Peptides and proteins are the principal components of most biologicalsystems. Determination of the amino acid sequence is an essential first step for structural characterization. The current generation of protein sequencers, which include an on-line HPLC and uv detector, all utilize basic technology and instrumentation developed 10-20 years ago. With only modest enhancements in recent years, protein sequence analysis has failed to keep pace with advancements in other methods of protein structural analysis, such as mass spectrometry, and protein purification techniques. While it is possible to purify and determine accurate masses of protein with femtomole amounts of sample, routine protein sequence analysis by Edman chemistry still requires 10 - 50 pmoles of sample. The objective of the proposed research is to develop a micro-scale protein sequencing system capable of routine analyses at sample levels well into the fmole range (5~500 fmole). The instrument would use standard Edman chemistry for N-terminal sequencing. The design would be sufficiently flexible that it could be adapted to other chemistries that utilize fluorescence detection or to C terminal sequence analysis. Improvements in sensitivity would be achieved by novel designs for the sequencer and HPLC systems, and the use of state-of-the-art absorbance detection utilizing a pulsed light source.
