Conformational studies of proteins by H/D exchange
and electrospray ionization mass spectrometry

Zbigniew Szewczuk

Faculty of Chemistry, University of Wrocław

szewczuk@wchuwr.pl

Electrospray mass spectrometry (ESI-MS) is a basic tool for studying biomolecules such as proteins, peptides and other non-volatile compounds. In contrast to other methods (NMR, FT-IR) ESI-MS enables simultaneous analysis of more than one substance in solution. This may be very practical in the case of a mixture of homologs which are difficult to separate (for example proteins from a biological source). Hydrogen/deuterium (H/D) exchange is one of the most important approaches to characterize protein dynamics. Mass spectroscopy measuring the changes in mass on H/D exchange, is an important method for analyzing the H/D exchange reaction (Fig 1). Therefore, ESI-MS is often used as a tool to study three-dimensional structure by monitoring H/D isotope exchange. Such experiments provide information about protein stability, dynamics and protein folding.

Fig. 1. A protein in a native conformation (A) has fewer protons available for deuteration than the same protein in an unfolded state (B). Since the deuteration increases the molecular mass of a protein, ESI-MS can be used to monitor the H/D exchange.

Taking advantage of the unique ability of ESI-MS to characterize mixtures of proteins without purification we analyzed conformational stabilities of a complex mixture of truncated cHMG1a proteins [1], monitor simultaneously the exchange kinetics of more than seven modified cytochrome c analogs with a different number of acetyl groups [2]. We also proved that electron capture dissociation (ECD) of electrosprayed H/D exchanged proteins is good method of analysis of protein 3D structures even at the residue level [3].

References:

1.     Petry, I.; Wiśniewski, J.R.; Szewczuk, Z. Conformational stability of six truncated cHMG1a-proteins studied in their mixture by H/D exchange and electrospray ionization mass spectrometry Acta Biochim. Polon. 2001, 48,1131-1136.

2.     Szewczuk, Z.; Konishi, Y.; Goto, J. A two-process model describes the hydrogen exchange behavior of cytochrom c in the molten globule state with various extents of acetylation. Biochemistry 2001, 40, 9623-9630.

3.     Stefanowicz, P.; Petry-Podgorska, P., Kowalewska, K., Jaremko, Ł., Jaremko, M., Szewczuk, Z. 2009, submitted.