FPLC System for Bioactive Peptides Research

点击翻译按钮获取中文摘要

Isolation, purification, functional characterization, and in vivo testing of bioactive peptides require qualitative and quantitative analysis using the fast protein liquid chromatography (FPLC) system. At the Department of Food and Human Nutritional Sciences, University of Manitoba, there is only one FPLC system, which was purchased in 2004 but is no longer serviceable because the model has been discontinued and there is no suitable FPLC system on the University of Manitoba campuses. Therefore, the aim of this proposal is to replace the non-serviceable 16-year old system with a new FPLC system that will provide critical tools for continued operation of my NSERC-funded Discovery research program on bioactive peptides. The FPLC provides the most rapid and cost-effective means of separating and isolating peptides in forms that can be characterized for structure-function properties or further purified on a higher pressure system (HPLC). Moreover, the FPLC facilitates rapid quantitative estimation of peptides of defined molecular sizes, which is a critical component of structural elucidation and a major goal of my NSERC-funded Discovery research program. The research program focuses on elucidation of the structure-function properties of food protein-derived bioactive peptides, which are usually contained in enzymatic digests of food proteins called protein hydrolysates. Because enzymatic hydrolysates contain a mixture (sometimes hundreds) of peptides, the initial rate-determining step for successful structure-function analysis is separation on an FPLC system. Thus, the FPLC is a required component of my Discovery research work and for successful training of highly qualified personnel (HQP). The FPLC will be used to separate bioactive peptides into fractions that are suitable for structural elucidation (amino acid sequencing) by liquid chromatography-tandem mass spectrometry. Knowledge of the amino acid sequence is required for the discovery of highly potent peptides that can serve as effective modulators of various metabolic pathways. Thus the FPLC system is a valuable tool in my quest to provide fundamental scientific information on the mechanism of action of bioactive peptides as well as the discovery of novel peptides that can reduce or prevent negative metabolic events. The system will also facilitate my research that seeks discovery of highly functional food ingredients that can enhance nutritional status of Canadians while promoting value-added utilization of Canadian crops and animal products. This is because results obtained from the instrument will provide researchers with the ability to design novel food ingredients that can be used to formulate functional foods and nutraceuticals. The FPLC system will be used by graduate and undergraduate students, postdoctoral fellows/research associates, and technicians on a daily basis for peptide fractionation and quantification. Therefore, the instrument is critical to the successful training of HQPs.