Elucidating Protein–Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen–Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion

Hydrogen–deuterium exchange coupled with mass
spectrometry
(HDX-MS) is a powerful technique for the characterization of protein–ligand
interactions. Currently, there is a growing need for breakthroughs
in the application of HDX-MS analysis to protein–ligand interactions
in highly complex biological samples such as cell lysates. However,
HDX-MS analysis in such systems suffers from extreme spectral complexity
as a result of high sample complexity and limited LC separation power
due to the traditional use of short LC gradients. Here, we introduced
protein thermal depletion (PTD) to reduce protein complexity in E. coli cell lysate for our subzero-temperature long gradient
UPLC-HDX-MS platform (PTD-HDX-MS) to facilitate high-throughput analysis
of protein–ligand interactions in cell lysates. We spiked bovine
carbonic anhydrase II (CaII) and its inhibitor acetazolamide (AZM)
into E. coli cell lysate as a model system in our
study. We demonstrated that PTD at 60 °C greatly reduces protein
complexity in cell lysates, while the AZM-targeted CaII complex remains
in solution due to improved thermal stability upon binding. Using
both PTD to reduce sample complexity and subzero-temperature long
gradient UPLC to boost LC separation power, we successfully elucidated
the interaction sites between AZM and CaII in E. coli cell lysate from the high-throughput HDX-MS analysis of thousands
of deuterated peptides from hundreds of proteins. Our results highlight
the great promise of the PTD-HDX-MS platform for the identification
of ligand targets and characterization of protein–ligand interactions
in highly complex biological samples such as cell lysates.