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 [4-7].
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 [4-7].
Read more about N- and C-terminal sequencing by MALDI ISD >>>
 Wellner et al: “Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine“, Proceedings of the National Academy of Sciences of the United States of America, 1990
 European Medicines Agency: “ICH Topic Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products“, 1999
 Fowler et al: “Removal of N‐Terminal Blocking Groups from Proteins“, Current Protocols in Protein Science, 1996
 Debois et al: “MALDI-In Source Decay Applied to Mass Spectrometry Imaging: A New Tool for Protein Identification“, Analytical Chemistry, 2010
 Demeure et al: “Rational Selection of the Optimum MALDI Matrix for Top-Down Proteomics by In-Source Decay“, Analytical Chemistry, 2007
 Hanisch, F. G.: “Top-down sequencing of O-glycoproteins by in-source decay matrix-assisted laser desorption ionization mass spectrometry for glycosylation site analysis“, Analytical Chemistry, 2011
 Debois et al: “MALDI In-Source Decay, from Sequencing to Imaging“, Applications of MALDI-TOF Spectroscopy, 2012