Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry

Abstract

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H(2)O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.

Figure 1

Elution profiles for myoglobin fragment peptides through an SFC Waters HILIC column (top), HPLC ProZap™ C₁₈ column (middle) and HPLC Jupiter™ C₅ column (bottom), with respective retention times of ∼0.5-1.2 min, ∼0.7-1.5 min, and ∼4.0-4.5 min.

Figure 2

Deuterium uptake for myoglobin peptides after exchange for 240 s and 900 s and separation with the Jupiter™ C₅ and ProZap™ C₁₈ columns with 1.5 min short gradient and SFC Waters HILIC columns.

Figure 3

LHRH isotopic distribution and back exchange under various conditions. A: LHRH natural abundance isotopic distribution from blank control (no deuterium exposure); B: fully exchanged LHRH (direct infusion after 56 hour HDX); C: fully exchanged LHRH after sham digestion procedure (direct infusion after 2 min incubation in quench buffer); D: fully exchanged LHRH after ProZap™ C₁₈ LC separation; E: fully exchanged LHRH after Jupiter™ C₅ LC separation.