H/D Exchange and Mass Spectrometry-Based Method for Biophysical Analysis of Multidomain Proteins at the Domain Level

Abstract

A protocol was developed to characterize the domain-specific thermodynamic stabilities of multidomain proteins using SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange). The protocol incorporates a protease digestion step into the conventional SUPREX protocol and enables folding free energy (DeltaGf) and cooperativity (m-value) measurements to be made on the individual domains of multidomain proteins in their native context (i.e., in the intact protein). Three multidomain protein systems (calmodulin, a Fyn construct, and transferrin) were used to validate the SUPREX-protease digestion protocol. The DeltaGf and m-value of each domain in the intact test proteins were measured in the absence and presence of ligands using the new protocol. Domain-specific thermodynamic parameters were obtained on each system; and the measured parameters were consistent with known biophysical properties of the test proteins. The known stabilization of the N-terminal domain of CaM in the context of the intact protein and the known binding affinity of a proline-rich peptide to the SH3 domain in the Fyn construct were successfully quantified using the new protocol. Qualitative information about the relative calcium binding affinities of the N- and C-terminal domains of CaM and about the relative iron binding affinities of the N- and C-terminal domains of transferrin was also obtained using the new protocol.

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

“…The HX-MS approach measures deuterium buildup in a protein over time courses and provides information regarding conformation and solution dynamics of the protein. 8 HX-MS has been successfully applied to conformation analysis of IgG1 for both unmodified and modified forms produced through deglycosylation or were acquired by MALDI-MS. 12,15 The SUPREX experiments described here used electrospray ionization mass spectrometry (ESI-MS) to acquire mass spectra of the peptic fragments of MAb1 after reversed-phase separation.…”

“…9,12,14 With the use of enzymatic digestion, SUPREX is also able to probe the unfolding stability of a specific domain of a multi-domain protein. 15 SUPREX is well suited to be a subclass of various H/D exchange mass spectrometry techniques since it uses H/D exchange and mass spectrometry readout. SUPREX was originally developed to study the stability of unpurified proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) without separation of the peptides.…”

Throughput and efficiency of a mass spectrometry-based screening assay for protein—Ligand binding detection

“…Recently, we reported on the use of a rapid (Ͻ2 min) protease digestion step to facilitate the SUPREX analyses of large multidomain proteins [29]. In this SUPREXprotease digestion protocol the H/D exchange properties of the individual domains of a protein are defined when the domains are in the intact protein.…”

Painting proteins with covalent labels: What’s in the picture?

“…Ultimately, the deuterium content (i.e., ⌬Mass value) is determined for the protein in each denaturant-containing H/D exchange buffer; these values are then used to generate a SUPREX curve (i.e., a plot of ⌬Mass versus [denaturant]) at a specific exchange time (see Figure 4). SUPREX experiments can also be performed using a strategy that incorporates a protease digestion step into the basic SUPREX protocol [61]. This protocol significantly expands the application of SUPREX to large multidomain protein systems.…”

“…Such protein systems must be analyzed by the SUPREX with protease digestion protocol outlined earlier [61]. The SUPREX behavior of peptide fragments generated in the SUPREX with protease digestion protocol can be used to report on the biophysical properties of the individual domains from which they were derived.…”