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This talk describes an approach to using mass spectrometry to analyze biochips—including enzyme-mediated reaction of immobilized biomolecules and protein-protein interactions. The method is based on self-assembled monolayers of alkanethiolates on gold that present proteins and small molecules with control over the densities, patterns, and orientations of these species. Of particular interest is the development of fusion protein capture strategies that give selective and covalent immobilization of proteins. In one example, the serine esterase cutinase reacts with phosphonate capture ligands to give an active-site covalent adduct. Application of a cutinase fusion protein with a monolayer presenting the capture ligand results in immobilization of the display protein, with strict control over both the orientation and the density of this reagent. The chips are compatible with matrix-assisted laser desorption ionization mass spectrometry, and therefore do not require fluorescent or radioisotopic labels for analysis. This technique, termed SAMDI MS, can efficiently monitor a broad class of enzyme activities, including kinase, protease, methyltransferase, and carbohydrate-directed modifications, and can detect proteins having molecular weights up to 100 kDa. The talk will describe examples in assays of endogeneous cellular activities and protein-protein interaction mapping.