Hydrogen Exchange Mass Spectrometry

Abstract

Hydrogen exchange (HX) methods can reveal much about the structure, energetics, and dynamics of proteins. The addition of mass spectrometry (MS) to an earlier fragmentation-separation HX analysis now extends HX studies to larger proteins at high structural resolution and can provide information not available before. This chapter discusses experimental aspects of HX labeling, especially with respect to the use of MS and the analysis of MS data.

Keywords: Epitope mapping; HDX-MS; HX-MS; Hydrogen exchange; Hydrogen–deuterium exchange; Mass spectrometry; Protein dynamics; Protein folding; Protein stability; Protein structure.

Figure 1

Classes of HX experiments.

Figure 2

(A) Flow apparatus used for short labeling times as described by Coales et al. (2010). Exchange begins when the sample is mixed with D₂O buffer at mixing T1 pushed by pumps 1 and 2. After an exchange time, determined by the flow rate and the volume of the exchange loop, quench buffer is added. The sample flows through a pepsin column and onto a trap column. From the trap column, peptides are eluted to the MS by a gradient supplied by a separate pump. Not shown are valves that allow the reloading of the sample, D buffer, and quench loops, and that control flow through the trap column. (B) Our system using a BioLogic SFM400 stopped-flow mixer for labeling and a separate cooled chamber (Fig. 3) for proteolysis and chromatography. In the stopped-flow mixer, the speed of each syringe is separately controlled. For a simple exchange experiment, syringe 1 is not used. Unlabeled protein from syringe 2 is mixed with D₂O buffer from syringe 3. After an exchange time determined by the volume of delay line 2 and the speed of flow exchange is quenched by syringe 4. The quenched sample flows to the injection loop of our proteolysis/HPLC system. When used for refolding experiments, we start with unfolded protein in syringe 1. Folding is started with a dilution into refolding conditions at mixer 1. From here, the labeling pulse is as above.

Figure 3

Our online digestion/HPLC system. The quenched sample is pushed through the protease column by a cold flow of pH 2.3 buffer (0.1% formic acid, 0.05% TFA). The produced peptides are bound by the small trap column and buffer salts are washed away. After switching the second valve peptides are eluted and separated by an H₂O/acetonitrile gradient through a C18 analytical column. The output of the C18 column goes directly to the electrospray. Whole protein can be desalted for ESI by omitting the protease and analytical columns.

Figure 4

EX2 exchange. (A, B) Mass spectra of a peptide produced by pepsin digest (RVALTEDRLPRL) as a function of exchange time. The top spectra are for an unexchanged, all H, sample and the bottom are from a fully exchanged, all D, sample used to calibrate back exchange. Intermediate time points show the progress of exchange for (A) the protein free in solution and (B) the protein bound to an antibody. The increase in centroid above the all H sample uncorrected for back exchange is plotted in (C), corrected for back exchange in (D). In the absence of back exchange, we could expect nine deuterons for an all D sample. The measured centroid increase for the all D sample is 7 D’s indicating 78% recovery. It can be seen that antibody binding apparently slows exchange for at least six positions by a factor of 1000 or more.

Figure 5

Example data from a pulsed labeling experiment following the folding of maltose binding protein (MBP) showing bimodal isotopic distributions. Three peptides are shown. Residues 21–43 (A), 76–89 (B), and 346–370 (C). Folding time before the labeling pulse is indicated. In this case, starting with all D unfolded protein, the heavy fraction reflects protein molecules that were protected from exchange during a 43-ms, pH 9 pulse. Different parts of the protein acquire protecting structure at different times after folding starts showing the progressive formation of the native structure. The data shown in gray are from unfolded and native control experiments and show that the protected and unprotected fractions behave like fully folded and unfolded protein. The fits are with binomials. A concerted EX1 exchange mechanism will yield similar data. From Walters et al. (2013).

Figure 6

Toward residue resolution. (A) Bars represent the nearly 300 overlapping peptides obtained for staphylococcal nuclease. The staggered ends of overlapping peptides can in principle locate D occupancy to higher resolution than the peptide level. Given this set of peptides, the potential resolution is indicated by the dot and dashed line along the bottom. Small spots indicate residues that should be resolvable. Bars represent groups or two or more residues that always appear together and cannot be resolved. The HDsite algorithm can provide D occupancy information for each of these “switchable” residues but cannot assign D values to a particular residue in the group. (B–D) The basis for the HDsite algorithm. Simulated isotopic distributions for three different D distributions, all with the same centroid, yield different MS isotopic distributions. (B) All eight sites 50% D. (C) Four sites 100% D, four sites 0% D. (D) Four sites 10% D, four sites 90% D. Adapted from Kan et al. (2013).