title: Microbial degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantification using liquid chromatography-mass spectrometry.
abstract: The bacterial strain Sphingosinicella sp. B-9 was originally discovered to have the ability to degrade cyanobacterial cyclic peptides (microcystins), and has three hydrolytic enzymes (MlrA, MlrB, and MlrC). The purpose of this study was to examine in detail the degradation of glucagon/vasoactive intestinal polypeptide (VIP) family peptides by B-9, and to investigate the substrate specificity of B-9 proteases and the possibility of using a B-9 protease as a novel protease for peptide quantification by using a surrogate peptide and mass spectrometry (MS). The effective use of inhibitors revealed the following hydrolytic capability of B-9: One of the B-9 proteases (presumably MlrB) that was not inhibited by ethylenediaminetetraacetic acid (EDTA) cleaved bioactive peptides into medium-sized peptides with broad selectivity, similar to neutral endopeptidase, and another protease that was not inhibited by phenylmethylsulfonyl fluoride (PMSF) corresponded to MlrC and cleaved the resulting medium-sized peptides to smaller peptides or amino acids. The former property was desirable to obtain a suitable surrogate peptide, which was used successfully to quantify peptide using liquid chromatography (LC)-MS. Thus, the present study verified that one of the B-9 proteases has broad cleavage selectivity and cleavage sites, not seen in commercially available proteases, and is applicable to protein and peptide quantification using LC-MS.
authors.affiliation: Laboratory Management Department, Sanwa Kagaku Kenkyusho Co., Ltd., Inabe, Mie 511-0406, Japan; Graduate School of Environmental and Human Science and Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan. Electronic address: a_miyachi@mb4.skk-net.com.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyachi
authors.affiliation: Department of Pharmacology, School of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Kondo
authors.affiliation: Graduate School of Environmental and Human Science and Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Kurita
authors.affiliation: Kanagawa Prefectural Institute of Public Health, Shimomachiya, Chigasaki, Kanagawa 253-0087, Japan.
authors.forename: Kiyomi
authors.initials: K
authors.lastname: Tsuji
authors.affiliation: Graduate School of Environmental and Human Science and Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Harada
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002853:Chromatography, Liquid; D006868:Hydrolysis / Q000187:drug effects; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D042301:Sphingomonadaceae / Q000201:enzymology / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D011480:Protease Inhibitors; D014660:Vasoactive Intestinal Peptide; D010664:Phenylmethylsulfonyl Fluoride; D010447:Peptide Hydrolases
keywords: B-9 strain; Glucagon/vasoactive intestinal polypeptide family peptide; Inhibitor; Protease; Surrogate peptide