A sentinel protein assay for simultaneously quantifying cellular processes

This presentation will describe a screening approach based on the concept of sentinel proteins, biological markers whose change in abundance characterizes the activation state of a given pathway or functional module in a cell. Sentinels are proteins, phosphorylation sites or protein degradation products that have been previously validated and are commonly assayed in molecular biology laboratories, typically one at a time by traditional antibody-based techniques. For example, sentinels include: specific phosphorylation events in the activation loop of MAP kinases as markers for signalling along different MAPK branches; or induction of the protein Atg8 (LC3 in mammals) as a marker for the activation of the autophagic response. By combining previous biochemical knowledge and computational prediction, a set of 309 sentinels was selected to probe the physiology of S. cerevisiae cells. An information-rich targeted proteomic fingerprint assay was assembled based on selected reaction monitoring-mass spectrometry (SRM-MS) that measures the complete sentinel set at high-throughput (one hour per sample), and reports on the activation state of 188 biological processes. To validate the approach, it was applied to a set of eight well-characterized environmental perturbations of yeast physiology. The approach recapitulates many known responses and enables the identification of novel cellular events, thereby demonstrating that the sentinel approach could be used to analyse the cellular responses to a large set of uncharacterized perturbations, such as a collection of drugs.