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 older 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-scr) 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-Sel-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-mylistoyl cleaving enzyme.