Protein microsequencing represents one of the cornerstones of biotechnology. In the automated Edman degradation process, one amino acid residue is released per cycle, and it is then identified by HPLC coupled with ultraviolet absorption spectrometry. The current methodology is limited by this mode of detection to the picomole range (i.e. micrograms of protein). An improvement in sensitivity to the femtomole level (i.e. nanograms of protein) should have a major impact on protein research, analogous to that offered by the introduction of the gas-phase sequencer several years ago. The approach to be taken is to use a fluorescent Edman-type reagent. The isothiocyanate derivatives of acridine and anthracene will be used in place of phenylisothiocyanate. The kinetics of the individual steps (i.e. coupling, cleavage and conversion) will be studied using the dipeptide leucyl-alanine. The thiocarbamyl, anilinothiazolinone and thiohydantoin derivatives of leucine will be synthesized with each fluorescent reagent. They will be used as calibration standards for reversephase HPLC which will be utilized for monitoring the progress of the reaction steps. The goal is to demonstrate that the modified Edman degradation can be made to proceed to near completion in an efficient manner and with minimal side reactions. A major obstacle to the successful implementation of femtomole level sequencing is expected to be the accumulation of reagent byproducts. This problem of interfering background will be addressed using an ion- exchange precolumn for trapping the desired derivatives and for removing excess reagent and reagent byproducts prior to the final analysis of the released amino acid.
