title: Removal of N-terminal blocking groups from proteins.
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.
authors.affiliation: Pfizer VMRD, Kalamazoo, Michigan.
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Leone
authors.affiliation: University of Maryland School of Medicine, Baltimore, Maryland.
authors.forename: Brian
authors.initials: B
authors.lastname: Hampton
authors.affiliation: AutoImmune, Lexington, Massachusetts.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fowler
authors.affiliation: Glaxo Research Institute, Research Triangle Park, North Carolina.
authors.forename: Mary
authors.initials: M
authors.lastname: Moyer
authors.affiliation: University of Texas Medical School, Houston, Texas.
authors.forename: Radha G
authors.initials: RG
authors.lastname: Krishna
authors.affiliation: University of Texas Medical School, Houston, Texas.
authors.forename: Christopher C Q
authors.initials: CCQ
authors.lastname: Chin
mesh_terms: D000215:Acylation; D001671:Biochemistry / Q000379:methods*; D003124:Colorimetry; D005561:Formates / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D017879:Isothiocyanates / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011751:Pyroglutamyl-Peptidase I / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis / Q000379:methods*; D012996:Solutions; D013387:Succinic Anhydrides / Q000378:metabolism
chemical_list: D005561:Formates; D017879:Isothiocyanates; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D012996:Solutions; D013387:Succinic Anhydrides; C005441:phenylisothiocyanate; C008817:peroxyformic acid; C031801:succinic anhydride; D006867:Hydrolases; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase; D011751:Pyroglutamyl-Peptidase I; D011761:Pyrrolidonecarboxylic Acid