The general protocol for a PLIMSTEX experiment (Fig. 11.1) starts with equilibration of the protein of interest with different concentrations of ligand in aqueous buffer solutions. D2O containing the same concentrations of buffer and salts as in the starting solution is then added to initiate H/D exchange. The protocol utilizes a high D/H ratio in the forward and a high H/D ratio in the back exchange, and carries the added advantage of in situ desalting. When the system reaches a near steady state (1-3 h of exchange) where the fast exchangeable hydrogens reach steady state while the slow exchangers have not (as determined by a kinetic study conducted previously), the exchange is quenched by decreasing the pH to @2.5. The solution is then loaded on a small C18 column (or C4 column for large
protein), cooled to 0 °C, and the labile, non-amide sites of the immobilized protein are back-exchanged to the H form. The solution is desalted by washing with ice-cold, aqueous formic acid (pH @ 2.5). The protein, which now bears an isoto-pic exchange ''signature'' in its amide linkages reflecting its state at the time of exchange, is introduced into a mass spectrometer, and its molecular weight is determined. Rapid elution (by an isocratic flow of solvent at 30-35 mL min-1 with high organic composition or with a fast pH 2.5 gradient) delivers the protein to an electrospray ionization (ESI) source. Although the initial studies used ESI (ion trap or quadrupole/time-of-flight analyzers) in the positive-ion mode, matrix-assisted laser desorption ionization (MALDI)-MS may also be an appropriate method.
Was this article helpful?