title: Determination of N-myristoyl peptide sequence both by MALDI TOF MASS and with an N-myristoyl cleaving enzyme (polymyxin acylase).
abstract: Polymyxin acylase isolated from Pseudomonas sp. M-6-3 was used as an N-myristoyl cleaving enzyme in order to determine a part of the N-terminal amino acid sequence of N-myristoyl proteins. The enzyme hydrolyzed a number of N-myristoyl oligopeptides at various hydrolysis rates but not N-myristoyl proteins. The oncogenic protein (N-myristoyl-pp60c-src) was isolated from human colon adenocarcinoma cell line COLO 320DM by two-dimensional polyacrylamide gel electrophoresis. The protein was digested with trypsin and the resultant tryptic N-myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Asn-Lys) was purified by HPLC and the structure was determined both by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MASS) and by a gas-phase protein sequencer before or after treatment with the polymyxin acylase. The results suggest that the N-myristoyl peptide sequence derived from N-myristoyl proteins was clearly determined by the combined use of MALDI TOF MASS and the N-myristoyl cleaving enzyme.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Misumi
authors.forename: M
authors.initials: M
authors.lastname: Tsuruta
authors.forename: K
authors.initials: K
authors.lastname: Furuishi
authors.forename: S
authors.initials: S
authors.lastname: Shoji
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009226:Myristates / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D009226:Myristates; D009842:Oligopeptides; D016392:Proto-Oncogene Proteins pp60(c-src); D000581:Amidohydrolases; C038758:polymyxin acylase