title: Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins.
abstract: The rates at which peptide amide hydrogens in folded proteins undergo isotopic exchange are reduced by factors of 10(0)-10(-8) relative to exchange rates at the same peptide linkages in unfolded proteins. To measure the isotopic exchange rates of the most rapidly exchanging peptide amide hydrogens in proteins, a flow-quench deuterium exchange-in step has been added to the protein fragmentation/mass spectrometry method (Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531). Isotopic exchange rates in eight short segments spanning the entire backbone of cytochrome c have been determined for exchange-in times of 0.2-120 s. These results show that the isotopic exchange rates of 10 of the peptide amide hydrogens in cytochrome c are similar to those expected for unfolded cyt c, while the exchange rates for 33 other non-hydrogen-bonded amide hydrogens are much less than expected for unfolded cyt c. Since the isotopic exchange rates of the most rapidly exchanging amide hydrogens in folded proteins are a direct measure of their access to the aqueous solvent, the ability to determine these isotopic exchange rates points to the possibility of using quenched-flow amide hydrogen exchange and mass spectrometry as a tool for identifying protein surfaces involved with binding.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
authors.forename: K
authors.initials: K
authors.lastname: Dharmasiri
authors.forename: D L
authors.initials: DL
authors.lastname: Smith
mesh_terms: D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D006859:Hydrogen / Q000737:chemistry*; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology
chemical_list: D000577:Amides; D003574:Cytochrome c Group; D007554:Isotopes; D006859:Hydrogen