IDENTIFICATION OF PHOSPHORYLATION SITES IN PROTEINS SEPARATED BY POLYACRYLAMIDE-GEL ELECTROPHORESIS

We report a fast, sensitive, and robust procedure for the identificati on of precise phosphorylation sites in proteins separated by polyacryl amide gel electrophoresis by a combination of matrix-assisted laser de sorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and o nline capillary liquid chromatography electrospray tandem ion trap mas s spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphory lation site was identified on as little as 20 ng (500 fmol) of the bac ulovirus-expressed catalytic domaine of myosin I heavy-chain kinase se parated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with greate r-than 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptide s are then separated and fragmented in an online LC/ESI ion trap mass spectrometer to identify the precise phosphophorylation sites. This pr ocedure eliminates any off-line HPLC separation and minimizes sample h andling. The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists. We applied this technique to identify two autophosphorylation sites and to assign at least anot her 12 phosphorylation sites to two tryptic peptides in a series of ex periments using a gel slice containing only 200 ng (3 pmol) of human d ouble-stranded RNA-activated protein kinase expressed in a mutant stra in of the yeast Saccharomyces cerevisiae.