N-terminal protein sequencing by Edman degradation is the method of choice for characterization of therapeutic proteins, monoclonal antibodies and protein vaccines, since it allows the exact identification of the N-terminal amino acids independently of known sequence information and enzymatic cleavage sites.
Using Procise instruments for automated sequencing, the N-terminal amino acids are sequentially removed from the protein strand and identified by their retention time. Identification and rough quantitation is performed in comparison to a standard chroma-togram containing the PTH-derivatives of each of the 19 proteinogenic amino acids (without cysteine).
Several picomoles of a purified protein with an unmodified N-terminus, either in solution or immobilized on PVDF membrane, are required in order to obtain useful sequence information. Depending on the sequence, as little as 1 pmol protein can be sufficient for sequence identification.
In case the protein N-terminus is blocked by pyroglutamate, enzymatically de-blocking and removal of the first amino acid can be performed to obtain a free N-terminus accessible for Edman degradation.
N-terminal sequencing allows to unambiguously identify isobaric amino acids such as leucine and isoleucine.
Samples can be applied from pure sample solution by immobilization onto PVDF filters (Prosorb) or specific protein bands previously blotted onto a PVDF membrane and stained with Ponceau S, Coomassie or Amido Black.
TOPLAB operates N-terminal sequencing in accordance with Good Manufacturing Practice (GMP). The certification by Regierung von Oberbayern covers the testing procedures for N-terminal sequencing of proteins, peptides and antibodies by Edman degradation.