# Mass defection for massive multiplexing

The topic du jour around here is stable isotopes, and in particular the mass defect that stable isotopes exhibit. A quick summary:

• Isotopes of an element have atoms that differ only in their neutron count.
• The difference between the mass of an atom and its isotope is an apparent neutron mass for that atom. E.g. $m_{^{34}S} - m_{^{32}S}$ = 1.9958 Da, meaning each “extra” neutron in 34S has a mass of 1.9958/2 = 0.9979 Da.
• The mass defect is the difference between the apparent neutron mass of various isotopes or elements. In the last post we saw the “extra” neutron in 13C was 1.0034 Da, meaning the carbon-vs-sulfur mass defect is 1.0034 – 0.9979 = 0.0055 Da or 5.5 millidaltons.

The natural follow-up question is “OK, who cares?” Well, modern mass spectrometers can resolve these very tiny differences. And that ability can be put to some very interesting ends.

First, it’s worth noting that millidalton resolution by itself is very useful, whether or not the tiny mass differences stem from isotopic substitutions. Old instruments would not be able to resolve such molecules, but new ones can, and they are finding that interesting samples like meteorites or rock oils, are in fact a complex brew of thousands more molecules than scientists of yesteryear could have guessed.

But here our focus is on isotopic substitutions. One category of applications of high-resolution mass spectrometry assumes that isotopic differences do not matter in the world at large…until samples get introduced in the mass spectrometer. This means you can fiddle with the isotopic composition of your samples before analysis, and detect these artificial perturbations after analysis to study the chemistry that happened between “before” and “after”. The most common of these applications use isotopes as “labels” or “tags”, convenient ways to tell samples apart using only mass.

Tagged or labeled samples can be mixed because the tag will tell you which molecules came from which sample post facto. So if your experiment compares condition A to condition B, you can separately tag the A samples with one tag and the B samples with another. Then, the samples are mixed and analyzed as a single mixture. That (a) saves precious instrument time required for analysis by twofold and (b) reduces error in the determination of the relative abundance of your analytes in the two classes. But you don’t need to resolve different isotopic mass defects for comparing merely two conditions. But what if you want to resolve more conditions? Like say twelve?

Today’s peptide-tagging reagents enable just that, by relying on differences in the mass defect of carefully substituted and synthesized tag molecules. A recent paper provides a nice example. In a three-dalton mass window, the authors cram twelve different tags, as shown in the graphic above. (By the way, eight isotopologues of $\textrm{C}_7\textrm{H}_{16}\textrm{N}^+$ , not just the reported four, lie in a mass range between 118.1348 and 118.1499 Da, suggesting there might be room to move beyond 12-plex tagging.) Other similar labeling reagents are already commercalized.

The core assumption of all labeling studies is that the isotopic substitutions don’t affect the reactivity of the label. In cases like the proteomics tagging reagents, that is probably a very good assumption. Later, we’ll examine a different application of isotopic fine structure mass spectrometry, one where isotopic reactivity differences are not assumed to be negligible. Here’s a preview.