Peptide sequencers, automated

Modem methods of peptide sequencing follow a strategy similar to that used to sequence insulin but are automated and can be carried out on a small scale A key feature is repetitive N terminal identification using the Edman degradation... 

Petir Victor Edman (1916-1977) was born in Stockholm, Sweden, and received an M.D. in 1946 at the Karolinska Institute. After a year in the United States at the Rockefeller Institute, he returned to Sweden as professor at the University of Lund. In 1957, he moved to St. Vincent s School of Medical Research in Melbourne, Australia, where he developed and automated the method of peptide sequencing that bears his name. A reclusive man, he never received the prizes or recognition merited by the importance or his work. 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Taylor, J. A., and Johnson, R. S. (2001). Implementation and uses of automated de novo peptide sequencing by tandem mass spectrometry. Anal. Chem. 73 2594-2604. 

Stevenson, T. I. Loo, J. A. Greis, K. D. 1998. Coupling capillary high-performance liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of peptides via automated microblotting onto membrane substrates. Anal. Biochem., 262,99-109. 

Peptides, for example, are needed for the production of antibodies and for epitope mapping proteins can be produced in radioactively labelled forms by in vitro translation for studies of protein-protein interactions and other applications. In vitro protein synthesis is a technique that can be readily carried out in any biochemistry laboratory, whereas peptide synthesis, like peptide sequencing, is a specialist technique using dedicated equipment, which is nowadays routinely automated. 

Most peptide sequencing is now done by Edman degradation, an efficient method of N-terminal analysis. Automated Edman protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by-products interferes with the... 

With the dissemination of multinuclear multidimensional NMR experiments, the automated NMR resonance assignment became feasible and indispensable, simultaneously.23,35,41,99 Typically, 10-20 individual multidimensional NMR spectra are measured for a large protein. The NMR-derived information quality definitely relies on the resonance assignment. The precious assignment has been based on and assessed by the sequential NOE continuity on the peptide sequence and consistency with the amino acid spin... 

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