N-terminal sequencing, often referred to as Edman sequencing or Edman degradation is an important technique in protein study and remains a unique approach that can yield new protein sequence data. It is also the preferred technique for quick protein expression confirmation.
The chemistry of this approach was first introduced in 1950 by P. Edman from Lund University in Sweden and further developed during his work in Melbourne, Australia to what is believed to be the first automated peptide sequencing device.
Fig 1. Edman degradation chemistry for N-terminal protein sequencing.
The chemical reactions that yield the hydrolized N-terminal labeled amino acid are shown in fig 1. Each cycle includes essentially 3 steps:
Sequencing cycle chemistry and work up is well controlled and yields perfectly defined species, there are a few by-products that are detected during analysis.
As shown in figure 2, they mainly come from PITC hydrolysis and methanolysis during the cycle and include diphenylthiourea (DPTU), N-phenyl,O-methyl-thiocarbonate (PMTC) and diphenylurea (DPU), by-products that co-extract and co-elute with PTH-amino acids.
Fig 2. Side reactions of Edman reagent chemistry yielding well characterized by-products N-terminal protein sequencing including diphenylthiourea (DPTU), N-phenyl, O-methyl-thiocarbonate (PMTC) and diphenylurea (DPU).