Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA. ekomives@ucsd.edu
J G
JG
Mandell
A M
AM
Falick
E A
EA
Komives
D000577:Amides / Q000032:analysis*; D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000577:Amides; D010446:Peptide Fragments; D006859:Hydrogen; D000803:Angiotensin I; D003903:Deuterium; D017868:Cyclic AMP-Dependent Protein Kinases; D010434:Pepsin A