Jeremiah Tipton - Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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      Publication Details (including relevant citation   information):

      Zhang, H.; Kazazic, S.; Schaub, T.M.; Tipton,   J.D.; Emmett, M.R. and Marshall, A.G., Analytical   Chemistry, 80 (23), 9034-9041 (2008)

      Abstract:

      Solution-phase hydrogen/deuterium exchange (HDX) monitored by   high-resolution Fourier transform ion cyclotron resonance (FTICR)   mass spectrometry offers a rapid method to study protein   conformations and protein-protein interactions. Pepsin is usually   used to digest proteins in HDX and is known for lack of cleavage   specificity. To improve digestion efficiency and specificity, we   have optimized digestion conditions and cleavage preferences for   pepsin and protease type XIII from Aspergillus saitoi. A dilution   series of the proteases was used to determine the digestion   efficiency for several test proteins. 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 from Rhizhopus.   Furthermore, protease type XIII exhibited much less   self-digestion than pepsin and thus is superior for HDX   experiments. Many highly overlapped segments from protease type   XIII and pepsin digestion, combined with high-resolution FTICR   mass spectrometry, provide high sequence resolution (to as few as   one or two amino acids) for the assignment of amide hydrogen   exchange rate. Our H/D exchange results correlate well with the   secondary and tertiary structure of myoglobin. Such assignments   of highly overlapped fragments promise to greatly enhance the   accuracy and sequence resolution for determining conformational   differences resulting from ligand binding or protein-protein   interactions.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/ac801417d