Improved HDX Workflow for On-line Digestion, Separation and Data Analysis — ASN Events
  1. Schedule
  2. Lunch & Poster Session One
  3. Improved HDX Workflow for On-line Digestion, Separation and Data Analysis

Improved HDX Workflow for On-line Digestion, Separation and Data Analysis (#114)

Asish Chakraborty 1 , Ying Qing Yu 1 , Michael Eggertson 1 , Keith Fadgen 1 , Weibin Chen 1 , Christopher Buck 2
  1. Waters Corporation, Milford, MA, USA
  2. Waters Australia, Burnside, VIC, Australia

Hydrogen Deuterium Exchange (HDX) experiments on proteins can reveal detailed information about protein tertiary structure (folding/packing) and quaternary structure (protein-protein interactions).  Several key components of a Hydrogen Deuterium Exchange Mass Spectrometry (HDX MS) workflow include reproducible sample preparation, on-line digestion, chilled chromatography, MS and dedicated informatics tools.  Each of these components have been improved in recent years and in this study we demonstrate optimizations in microscale chromatography that improve both pepsin digestion efficiency and the resolution of the reverse phase peptide separation.  The practical results are improved protein sequence coverage and more reproducible HDX experiments.

  1. Ahn J, Jung MC, Wyndham K, Yu YQ, Engen JR. Pepsin immobilized on high-strength hybrid particles for continuous flow online digestion at 10,000 psi. Anal Chem. 2012, 84(16), 7256-62
  2. Wu Y, Kaveti S, Engen JR. Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry. Anal Chem. 2006, 78(5), 1719-23.
  3. Wales TE, Fadgen KE, Gerhardt GC, Engen JR. High-speed and high-resolution UPLC separation at zero degrees Celsius. Anal Chem. 2008, 80(17), 6815-20.