Selection and optimization of enzyme reporters for chemical cytometry

Abstract Chemical cytometry, sensitive analytical measurements of single cells, reveals inherent heterogeneity of cells within a population which is masked or averaged out when using bulk analysis techniques. A particular challenge of chemical cytometry is the development of a suitable reporter or probe for the desired measurement. These reporters must be sufficiently specific for measuring the desired process; possess a lifetime long enough to accomplish the measurement; and have the ability to be loaded into single cells. This chapter details our approach to rationally design and improve peptide substrates as reporters of enzyme activity utilizing chemical cytometry. This method details the iterative approach used to design, characterize, and identify a peptidase-resistant peptide reporter which acts as a kinase substrate within intact cells. Small-scale, rationally designed peptide libraries are generated to rapidly and economically screen candidate reporter peptides for substrate suitability and peptidase resistance. Also detailed are strategies to characterize and validate the designed reporters by determining kinetic parameters, intracellular substrate specificity, resistance to degradation by intracellular peptidases, and behavior within lysates and intact cells.