Mapping protein-protein interactions by affinity-directed mass spectrometry.
A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.
Zhao
Y
Y
Rockefeller University, New York, NY 10021, USA.
Muir
T W
TW
Kent
S B
SB
Tischer
E
E
Scardina
J M
JM
Chait
B T
BT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2