High-throughput nonradioisotopic detection of picomole levels of phosphothreonine and phosphoserine containing peptides via biotinylation and enzyme-linked immunosorbent assay

Determination of phosphorylation sites in proteins is usually accomplished using [gamma-P-32]ATP. For low-abundance phosphoproteins, in vivo and intact cell studies usually require millicurie levels of P-32 for a single experiment, making multiple experiments prohibitive. Here we describe a low picomole sensitivity, nonradioisotopic, high-throughput method for tracing phosphorylation sites in proteins and peptides. The method is based on in situ enzyme-linked immunosorbent assay (ELISA) plate biotinylation of nonphospho- and phosphopeptides, streptavidin capture, and ELISA detection using recently available antiphospho-Thr and anti-phospho-Ser antibodies. (C) 1999 Academic Press.