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Internal and Complete sequence analysis

N-terminal sequence analysis of a protein by automated Edman degradation will in general result
in the first ±40 residues, due to incomplete coupling and cleavage reactions (±94% efficiency). Consequently, other strategies has to be applied if either a specific part more towards the C-terminus needs to be investigated or even the complete sequence needs to be confirmed or determined.

Such strategies rely on proper selection and application of fragmentation methods of the protein,
either enzymatic or by chemical cleavage methods, followed by isolation and purification of the
resulting peptide fragments which can be sequenced completely or at least to the extend necessary.  

In case of complete sequence analysis of a de novo protein, this implies also the performance of a
second, different fragmentation pattern (or even more), to obtain the necessary “overlaps” in order
to determine the sequences of the individual fragments obtained from the first fragmentation. A
second fragmentation and isolation of fragments is not strictly necessary if one already has DNA
sequence information.

Eurosequence has extensive experience in establishing the most cost-favourable and fastest
strategy to elucidate or to confirm the complete sequence of your protein chain of concern. Such a
strategy is in principle based on the generation and extensive sequence analysis of relative large fragments, which can be visualized as follows: