The process of determining the amino-acid sequence of a protein or its component polypeptides. The technique traditionally used is Edman degradation (devised by Pehr Edman), in which the terminal amino-acid residues are removed sequentially and identified chromatographically. Each step is automated and the whole process can now be performed by a single machine—the sequenator. Large polypeptides must be cleaved into smaller peptides before sequencing. The identification of unknown protein samples, determination of protein structure, and detection of post-translational modifications, as well as amino acid sequencing now commonly employ mass spectrometry techniques, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). For large proteins, this can be complemented by peptide mapping, in which the polypeptide chain is cleaved at specific peptide links.

The results of chemical sequencing can often be compared with the amino-acid sequence deduced by DNA sequencing. The gene coding for the protein under investigation may be found by screening a DNA library, for example by western blotting. However, the base sequence of the gene gives only the amino-acid sequence of the nascent protein, i.e. before post-translational modification. The sequence of the functional protein can only be found by chemical analysis.