Mass spectrometric measurement of protein amide hydrogen exchange rates of apo- and holo-myoglobin.
Measurement of backbone amide hydrogen exchange rates can provide detailed information concerning protein structure, dynamics, and interactions. Although nuclear magnetic resonance is typically used to provide these data, its use is restricted to lower molecular weight proteins that are soluble at millimolar concentrations. Not subject to these limitations is a mass spectrometric approach for measuring deuterium incorporation into proteins that are subsequently proteolyzed by pepsin; the resulting peptide masses are measured using a flowing-fast atom bombardment ionization source (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). In the current study, amide deuterium incorporation for intact apo- and holo-myoglobin was measured using liquid chromatography coupled directly to an electrospray ionization (LC/MS) source. Electrospray ionization provided a more complete coverage of the protein sequence and permitted the measurement of deuterium incorporation into intact proteins. Tandem mass spectrometry was used to rapidly identify the peptic peptides. It was found that within 30 s, the amides in apo-myoglobin were 47% deuterated, whereas holo-myoglobin was 12% deuterated. Peptic digestion and LC/MS demonstrated that regions represented by peptic peptides encompassing positions 1-7, 12-29, and 110-134 were not significantly altered by removal of the heme. Likewise, destabilized regions were identified within positions 33-106 and 138-153.
Johnson
R S
RS
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Walsh
K A
KA
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D003903:Deuterium / Q000378:metabolism; D006736:Horses; D006859:Hydrogen / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000378:metabolism*; D010434:Pepsin A / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation
D000577:Amides; D001059:Apoproteins; D009211:Myoglobin; D010446:Peptide Fragments; C010857:apomyoglobin; D006859:Hydrogen; D003903:Deuterium; D010434:Pepsin A