Abstract: A protein sequencing method utilizing a composition of matter which comprises a D-C-B-A reagent wherein A is a moiety which can react with and bind to a terminal amino acid of a protein and can result in removal of the terminal amino amino acid, B is a moiety which provides steric separation between C and A, C is a nucleophilic moiety which can be detected, and D is a moiety which protects the C moiety from degradation or other modification, and is labile in acidic media and stable in neutral or basic media. The protein sequencing method and reagent can be used in micro sequencing apparatus.