Preparation and Analysis of Phosphorylated Proteins

摘要

The function of cells of the immune system is regulated in part by engagement ofspecific receptors on the cell surface by soluble ligands or ligands presented on the surface of other cells. One of the major mechanisms by which cell-surface receptor engagement influences cellular function is by activating protein kinases and increasing the phosphorylation of critical cellular proteins. There are two major categories of protein kinases: serine/threonine kinases which phosphorylate both serine and threonine residues; and tyrosine kinases which phosphorylate tyrosine residues exclusively. By metabolically labeling cells with 32p (first basic protocol), the phosphorylation state of cellular proteins can be analyzed and changes occurring with receptor stimulation can be examined. Phosphoamino acid analysis (second basic protocol) can be used to determine whether serine, threonine, or tyrosine residues are phosphorylated, thereby indicating which category of protein kinase is responsible. Phosphopeptide mapping (third basic protocol) produces a fingerprint of the peptides that are phosphorylated providing information regarding the sites of phosphorylation.