Affinity mass spectrometry-based approaches for the analysis of protein-protein interaction and complex mixtures of peptide-ligands

Combined applications of affinity purification procedures and mass-spectrom etric analyses (affinity mass spectrometry or affinity-directed mass spectr ometry) have gained broad interest in various fields of biological sciences . We have extended these techniques to the purification and analysis of clo sely related peptides from complex mixtures and to the characterization of binding motifs and relative affinities in protein-protein interactions. The posttranslational modifications in the carboxy-terminal region of porcine brain tubulin are used as an example for the applicability of affinity mass spectrometry in the characterization of complex patterns of related peptid es. We also show that affinity mass spectrometry allows the mapping of sequ ential binding motifs of two interacting proteins. Using the ActA/Mena prot ein-protein complex as a model system, we show that we can selectively puri fy Mena-binding peptides from a tryptic digest of ActA. The results from th is assay are compared to data sets obtained earlier by classical methods us ing synthetic peptides and molecular genetic experiments. As a further expa nsion of affinity mass spectrometry, we have established an internally stan dardized system that allows comparison of the affinities of related ligands for a given protein. Here the affinities of two peptide ligands for the mo noclonal tubulin-specific antibody YL1/2 are determined in terms of half-ma ximal competition. (C) 1999 Academic Press.