Wait. Where does thermolysin cut? Does everyone know that and that's why it isn't listed in the methods section of the paper? It's so common knowledge that the Wikipedia page doesn't list them explicitly either? Gemini, which is apparently now installed in my browser without my permission says it's Leu, Phe, Ile, Val, Ala, and Met, so I'll bet you that is NOT where it cuts. However, this is really cool, for real.
I love fast cheap enzymes for proteomics! Let's go. However, the reason this paper is great is because they went the extra mile and developed a stable isotope labeled protein standard for this enzyme!
You chuck in the protein they set up and then you throw in your thermolysin and do your fast digestion and now you've got a pile of internal heavy labeled peptide standards. If you don't see them...the digestion didn't work! If you do see them, you can use them for your quantification! This group does a pile of targeted PRMs on an Orbitrap Exploris with an EvoSep on it. So high resolution discriminatory power of the mass spec and reproducible run times with no tinkering allowed.
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