Structural characterization of purified natural and recombinant proteins traditionally includes N-terminal Edman sequencing to confirm 5-15 amino acid residues from the amino terminus. N-terminal sequencing is a requirement according to the ICH Q6B Guideline. This includes the characterization of recombinant proteins for clinical testing, and demonstration of comparability and consistency between cGMP batches.
N-terminal Edman sequencing does not work for N-terminally blocked proteins, and the analysis is time-consuming with a cycle time of 40 minutes per amino acid. An alternative technique, ISD MALDI MS has been developed and demonstrated in several publications for top-down sequencing of intact peptides and proteins. MALDI ISD offers several key benefits compared to Edman sequencing:
- Obtain both N- and C-terminal sequences of 20-50 residues.
- Sequence N-terminally modified and blocked proteins (acetylated, pyroglutamate, PEGylated).
- Fast data acquisition.
- Obtain very long sequence reads with up to 80 residues from one single ISD MALDI mass spectrum.
To sum it up:
MALDI ISD analysis can thus confirm expression and purification of the full length protein sequence. You also use it to detect unexpected truncations and modifications of the termini. And especially important, you can even apply the MALDI ISD analysis to N-terminally blocked and PEGylated proteins. Read more about n- and c- terminal sequencing by MALDI ISD.