Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment

Abstract

The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2%, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90% ± 5%.

Fig 1

Dependence of HX rates on pH and ionic strength at 0 °C. A. Expected pH dependence for the hypothetical peptide GGVALISTDENQRHKCMFTW. The rate at any pH is the sum of the H₃O⁺ ion catalyzed reaction (red) and the OH⁻ ion catalyzed reaction (blue), each of which varies by 10-fold per pH unit. The additional pH-independent contribution due to water catalysis is shown with hatch marks. The averaged fragment-level HX rate constant shown is taken as the geometric mean (log averaged) of the 20 amides, each of which exchange with somewhat different rate constants. B. Cumulative population distribution pH series. C. Slices taken across B at given population percentiles. D. Effect of ionic strength at pH 2.5.

Fig 2

The dependence of D-label recovery on transfer tube temperature.

Fig 3

Exchange on the column. A. Observed D-recovery for all peptides across the delay time series. The inset places the variable delay time during sample preparation. B. Recovery for the various peptides after a 20 minute delay on the trap column. C – E. Observed (data points) and theoretical (dashed lines) D-label recovery. C. Some peptides with normal recovery. D. A peptide with large slowing on the column due to structure formation and two component peptides with normal recovery. E. Some histidine-containing peptides with accelerated early loss.

Fig 4

Minimizing preparation time by increasing flow rates (termed the fast condition, see text) increases D-recovery from the green to the colored distributions, which also show the effect of sharper elution gradients. Fig 4 Change in sample exposure time. Sharper elution gradients provide little reduction in back exchange and sacrifice peptide fragment yield. Minimizing preparation time by increasing flow rates (termed the fast flow condition, see text) increased recovery levels from the green to the colored distributions.

Fig. 5. Summary of gains in D-label recovery

Arrows show the improvement made by manipulating sample ionic strength at pH 2.5 (blue to green) and increasing system flow rates during the prep/wash (green to red) and elution (red to black).