Subzero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of deuterium label (back-exchange) during the course of the analysis. A method to limit loss of label during the separation stage of the analysis using sub-zero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as −30°C with negligible loss of deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of HDX MS in terms of mixture complexity, and the magnitude with which changes in deuteration level can be quantified.

Isotope-Coded Labeling for Accelerated Protein Interaction Profiling Using MS

“…No major differences in the extent of labeling could be discerned between the three aforementioned lysines, suggesting that all three Flag repeats are similarly blocked by the M2 anti-Flag mAb. This result is in agreement with data obtained on the complex using hydrogen deuterium exchange mass spectrometry 50 . The disappearance of the difference signal at 300s is consistent with competitive elution of FLAG tagged proteins with FLAG peptide in protein purification applications and reported off-rates 51,52 .…”

Measuring the Hydrogen/Deuterium Exchange of Proteins at High Spatial Resolution by Mass Spectrometry: Overcoming Gas-Phase Hydrogen/Deuterium Scrambling

“…This concept of supercooling in mixed aqueous/ organic solvents to reduce back-exchange (i.e., unintended loss of deuterium) was later used in top-down and bottom-up HDX-MS applications. 15,16 On the basis of an earlier systematic study by Bai et al, 17 the intrinsic exchange rates for amides in unstructured peptides can be predicted. Interestingly, we found that while the slow exchange of amides in the C-terminal half was in good accordance with expected primary structure effects, the amides of the His-containing N-terminal half exchanged considerably faster than predicted under the conditions used for direct infusion.…”

Protein charge ladders reveal that the net charge of ALS‐linked superoxide dismutase can be different in sign and magnitude from predicted values

“…6). Mass spectrometry allowed us to simultaneously measure the rate of amide H/D exchange of each rung under identical solution conditions and abolished the artifactual variations in measured rates that can occur in parallel experiments (because of variations in D/H backexchange 51 ).…”