Grant: Research Tools and Instruments - Category 1 (<$150,000)
Université du Québec à Montréal
$104,298.00
Sample fractionation and HPLC equipment for investigating protein covalent binding by reactive drug metabolites
FY 2008-09
Summary
This grant will be used for the purchase of two pieces of equipment:
1) ) an off-gel (in-solution) isoelectric focusing (IEF) fractionator, and
2) ) an ultra-high pressure liquid chromatography (uHPLC) system with UV detection and fraction collection.
Our research program will be centered on studying different drugs known to be metabolized into chemically-reactive species and covalently bind to proteins. We are seeking to identify critical protein targets as well as pinpointing the precise modifications occurring on these proteins. The formation of reactive drug metabolites and their covalent binding are often linked to toxicity in the cell. However, the exact mechanism causing this binding to induce drug toxicity remains elusive.
In vitro incubations will be performed to study these reactive metabolites and how they bind to proteins. Both metabolite and protein fractions will be characterized by liquid chromatography-mass spectrometry (LC-MS). Protein modifications will be assessed by protein digestion and LC-MS/MS, however, sample preparation steps must precede these analyses in order to discern these changes in complex samples. The equipment for which funds are requested will enable the preparation of drug metabolism samples.
For analyzing protein modifications, we will initially separate samples based on protein isoelectric point. Solution-IEF fractionation is ideal since the resulting soluble fractions are more amenable to LC-MS. Resulting fractions will be less complex and we will have a better chance of visualizing as many peptides or proteins in a single experiment. We can also separate digested peptides prior to LC-MS. The uHPLC system will allow for the separation of metabolites or protein samples with UV detection as well as the possibility to collect fractions. This system will be used for monitoring samples and yielding purified fractions of unknown metabolites or modified proteins for additional characterization. This equipment is essential for our research program using advanced bioanalytical techniques for studying reactive metabolites and protein binding.
Program
Research Tools and Instruments - Category 1 (<$150,000)
Recipient
Location
Quebec, Canada
Recipient Type
Academia
Type
Grant
Source
NSERC's Awards Data
As published
Application id
359667-2008
Name nom
Sleno, Lekha
Department département
Chimie
Organization id
57
Institution établissement
Université du Québec à Montréal
Province en
Québec
Province fr
Québec
Country en
CANADA
Country fr
CANADA
Fiscal year exercice financier
2008
Competition year année de concours
2008
Award amount
104298
Program id
EQPEQ
Program nane 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
26
Committee name en
Anal. & Phys. Chemistry
Committee name fr
Chimie analytique et physique
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
3504
Research subject group en
Analytical chemistry
Research subject group fr
Chimie analytique
Research subject en
Bioanalytical chemistry
Research subject fr
Chimie bioanalytique
Partie
1
Nb partie
1
Application title
Sample fractionation and HPLC equipment for investigating protein covalent binding by reactive drug metabolites
Keyword
NA
Application summary
This grant will be used for the purchase of two pieces of equipment:
1) ) an off-gel (in-solution) isoelectric focusing (IEF) fractionator, and
2) ) an ultra-high pressure liquid chromatography (uHPLC) system with UV detection and fraction collection.
Our research program will be centered on studying different drugs known to be metabolized into chemically-reactive species and covalently bind to proteins. We are seeking to identify critical protein targets as well as pinpointing the precise modifications occurring on these proteins. The formation of reactive drug metabolites and their covalent binding are often linked to toxicity in the cell. However, the exact mechanism causing this binding to induce drug toxicity remains elusive.
In vitro incubations will be performed to study these reactive metabolites and how they bind to proteins. Both metabolite and protein fractions will be characterized by liquid chromatography-mass spectrometry (LC-MS). Protein modifications will be assessed by protein digestion and LC-MS/MS, however, sample preparation steps must precede these analyses in order to discern these changes in complex samples. The equipment for which funds are requested will enable the preparation of drug metabolism samples.
For analyzing protein modifications, we will initially separate samples based on protein isoelectric point. Solution-IEF fractionation is ideal since the resulting soluble fractions are more amenable to LC-MS. Resulting fractions will be less complex and we will have a better chance of visualizing as many peptides or proteins in a single experiment. We can also separate digested peptides prior to LC-MS. The uHPLC system will allow for the separation of metabolites or protein samples with UV detection as well as the possibility to collect fractions. This system will be used for monitoring samples and yielding purified fractions of unknown metabolites or modified proteins for additional characterization. This equipment is essential for our research program using advanced bioanalytical techniques for studying reactive metabolites and protein binding.
