Hydrogen-deuterium exchange mass spectrometry (HDX-MS) with my grad school Ago work as an example

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is kinda like giving wetsuit-wearing proteins a bath and seeing where they get wet. You bathe them in heavy water and exposed unstructured regions of the protein get heavier, whereas the structured regions are hidden under the wetsuit so they stay dry. First an overview then some more detail. So here’s the gist...
blog form: bit.ly/hdxmassspec  (updated from grad school)
Proteins are long, folded-up chains of letters called amino acids, which are themselves made up of atoms (individual units of carbon, oxygen, hydrogen, etc.). There are 20 (common, genetically-encoded) amino acids and they have different properties (size, charge, etc.). Different proteins have different combinations of amino acids, so, in order to satisfy these amino acids’ desires, they fold and act differently from one another. Ideal folding usually involves grouping together water-excluded (hydrophobic) regions in the center of the protein, away from water (although water really runs throughout the protein through channels so they can never really escape!), and letting water-loving (hydrophilic) regions stay on the outskirts where they can hang out with the watery solvent (liquid they’re dissolved in). For reasons I’ll get into more later, hydrogens tend to come and go a lot easier than other atoms. Thus, when molecules hang out with water, if they’re not too tied up interacting with other things, they can sometimes swap hydrogens with the water. And if that water is “labeled” this can label “unprotected” regions of the protein.  
 
To understand how, we need to look even smaller than amino acids, and even smaller than the units those amino acids are made up of - atoms. We need to go subatomic!  
 
Atoms are made up of smaller parts called subatomic particles: protons (+ charged), electrons (- charged), & neutrons. Different elements are defined by how many protons they have (e.g. carbon always has 6, oxygen always has 8, and hydrogen always has 1), but the number of electrons & neutrons is more flexible and we call versions of an element with different numbers of neutrons nuclear isotopes. Deuterium (D) is a version of hydrogen (a hydrogen isotope) which has 1 more neutron than “normal hydrogen” so it’s heavier → when you bathe your protein in D₂O the protein can swap out H for D and this makes the protein heavier. If you then cut the protein up into pieces and weigh those pieces individually you can see where swapping occurred and didn’t occur, telling you about how accessible &/or structured those regions are. It’s often used to see if regions become more or less swappable under different conditions or after adding a binding partner.
If H is held tightly, it won’t get swapped, so it will stay “light.” But if H is in a solvent accessible region (in contact with the liquid its dissolved in) and it’s not tied up with bonds to other things, it will get swapped out with the heavier version, deuterium. So when you then cut the protein up into pieces, the piece that was accessible will be heavier.
The “weighing” is done by mass spectrometry or “mass spec.” It’s a technique that can be used to identify proteins and identify modifications to proteins - all based on how heavy and charged they (or at least parts of them) are. Not going into the technical stuff, the principal is that you use endoproteases (protein scissors) to cut up proteins into little pieces, then you charge those pieces - turn them into ions using electrospray - measure the weight of those pieces and, because different protein letters weigh different amounts, you can figure out what letters are in each piece and then match that up to the letters in protein sequences in a big database.
It’s kinda like “reverse-redacting” - you know the full text and you’re trying to see what parts of that text are covered (and covered in the sense that those letters were detected - not blacked out :P). Mass-spec results come out as a series of peaks on an m/z graph, where m is mass (heaviness) and z is charge. Increased accessibility leads to increased deuteration leads to increased mass leads to rightward shift. Decreased accessibility leads to decreased deuteration leads to decreased mass leads to leftward shift.
Different protein letters have different masses because they’re made up of different combinations of elements. All protein letters have a generic backbone (although proline’s is slightly different since it’ side chain kinda curves back to hog the N). But they have different side chains, which have different numbers and arrangements of atoms of carbon, oxygen, nitrogen, and/or sulfur. One thing they all have - hydrogen.
Hydrogen’s often “ignored” - sometimes it’s not even drawn in, its presence is just implied. Because it’s not very reactive. And speaking of activeness - hydrogen has heavy form that are NOT radioactive.
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