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Updated: Jun 3, 2026

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens
Published on: February 24, 2021
Removal of N-terminal blocking groups from proteins
Joseph W Leone1, Brian Hampton2, Elizabeth Fowler3
1Pfizer VMRD, Kalamazoo, Michigan.
Abstract:
Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.
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