Wednesday, May 20, 2020

Phosphoproteomics of SARS-CoV-2 infection suggests new treatment options!!

I'm guessing you've probably seen it, but if you haven't, you should stop and read this.

As a field, we've been messing around with phosphopeptide enrichments at least 12? 15 years? And -- yeah -- it's cool that we can pull down phosphorylated peptides and identify/quantify a bunch of them, and put them into big databases. Sometimes, though, it feels like we're just going through the research motions.

This is an actual real-life application of phosphoproteomics that shows -- it's actually useful. Not to put down anybody else or the thousands of publicly deposited studies, but this group just used our very standard research experiments to find new ways that we could potentially inhibit the replication of a virus that is fully confirmed to have killed 1,425 people in my country yesterday.

HOLY COW. Wait. Can proteomics be helpful to the world? Is this one of the tiny silver linings in this global catastrophe?

This blog is supposed to be more on the technical levels of how to do this stuff. Or just the ramblings of some guy who reads too many scientific papers while experimenting with the LD50 of espresso.

How'd they do it?
SP3 based digestion
TMT10-plex labeling of 125ug of peptides per channel (to 1.25mg total peptide)
250ug of the total peptide amount was fractionated (high pH reversed phase with a big (4.6mm Waters Xbridge column -- [I think the same one that Jesper Olssen's lab uses for the high resolution fractionations, but don't quote me -- I always use the 2.1 mm one, but I may need to scale up, both group's data might be better than mine...but both have better instruments....worth investigating....]) into 96 fractions over 70 minutes
The 96 fractions were concatenated to 24 for LCMS

The remaining 1mg of peptide was enriched with Fe-NTA and then fractionated differently. They used C-18 stage tips, fractionated to 8 and then concatenated to 4.

Two different LCMS methods were used on the Fusion Lumos --- SPS MS3 for the whole proteomics (Ion Trap on Turbo, MS3 at 50k resolution)

MS2 based quan at 50k resolution was used for the 4 phosphoproteomics files.

At first I was a little thrown, but the real reason that SPS MS3 is useful is coislation interference and ratio suppression. When you do a relatively simple mixture, I get a lot less improvement from MS3 over MS2.

Also -- it's phosphoproteomics. Do biological systems function with a 1.8-fold upregulatation of a kinase changing anything? I dunno, but it sounds unlikely. When I see a real phosphorylation event it's typically 5-fold minimum. A lot of the time it's a huge difference.

PD 2.4 for data processing. I'm a little lost on how the data was combined with ZScores, but Siri is telling me that I have work to do, so I'm gonna wrap this up.

So.....the phosphoproteomics points them to some things that they could inhibit to reduce the viral replication -- so....they




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