Hydrogen / Deuterium Exchange Monitored by Electron Transfer Dissociation Mass Spectrometry for Studying Protein Structures and Interactions

Sabine Amon (Foredragsholder)

Aktivitet: Foredrag og mundtlige bidragForedrag og præsentationer i privat eller offentlig virksomhed

Beskrivelse

The structure and dynamics of proteins as well as their interaction with other proteins or ligands is of great interest. An effective tool in this research area is hydrogen / deuterium exchange (HDX). HDX utilises the fact that the exchange rates of amide hydrogens in proteins are strongly dependent on the local surrounding – protection of these hydrogens either due to the three-dimensional structure or by involvement in hydrogen bonding leads to reduced exchange rates. This is studied by dissolving a fully deuterated protein (complex) in protiated buffer and thereby inducing back-exchange.

Besides nuclear magnetic resonance (NMR), mass spectrometry (MS) is increasingly used for monitoring the hydrogen isotope exchange. Most commonly, this is done by digesting proteins that have been subjected to HDX into peptides and analysing them by means of liquid chromatography (LC) coupled to MS. Protection of certain regions of the protein or the protein complex is deduced from the deuterium incorporation into the different peptides. However, the enzymatic digestion step causes back-exchange of labile deuteriums during the downstream analysis. Furthermore, exact localisation of the deuteriums along the peptide backbone is impossible when using collision induced dissociation (CID) for fragmentation due to the occurrence of hydrogen scrambling.

Recently, we have been investigating electron transfer dissociation (ETD) as a promising, complementary fragmentation technique. We have previously shown that due to the mechanism of ETD the deuterium distribution along the peptide backbone is conserved. Therefore, characterisation of the HDX pattern down to the single amino acid residue level is within reach. An even more promising strategy may be the fragmentation of intact proteins in a so-called top-down approach and thereby avoiding the enzymatic digestion step altogether. We will present first results in this direction and discuss the potential and current limitations of this method.

Periode11. sep. 2009
Begivenhedstitel4th Annual Meeting of the MC-RTN ”DNA Enzymes”
BegivenhedstypeKonference
PlaceringBudapest, Ungarn