Enhancing the quality of H/D exchange measurements with mass spectrometry detection in disulfide-rich proteins using electron capture dissociation

Abstract

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a potent technique to probe higher-order structures, dynamics, and interactions of proteins. While the range of proteins amenable to interrogation by HDX MS continues to expand at an accelerating pace, there are still a few classes of proteins whose analysis with this technique remains challenging. Disulfide-rich proteins constitute one of such groups: since the reduction of thiol-thiol bonds must be carried out under suboptimal conditions (to minimize the back-exchange), it frequently results in incomplete dissociation of disulfide bridges prior to MS analysis, leading to a loss of signal, inadequate sequence coverage, and a dramatic increase in the difficulty of data analysis. In this work, the dissociation of disulfide-linked peptide dimers produced by peptic digestion of the 80 kDa glycoprotein transferrin in the course of HDX MS experiments is carried out using electron capture dissociation (ECD). ECD results in efficient cleavage of the thiol-thiol bonds in the gas phase on the fast LC time scale and allows the deuterium content of the monomeric constituents of the peptide dimers to be measured individually. The measurements appear to be unaffected by hydrogen scrambling, even when high collisional energies are utilized. This technique will benefit HDX MS measurements for any protein that contains one or more disulfides and the potential gain in sequence coverage and spatial resolution would increase with disulfide bond number.

Figure 1

Identification of disulfide-linked peptide dimers by LC/MS/MS. The top diagram shows the total ion chromatograms (TIC) of the peptic fragments derived from Tf digested under conditions identical to those used in HDX MS experiments. The large TIC corresponds to the long elution gradient used to enhance separation of proteolytic fragments and identify the highest possible number of peptides and to increase the time window for MS/MS measurements. The smaller TIC inset above is an example TIC generated during HDX MS/MS data collection. Panels labeled A–D illustrate the identification of disulfide-linked peptide dimers by observing distinct peptide monomers following fragmentation of a precursor ion with ECD (extracted ion chromatograms for these four peptide dimers are indicated by corresponding letters A–D in both upper TIC traces).

Figure 2

HDX MS and HDX MS/MS data for the four overlapping peptide dimers (structures are shown in the left column). The “MS1” column illustrates HDX MS measurements for each dimer (top trace: isotopic distribution of an unlabeled peptide ion, middle trace: isotopic distribution of the peptide following 1 min of exchange in solution, and bottom trace: end-point of the exchange reaction). The two “MS2” columns illustrate HDX MS/MS data for each monomeric constituent of the peptide dimer (“Pep 1” and “Pep 2”). The far right column shows kinetic plots for HDX MS (black trace) and HDX MS/MS (blue and red) measurements.

Figure 3

Location of the disulfide-linked peptide dimers characterized by HDX MS/MS within the crystal structure of Tf (PDB ID: 2HAV).