A STUDY OF SRC SH2 DOMAIN PROTEIN-PHOSPHOPEPTIDE BINDING INTERACTIONSBY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

The noncovalent binding of various peptide ligands to pp60(src) (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry ( ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs i n proteins are critical in the signal transduction pathways of the tyr osine kinase growth factor receptors and recognize phosphotyrosine-con taining proteins and peptides. ESI-MS with a magnetic sector instrumen t and array detection has been used to detect the protein-peptide comp lex with low-picomole sensitivity. The relative abundances of the mult iply charged ions for the complex formed between Src SH2 protein and s everal nonphosphorylated and phosphorylated peptides have been compare d. The mass spectrometry data correlate well to the measured binding c onstants derived from solution-based methods, indicating that th mass spectrometry-based method can be used to assess the affinity of such i nteractions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of co ncentration for construction of a Scatchard graph. ESI-MS of a solutio n containing Src SH2 with a mixture of phosphopeptides showed the expe cted protein-phosphopeptide complex as the dominant species in the mas s spectrum, demonstrating the method's potential for screening mixture s from peptide libraries. (C) 1997 American Society for Mass Spectrome try.