HIGH SAMPLE THROUGHPUT PHOSPHOAMINO ACID ANALYSIS OF PROTEINS SEPARATED BY ONE-DIMENSIONAL AND 2-DIMENSIONAL GEL-ELECTROPHORESIS

Studies on protein phosphorylation usually involve radiolabelling tech niques and visualisation on gels; this limits studies to those on tiss ues which can be P-32 labelled. With the advent of reproducible microp reparative two dimensional gel electrophoresis, the proteome (protein complement) of a subcellular fraction, cell or tissue can be displayed . We have investigated the sensitivity of limited protein hydrolysis t o detect in vivo phosphorylation in proteins blotted from one or two-d imensional polyacrylamide gels onto polyvinylidene difluoride (PVDF) m embranes. The method uses 9-fluorenylmethyl chloroformate (FMOC) deriv atisation chemistry and a modified HPLC AMINOMATE system. Conditions w ere established for hydrolysis of the PVDF-blotted protein (5.7 M HC1 at 110 degrees C for 4 h) which resulted in the recovery of phosphoser ine (Ser(P)), phosphothreonine (Thr(P)) and phosphotyrosine (Tyr(P)). The chromatography was carried out on the same system routinely used f or amino acid compositional analysis using a gradient elution modified from that used for separation of 16 amino acids. A chromatographic wi ndow was designed where all 3 phosphoamino acids are separated with ba seline resolution in the order of Ser(P), Thr(P) and Tyr(P), and elute d before the normal protein amino acids. The total separation time is 13 min and includes the elution of excess FMOC, its derivatives and in completely hydrolysed peptides. The FMOC fluorescence of these three s tandard phosphoamino acids is linear in the range 10-100 pmol. The tec hnique is shown to be successful in finding phosphoproteins separated by two-dimensional gel electrophoresis. The method is sensitive (30 pm ol of a single site phosphorylated protein, 3.8 pmol of Ser(P) detecte d) and allows automated sample throughput. This presents an option for rapid screening of protein phosphorylation of large numbers of protei ns separated by two-dimensional polyacrylamide gel electrophoresis.