Protein Sequence analysis
Micro-sequencing of proteins and peptides.
N-terminal sequencing, the generally accepted and most
reliable method to determine the amino acid sequence (the primary structure) of a protein is performed by automated
Edman degradation [1,2]. The automated equipment used are Applied Biosystems Model 494 HT Procise Protein Sequencing Systems . Proteins or peptides are sequentially broken down from the N-terminus and the derivatised residues are separated and identified by on-line coupled RP-HPLC.
It is used both during drug discovery for de novo sequencing
of novel proteins and used throughout all stages of drug discovery, to demonstrate comparability and consistency
between batches for release testing during manufacturing and
to check for the presence of impurities or truncated forms.
It is a key part of the ICH Q6B guidelines for characterisation
and confirmation of biopharmaceuticals in support of new marketing applications.
Samples can be sent either in solution, dry, as a Coomassie stained protein band in a piece of SDS-PAGE gel, or as electro-blotted sample on to PVDF (see separate info on the latter). Routinely, 20 to 30 residues can be easily identified, even for PVDF samples.
See for further details and information the technical / background information.