Figure 2: A schematic representation of the workflow for a continuous labeling H/DX–MS experiment (arrows indicate the order in which each step in an H/DX-MS experiment is carried out). In the initial step, a protein solution is incubated at ambient temperature in its formulated buffer. The protein is then diluted ~20 fold with deuterated formulation buffer. The protein is then incubated and at predetermined time points, a sample is taken from the reaction and quenched by dropping the pH to ~2.5-2.6 and the temperature to 0 °C. This quenched protein is then digested with an acidic protease (pepsin), and the resulting peptides are separated at 0 °C under acidic conditions chromatographically before they are introduced into the mass spectrometer. The mass of each peptide is determined for each deuterium time point, and the deuterium incorporation is plotted versus time. If the structure of the protein is known, it can be plotted onto this structure, otherwise different data analysis and display formats can be used to interpret the H/DX–MS data.

The insertion of a digestion step just after quenching (17), but prior to LC–MS (see Figure 2) cuts the protein into smaller pieces (peptides). During the digestion step, quench conditions must be maintained to preserve the deuterium label; therefore an acid protease (usually pepsin) is used for proteolytic digestion. After digestion, the peptic peptides are separated by reversed phase LC, and the mass of each peptide is measured by the mass spectrometer. Both digestion and chromatographic separation steps are carried out on–line. The reversed phase chromatography step not only functions to separate peptides, it also serves the important function of removing buffer salts and other excipients not compatible with MS.

Following the chromatographic separation, the peptides are analyzed by mass spectrometry, and the amount and location of the deuterium are determined (electrospray is most commonly used, although MALDI has also been used (21)). Typically, the protein and amino-acid sequence are known, and the resulting pepsin peptides are unambiguously identified by tandem MS. The solvent used in the digestion and chromatography steps are pure (100%) H² O. As a result, deuterium that was incorporated in the protein starts to exchange back to hydrogen during these steps. To minimize this, and to supply enough time for analysis, the temperature of the quench, digestion, and separation steps is dropped, and the chromatography is done as quickly as possible under acidic pH conditions. At 0 °C and pH 2.5–2.6, in a properly controlled experiment, only about 20%–30% of the deuterium is lost during analysis. If desired, control experiments can be performed to correct for this loss (17).