Improved high-throughput hydrogen-deuterium exchange-mass spectrometry system and method

A method of characterizing the binding of a ligand to a protein in a sample of live cells, comprising the steps of:

• providing a sample of live cells;
• treating the sample of live cells with a ligand and incubating the treated sample of live cells to allow the ligand to bind to a target protein, thereby forming a ligand-target protein complex;
• subjecting the treated sample of live cells to a lysing step to from a cell lysate;
• heating the cell lysate to a predetermined temperature, thereby forming a heated mixture, wherein the predetermined temperature is the maximum temperature to which the ligand-target protein complex in the heated mixture can be exposed without causing denaturation of the target protein thereof;
• reducing the temperature of the heated mixture to about room temperature or below;
• treating the heated mixture to remove denatured proteins therefrom, forming a depleted protein mixture;
• subjecting the depleted protein mixture to hydrogen-deuterium exchange followed by a protein digestion step to form a peptide mixture;
• subjecting the peptide mixture to liquid chromatography forming eluted peptides;
• and
• using mass spectrometry to analyze the eluted peptides.