title: Determination of angiotensin-converting enzyme inhibitory peptide Leu-Lys-Pro-Asn-Met (LKPNM) in bonito muscle hydrolysates by LC-MS/MS.
abstract: Proteolytic digestion of dried bonito muscle with thermolysin produces a hydrolysate with strong angiotensin-converting enzyme (ACE) inhibitory activity and is the basis of a dietary supplement with antihypertensive activity. A major portion of the ACE activity was shown previously to arise from the peptide Leu-Lys-Pro-Asn-Met (LKPNM). A straightforward method to quantify this peptide was developed using one-step C18 solid-phase extraction (SPE) followed by LC-MS/MS quantification. The SPE step resulted in a hydrolysate that was still crude, as illustrated by combined size-exclusion chromatography/multi-angle laser light scattering detection that showed that a major fraction of oligopeptides were in the 2-20 kDa range. This fraction has a weight-average molecular weight (M(w)) of approximately 5.0 kDa. Method validation for specificity, linearity, accuracy, precision, and reproducibility showed that standard additions of synthetic LKPNM to bonito extract with SPE enrichment followed by LC-MS/MS is a suitably robust procedure for the determination of LKPNM content. The method was also successful for encapsulated powders in which the excipients used are insoluble in water and could be removed by centrifugation.
authors.affiliation: Ocean Nutrition Canada Ltd., 1721 Lower Water Street, Halifax, Nova Scotia, Canada, B3J 1S5. jcurtis@ocean-nutrition.com
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Curtis
authors.forename: Dorothy
authors.initials: D
authors.lastname: Dennis
authors.forename: David S
authors.initials: DS
authors.lastname: Waddell
authors.forename: Tanya
authors.initials: T
authors.lastname: MacGillivray
authors.forename: H Stephen
authors.initials: HS
authors.lastname: Ewart
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000032:analysis*; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D009124:Muscle Proteins / Q000032:analysis; D009132:Muscles / Q000737:chemistry*; D009842:Oligopeptides / Q000032:analysis*; D010473:Perciformes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D009124:Muscle Proteins; D009842:Oligopeptides; C463485:leucyl-lysyl-prolyl-asparagyl-methionine