Cell-free bead-based detection of total and phosphorylated proteins in plasma and cell lysates: detection of FLT3.

Abstract

Frequently direct measurement of proteins or their phosphorylation in intact cells is not possible, for instance, when cells are too few, frozen, or subject to degradation. We have demonstrated that tumor cells pour their DNA, RNA, and protein content into circulation because of turnover and breakdown of cell structures. Proteins in solution most likely circulate as complexes, which protects them from degradation. We describe a cell-free, bead-based method that takes advantage of this phenomenon. Our approach is based on immunoprecipitation of the protein of interest on the surface of beads, followed by detection of the protein or its modification (phosphorylation) using a secondary antibody labeled with phycoerythrin at a 1:1 ratio. Fms-like tyrosine kinase-3, which is mutated in majority of cases of acute myeloid leukemia, is used as an example. This method could be applied to the quantitation of several other proteins without the need for intact cells.