR paper....

Yeah....okay...so....ignoring the biological implications that sound like this would be completely and totally biologically impossible (all the proteins we eat get chopped up by that stomach acid stuff, right?), let's look at the amount of work that went into this ---

6 humans donated milk.
This milk was separated into 6 fractions by SDS-PAGE
These SDS-PAGE fractions were analyzed using a passive split flow Agilent nanoLC system in what looks like around 45 minute gradients on a Q Exactive Plus system
MS1 resolution of 35,000
Top10 experiment
MS/MS resolution of 17,500 with a 120ms fill time.

The data processing was done this way --

--I've highlighted the things that I find interesting here, and that I would have done differently.

Wooooooo.....okay.... In one sentence -- This is a really big search matrix searched against masses obtained at medium level resolution, using search tolerances that are strikingly high and searched against a software package version that has been lampooned for it's inability to accurately quantify FDR when matrices get really complex. Yo. If you're Q Exactive Plus is off by 48ppm, get a fire extinguisher. No QE+ that isn't currently on fire or under water is off by that much.

EDIT/Clarification: Proteome Discoverer 1.4 only controls FDR at the PSM level. All later versions of PD were designed to control FDR at multiple levels in order to more accurately control data quality with extremely large numbers of spectra or with high complexity search space.

Sorry, but this seems like an invitation to a list of proteins that are gonna be bad.


If you do your searching of your global and then validate the results with Parallel Reaction Monitoring (PRM) -- and it works -- maybe I should shut up.

Particularly, I guess, if you make heavy isotopic standards as well for your PRMs and this stuff looks legit. And if you go to the supplementary info -- it looks on point --

-I should probably definitely shut up.

In the end -- I'm still confused. WHY ARE THERE DOG PEPTIDES IN HUMAN MILK?!?!?

But the work looks solid.....

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