Analytical Aspects of Hydrogen Exchange Mass Spectrometry

Abstract

This article reviews the analytical aspects of measuring hydrogen exchange by mass spectrometry (HX MS). We describe the nature of analytical selectivity in hydrogen exchange, then review the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in HX MS depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that can be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics.

Keywords: biopharmaceutical; deuterium; fragment separation method; protein conformation; protein dynamics.

Figure 1

Hydrogen exchange is spontaneous. In this diagram, hydrogen or deuterium at backbone amide positions are represented by blue or red balls, respectively. When a protein with all hydrogen is placed in a solvent with a different isotope (here at left, D₂O), exchange occurs spontaneously. If a partially deuterated protein were to suddenly find itself in a pure H₂O solvent, the deuterium in the protein exchanges back to the solution (right). Options for measuring the amount of labeling include 1) measure deuteration before the protein is placed in pure H₂O, or (2) slow the labeling (with quench conditions) to limit-the back-exchange in H₂O and allow time to make the measurement.

Figure 2

Improving HX MS chromatographic separations at 0 °C. Panels (a–c) are taken from Ref. (132), with permission, and correspond to separation of a peptic digestion of a 52 kD protein with 20, 3.5, and 1.7 μm diameter particles, respectively. Panels (d–g) are separations of peptic digestion of monoclonal antibody (IgG) using a 1×50 mm HSS T3 1.8 μm column and a 5–35% water:acetonitrile gradient with changes to gradient time, flow rate, and backpressure: (d) 12 minute separation, 65 μL/min., backpressure 8000 psi; (e) 12 minute separation, 100 μL/min., backpressure 12000 psi; (f) 6 minute separation, 100 μL/min., backpressure 12000 psi, with addition of ion mobility separation. Separation with >2 μm particles (black traces, panels a,b) is inferior to separations with sub-2 μm particles (brown traces, panels c,d,e). Separation with both chromatography and ion mobility (purple trace, panel f) greatly enhances peak capacity. (g) Ion mobility separations require milliseconds and fit nicely between the time scale of liquid chromatography (minutes) and that of time-of-flight MS detection (microseconds).

Figure 3

Variables in HX MS. Each step of the experiment from earliest (top) to latest (bottom) is in a different colored box with the variables associated with that step indicated to the right.

Figure 4

Quantitation of deuterium by mass change. (a). An undeuterated spectrum (black) is compared to deuterated spectra where the increase in mass may be small (grey spectrum), intermediate (green spectrum) or large (red spectrum). Improper control of experimental conditions could produce an isotope cluster with an average m/z higher or lower than would be found with proper control of conditions, in which case changes could be falsely attributed to changes in the protein that were in fact due to undesirable changes in experimental conditions. (b) in EX1 kinetics, there can be a broadened isotope cluster which may resolve to two distributions (blue spectra): a higher-mass envelope representing a more unfolded/unprotected species and a lower-mass envelope representing a more folded/protected species. Quantitation in EX1 kinetics can be done by finding the centroid of each distribution (gray or orange bars) or the centroid of the entire distribution (purple bar).

Figure 5

Loss of deuterium incorporated at the penultimate backbone amide hydrogen position of peptides, as calculated using (73). All 400 combinations of the twenty common amino acids at the N-terminus (along the top of each panel) and penultimate position (vertically along the side) are shown. Deuterium loss after 1 min, 3 min or 10 min at pH 2.5 and 0 °C is colored using the gradient scale indicated. Note that proline has no backbone amide hydrogen (colored light grey).

Figure 6

Examples of how changes to various experimental parameters affect the deuterium level of peptic peptides. Three random sequences were chosen (blue, MYSLCEQTVNFK; red, QCSVFMTNYEKL; green, IHGASDFWVWER) and the deuterium level after forward exchange, 100% H to D (a, b) or back-exchange, 100% D to H, (c, d) was calculated using (73) for various conditions. (a) labeling at 25 °C for 1 second at variable pH (x-axis). (b) Same as panel a but labeling for 2 seconds. (c) Back-exchange for 5 minutes at 0 °C with variable quench pH. (d) Back-exchange for 5 minutes at pH 2.5 with variable quench temperature. Differences in deuterium levels at specific conditions are highlighted with numbers in colored boxes.

Figure 7

The five main types of HX MS experimental replication. (a) The major steps of the HX MS experiment are shown down the central spine with colored boxes. Replication can be one of five types, shown with the proposed nomenclature (colored lines and text). Some experiments (e.g., binding, pulse-labeling) require a(n) additional step(s) (manipulation, blue box and shading) just before labeling in which sample volumes, concentrations and other conditions are manipulated; simple, one-protein HX MS generally does not have a manipulation step. The final relative error for each type of replicate, as determined by the spread of the replicate data points during final data interpretation, is shown at the left (+++++ is most error, + is least error). (b) Examples of two types of replication experiments. In the analysis quadruplicate, a single sample of protein is labeled and quenched, and the quenched material is divided into four separate tubes for four independent LC/MS analyses and processing. In the other example, protein is overexpressed/isolated three independent times and then divided into three separate aliquots per biological preparation for independent labeling, analysis and processing. A total of nine replicates comprises this final data set.