Online, High-Pressure Digestion System for Protein Characterization by Hydrogen/Deuterium Exchange and Mass Spectrometry

摘要

The rapid and complete digestion of proteins is importantnwhen protein characterization by hydrogen-deuteriumnexchange (HDX) is coupled with mass spectrometry. Wendeveloped a single-pump, online, high-pressure digestionnsystem that relies on UPLC technology to aid in thendigestion of proteins. Two model proteins, amyloid u0001-peptiden1-42 (Au0001 1-42) and an HIV-1 capsid mutant proteinn(NBSA), were used to demonstrate the efficacy of the highpressurensystem. Both model proteins readily aggregatenand are difficult to digest under normal conditions. Ournhigh-pressure system successfully digests these proteinsninto small, overlapping peptides. The extra informationnafforded by overlapping peptides allows us to pinpointnHDX protection to protein segments smaller than thendigested peptide. The calculated average segment lengthn(ASL) for both model proteins decreased by 2-fold fornhigh-pressure digestion compared to digestion at ambientnpressure.