Grant: Research Tools and Instruments - Category 1 (<$150,000)
Carleton University
$42,782.00
High-throughput fluorescence HPLC detection of low abundance metabolites and functional groups
FY 2007-08
Summary
One of the challenges of modern day biochemistry is the detection of trace amounts of compounds in complex biological mixtures. Such compounds include metabolic intermediates that are either transitory in nature or present in undetectable levels. Derivatization of compounds with fluorescent adducts greatly enhances the ability to detect and to quantify them as well as preventing their metabolic turnover. The requested equipment will support three research programs with an interest in detection of trace compounds in biological samples. These includes the detection of sulfhydryl groups present on a) proteins, b) low molecular weight sulfhydryl containing antioxidants such as glutathione, cysteine and homocysteine and c) mixed disulfides between low molecular weight sulfhydryl compounds and proteins. Fluorescence detection of free thiols can be performed by High Performance Liquid Chromatography (HPLC) after derivatizing reduced sulfhydryl groups with either N-(1-pyrenyl)maleimide or monobromobimane. This technique allows for detection of micromolar (ppm) amounts of free thiols. Fluorescence HPLC will also be utilized to detect fluorescent metabolites of fatty acids. The requested equipment will build upon a previously purchased Varian HPLC system with electrochemical and photodiode array detectors. The equipment purchased with this grant will be used on a daily basis in the research and training of undergraduates, graduate students, postdoctoral researchers and visiting scientists in the field trace compound detection. The equipment includes a fluorescence detector for the HPLC as well as an autosampler for high throughput processing of samples. These additions to the HPLC will enable us to determine the reduced vs. oxidized state of low molecular weight as well as protein thiols within the cell as well as metabolic intermediates of biosynthetic (lipids) pathways. It will also be used for cross-disciplinary studies of fluorescent compounds contained within complex animal and plant samples.
Program
Research Tools and Instruments - Category 1 (<$150,000)
Recipient
Location
Ontario, Canada
Recipient Type
Academia
Type
Grant
Source
NSERC's Awards Data
As published
Application id
359782-2008
Name nom
Willmore, William
Department département
Biochemistry, Institute of
Organization id
23
Institution établissement
Carleton University
Province en
Ontario
Province fr
Ontario
Country en
CANADA
Country fr
CANADA
Fiscal year exercice financier
2007
Competition year année de concours
2008
Award amount
42782
Program id
EQPEQ
Program name en
Research Tools and Instruments - Category 1 (<$150,000)
Program name fr
Outils et instruments de recherche - catégorie 1 (<150 000$)
Group en
Discovery grants programs
Group fr
Programmes de subventions à la découverte
Committee code
32
Committee name en
Cell Biology
Committee name fr
Biologie cellulaire
Area of application code
1201
Area of application group en
Advancement of knowledge
Area of application group fr
Avancement des connaissances
Area of application en
Life sciences (including biotechnology)
Area of application fr
Sciences de la vie (y compris biotechnologie)
Research subject code
5400
Research subject group en
Biochemistry
Research subject group fr
Biochimie
Research subject en
Biochemistry
Research subject fr
Biochimie
Partie
1
Nb partie
1
Application title
High-throughput fluorescence HPLC detection of low abundance metabolites and functional groups
Keyword
NA
Application summary
One of the challenges of modern day biochemistry is the detection of trace amounts of compounds in complex biological mixtures. Such compounds include metabolic intermediates that are either transitory in nature or present in undetectable levels. Derivatization of compounds with fluorescent adducts greatly enhances the ability to detect and to quantify them as well as preventing their metabolic turnover. The requested equipment will support three research programs with an interest in detection of trace compounds in biological samples. These includes the detection of sulfhydryl groups present on a) proteins, b) low molecular weight sulfhydryl containing antioxidants such as glutathione, cysteine and homocysteine and c) mixed disulfides between low molecular weight sulfhydryl compounds and proteins. Fluorescence detection of free thiols can be performed by High Performance Liquid Chromatography (HPLC) after derivatizing reduced sulfhydryl groups with either N-(1-pyrenyl)maleimide or monobromobimane. This technique allows for detection of micromolar (ppm) amounts of free thiols. Fluorescence HPLC will also be utilized to detect fluorescent metabolites of fatty acids. The requested equipment will build upon a previously purchased Varian HPLC system with electrochemical and photodiode array detectors. The equipment purchased with this grant will be used on a daily basis in the research and training of undergraduates, graduate students, postdoctoral researchers and visiting scientists in the field trace compound detection. The equipment includes a fluorescence detector for the HPLC as well as an autosampler for high throughput processing of samples. These additions to the HPLC will enable us to determine the reduced vs. oxidized state of low molecular weight as well as protein thiols within the cell as well as metabolic intermediates of biosynthetic (lipids) pathways. It will also be used for cross-disciplinary studies of fluorescent compounds contained within complex animal and plant samples.
