VISUALIZATION OF PROTEINS BY MODIFICATION OF LYSINES, CYSTEINES, AND PHOSPHORYLATED SERINES FACILITATES SAMPLE PREPARATION FOR MICROSEQUENCING

A procedure for visualization and sensitive detection of protein durin g sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent, or visible tags for certa in amino acids in protein molecules, e.g., lysines modified with dansy l/dabsyl chloride and cystines/cysteines or phosphorylated serines mod ified with iodoacetamidofluorescein (I-15) after proper sample pretrea tments. Modifications are performed prior to SDS-PAGE, eliminating the need for fixing, staining, and destaining as required for the convent ional procedures. After electrophoresis, the fluorescent or visible ba nds are excised from the gel, homogenized in microcentrifuge tubes, an d soaked in an appropriate buffer to release the separated proteins in to solution, Enzymatic digestion can then be carried out in solution f or better efficiency of digestion and recovery. The subsequent HPLC ma pping and collection of protein digests are performed on PE Applied Bi osystems Model 173A MicroBlotter. The separated peptides containing ta gged amino acids are visible on the PVDF membrane and can be excised f or direct sequence analysis. This approach has been employed for selec tively isolating the lysine, cysteine, or phosphorylated serine contai ning peptides using model proteins. The sequencing results of the pept ides generated from premodified proteins demonstrate that this approac h facilitates sample preparation for microsequence analysis at low pic omole level, Overall. recoveries of 20-30% by sequencing initial yield s have been achieved using our model proteins. (C) 1998 Academic Press .