Low-Voltage Separation of Phosphoamino Acids by Silica Gel Thin-Layer Electrophoresis in a DNA Electrophoresis Cell

A major objective in the characterization of protein kinase activity is to determine phosphoamino acid identity in substrate proteins. Several fundamentally different phosphoamino acid analysis (PAA) procedures are available to identify phosphorylated amino acids (2,3). Phosphoamino acids can be released from proteins by partial hydrolysis (4) and identified directly by oneor two-dimensional high-voltage thin-layer electrophoresis (HV-TLE) and/or thin-layer chromatography (TLC) (1,3,5). PAA by conventional HV-TLE (≥1000 V) is quick but requires the use of expensive power supplies and temperature-regulated equipment not readily available to most laboratories. PAA by TLC does not require elaborate equipment but is timeconsuming, requiring 7–12 h for sample migration (3,5). Low-voltage thin-layer electrophoresis (LV-TLE) (≤500 V) is relatively quick and eliminates the need for cooling the TLE unit and other expensive equipment. Although the separation of complex mixtures of amino acids was originally achieved by LV-TLE (6), it is generally not accepted that separation of phosphoamino acids can be achieved at lower voltages because detailed methods for PAA by LV-TLE are unavailable. Several simple units designed for LV-TLE have been described (6); their similarity to modern agar electrophoresis equipment prompted us to investigate whether this equipment could be used for PAA. We have developed a simple one-dimensional LV-TLE procedure that will resolve the three most common eukaryotic phosphoamino acids. Our technique uses a standard method for partial acid hydrolysis of proteins (4) but includes significant modifications of existing HV-TLE protocols (1) that result in a simplified PAA technique that most laboratories are equipped to perform. Specifically, electrophoresis is performed on silica gel plates using a commonly used PAA buffer (2) in a DNA electrophoresis cell. The thickness and type of thin-layer sorbent used and the electrophoresis voltage differ from published procedures (1,2). The advantages of our technique over conventional HVTLE and/or TLC are that it is economical, it utilizes standard laboratory equipment and it allows rapid separation of phosphoamino acids. We describe our LV-TLE protocol in detail and demonstrate that it can resolve phospho-serine (pSer), threonine (pThr) and tyrosine (pTyr) residues in protein hydrolysates as discrete spots. We have purified a maize protein kinase, ZmMEK1, as a glutathione Stransferase (GST) fusion expressed in E. coli. During in vitro kinase reactions with [γ-32P]ATP, ZmMEK1 is capable of autophosphorylation on the kinase portion of the fusion product (our unpublished observations) and phosphorylation of myelin basic protein (MBP) (Figure 1). These reaction products