High sample throughput phosphoamino acid analysis of proteins separated by one- and two-dimensional gel electrophoresis

Abstract

Studies on protein phosphorylation usually involve radiolabelling techniques and visualisation on gels; this limits studies to those on tissues which can be 32P labelled. With the advent of reproducible micropreparative 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 to detect in vivo phosphorylation in proteins blotted from one or two-dimensional polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes. The method uses 9-fluorenylmethyl chloroformate (FMOC) derivatisation chemistry and a modified HPLC AMINOMATE system. Conditions were established for hydrolysis of the PVDF-blotted protein (5.7 M HCl at 110°C for 4 h) which resulted in the recovery of phosphoserine (Ser(P)), phosphothreonine (Thr(P)) and phosphotyrosine (Tyr(P)). The chromatography was carried out on the same system routinely used for amino acid compositional analysis using a gradient elution modified from that used for separation of 16 amino acids. A chromatographic window was designed where all 3 phosphoamino acids are separated with baseline resolution in the order of Ser(P), Thr(P) and Tyr(P), and eluted before the normal protein amino acids. The total separation time is 13 min and includes the elution of excess FMOC, its derivatives and incompletely hydrolysed peptides. The FMOC fluorescence of these three standard phosphoamino acids is linear in the range 10–100 pmol. The technique is shown to be successful in finding phosphoproteins separated by two-dimensional gel electrophoresis. The method is sensitive (30 pmol of a single site phosphorylated protein, 3.8 pmol of Ser(P) detected) and allows automated sample throughput. This presents an option for rapid screening of protein phosphorylation of large numbers of proteins separated by two-dimensional polyacrylamide gel electrophoresis.