engleski

Multiple overlapping protein peptides to increase spatial resolution of hydrogen deuterium exchange method

Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution FT-ICR mass spectrometry offers a rapid method to study protein structure and protein-protein interactions. Pepsin is usually used to digest proteins due to its activity under HDX quench conditions, but sequence coverage is usually not complete. Two other proteases: protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus were introduced by Cravello et al.1 in 2003. Combination of the digest results from three separate digestions of apomyoglobin with pepsin, protease type XIII, and protease type XVIII resulted in 100% sequence coverage and generated a large number of highly overlapped fragments. To better predict the digest results, we established general cleavage preferences for each enzyme. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII. Furthermore, protease type XIII exhibited much less self-digestion than pepsin, and thus is superior for HDX experiments. Analysis of deuterium incorporation kinetics for multiple overlapping peptide fragments could help to increase the spatial resolution of our H/D exchange results. In our experiment with apomyoglobin, overlapping peptides analysis is performed by hand in order to establish the method of analysis and the results correlate well with the secondary and tertiary structure of apomyoglobin. Further work to confirm established procedure by testing on other proteins with known structure is needed and building the software tool for analysis is necessary in order to maximize the impact of this effort on widening the scope of HDX method application. Reference: 1. Laetitia Cravello et al., Rapid Commun. Mass Spectom. 2003 ; 17:2387-2393

hydrogen/deuterium exchange (HDX); FT-ICR mass spectrometry

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