Sunday, August 9, 2020

PTM-Shepherd -- and a lot of implications for our experiments!


Whew. Okay. That was a long 6 weeks for me. The International Organization for Standardization does not mess around. And if you want to develop some methods and get them to sign off on those should plan to do nothing else at all for a while. 

When I was last typing in this box, the PTM-Shepherd paper had just posted to BioRXIV and I really wanted to get back to it. 

A thing that I've rambled about for years is -- "WHAT ARE ALL THESE OTHER MS/MS SPECTRA?!?!?"  

One of the biggest behind-the-scenes developments for turbocharging proteomics occurred when the Orbitrap systems rolled out en masse and featured  "Monoisotopic Precursor Selection" also called "MIPS" or, later, "Peptide Match". You can use these functions without ever thinking about them, because they just work well, and help your instrument avoid things that obviously aren't peptides. And....when you end up fragmenting 100,000 things and you only identify 10% of them (probably more like 20-30% if your organism is really well characterized) it'll start to drive you crazy. 

The first Open Searching tools started pointing us toward what these things are. And PTM-Shepherd is a new tool that helps push that understanding even further. 

PTM-Shepherd is used in this study on several different datasets from different instruments and experimental designs. There are some really interesting mass shifts in nearly all the reanalyses, but the CPTAC-3 control set (that's the new Lumos TMT-11plex work) might be the one that is the most revealing about what we don't know about the chemical behavior of our most beloved workflows and protocols. 

Like....I dunno...what the hell is a sodium adduct doing on my TMT tagged peptide?!?!? be honest, this is kind of funny from a small molecule perspective. Sodium adducts are so common in metabolomics that Compound Discoverer doesn't even show you when that is the actual ion that you've isolated for your metabolite (your metabolite + Na). Of course there are sodium adducts in proteomics, right? But who the heck is searching for them? Are you putting it in as one of your 7 possible modifications in SeQuest? No you aren't, because if you did, you'd have to subtract off one of the +H....and...if you've got that button, that's super cool, cause I sure don't. 

Even cooler? It's great to see these things pop up with PTM-Shepherd because it's easy to start to think the lack of sunlight in Madison has been getting to everyone up there, since MetaMorpheus has been replacing +H with +Na (and similar) as a possibility in the searches, but now we see some nice charts from a separate group showing that these things are happening! 

There is a big difference between finding PTMs and localizing PTMs, and PTM-Sheperd has a simple and elegant way of doing both. 

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