Journal Article

Fast Reversed-Phase Liquid Chromatography to Reduce Back Exchange and Increase Throughput in H/D Exchange Monitored by FT-ICR Mass Spectrometry

Bou-Assaf, George M., Marshall, Alan G., Zhang, Hui-Min, Emmett, Mark R.

2009

Overview

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 2O 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. A 2-min sham digestion indicates no backbone back exchange during digestion for fully exchanged LHRH. Rapid chromatography with a ProZap™ C 18 column reduces back exchange by about one third compared to a Jupiter™ C 5 column.

• Publisher: Elsevier Inc,Springer-Verlag,Elsevier,Springer Nature B.V
• Subject: Analytical Chemistry / Analytical, structural and metabolic biochemistry / Animals / Application Note / Backbone / Bioinformatics / Biological and medical sciences / Biotechnology / Chemistry / Chemistry and Materials Science / Chromatography / Chromatography, Liquid - methods / Desalination / Deuterium / Deuterium - chemistry / Deuterium Exchange Measurement - methods / Exchanging / Fragments / Fundamental and applied biological sciences. Psychology / General aspects, investigation methods / Gonadotropin-Releasing Hormone - chemistry / High-performance liquid chromatography / Horses / Hydrogen - chemistry / Hydrogen-Ion Concentration / Ions / Mass spectrometry / Mass Spectrometry - methods / Myoglobin - chemistry / Organic Chemistry / Phase separation / Proteins / Proteins - chemistry / Proteomics / Quenching / Reproducibility / Temperature