Figure 1.

Schematic depicting typical real versus false EX1 signatures in HX MS. Real EX1 (a) involves two populations: a lower mass species (peak 1, red) that has not yet unfolded to become deuterated and a higher mass species (peak 2, green) that has undergone cooperative unfolding and has become deuterated. In False EX1 (b), there are two distributions, but the lower mass distribution (1) is the result of some phenomena other than EX1 kinetic unfolding, including aggregation, abnormal backexchange or sample carryover. If part of the protein population exists as an aggregate, that part of the population may not become deuterated (peak 1, red) while the rest of the population will (peak 2, blue). Abnormal total-backexchange [13] can result when all the deuterium from a portion of the population (peak 1, red) is backexchanged during analysis. False EX1 as a result of carryover occurs when some of the sample does not elute from the LC column, but elutes later, typically with the solvent gradient for the next injected sample. Because the protein from the first injection (indicated here with a *) has been backexchanging longer than that from the second injection (indicated with a ▲), an artifactual lower mass distribution (peak 1, red) appears. Note that * and ▲ represent the same protein/peptides but are indicated here with different symbols to distinguish them in injection 2. This figure is meant to be illustrative and does not address all the types of intensities, mass differences and peaks shapes that are possible.