Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins.
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.
Dharmasiri
K
K
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Smith
D L
DL
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
D000577:Amides; D003574:Cytochrome c Group; D007554:Isotopes; D006859:Hydrogen