Online, high-pressure digestion system for protein characterization by hydrogen/deuterium exchange and mass spectrometry

Abstract

The rapid and complete digestion of proteins is important when protein characterization by hydrogen-deuterium exchange (HDX) is coupled with mass spectrometry. We developed a single-pump, online, high-pressure digestion system that relies on UPLC technology to aid in the digestion of proteins. Two model proteins, amyloid beta-peptide 1-42 (Abeta 1-42) and an HIV-1 capsid mutant protein (NBSA), were used to demonstrate the efficacy of the high-pressure system. Both model proteins readily aggregate and are difficult to digest under normal conditions. Our high-pressure system successfully digests these proteins into small, overlapping peptides. The extra information afforded by overlapping peptides allows us to pinpoint HDX protection to protein segments smaller than the digested peptide. The calculated average segment length (ASL) for both model proteins decreased by 2-fold for high-pressure digestion compared to digestion at ambient pressure.

Figure 1

Schematic of the on-line, high-pressure digestion system. The on-line digestion system has three modes, (a) injection, (b) digestion, and (c) detection. The blue line indicates the continuous flow through the system. The red lines indicate the flow that is blocked at position B4.

Figure 2

Segment length determination. (a) Means to determine the segment length: the observed peptides are shown with black lines; the dashed red lines show the delineation of the segments based on the amount of overlap of the peptides. The numbers show the lengths of each segment. Peptide IJ is considered a non-deducible segment. (b) Digestion coverage of Aβ1–42 with (red lines) and without (black lines) high pressure. The amino acid at the beginning and end of some peptides are highlighted to reduce ambiguity.

Figure 3

Coverage map of NBSA. Digestion coverage of NBSA with (red lines) and without (black lines) high pressure. The amino acids at the beginning and end of some peptides are highlighted.