From Edman Degradation to Mass Spectrometry: A Short History of Peptide Sequencing

Knowing a peptide's mass confirms its overall composition, but not necessarily the order of its amino acids. Sequence determination answers that second question.

Edman's stepwise method

In 1950 Pehr Edman published Method for Determination of the Amino Acid Sequence in Peptides in Acta Chemica Scandinavica. The method removes the N-terminal amino acid as a derivative that can be identified, leaving the rest of the chain intact. Repeating the reaction reads the sequence one residue at a time from the N-terminus.

Strengths and limits

Stepwise degradation is direct and does not rely on prediction. It requires a free N-terminus, however, so peptides with blocked or modified N-termini cannot be read this way without additional steps, and efficiency limits the length that can be read in a single run.

The mass spectrometry era

Since soft ionisation techniques such as electrospray (Fenn and colleagues, Science, 1989) became established, peptide sequences are frequently confirmed by tandem mass spectrometry, in which ions are fragmented and the fragment masses are compared with those expected from the sequence.

Practical relevance

For routine research peptides, identity is usually confirmed by molecular mass. Sequence-level confirmation is more relevant where isomeric or closely related sequences are possible — for instance where a deletion of one residue would be offset by another modification of similar mass.