NMR-based metabolite screening platform
Methods that enable one to specifically measure the metabolic product of a particular molecule in relatively few cells, e.g. primary cells, are described. The methods involve optionally preloading cells with labeled substrate (e.g. labeled by 13 C, 15 N, or 31 P). The methods allow for easy identification of metabolites that are differentially generated in cells of different phenotypes. The new methods for unbiased multi-dimensional NMR screening and rapid and efficient analysis of the NMR screening identify differentially expressed metabolites in different cell or tissue types. Analysis of the differentially expressed metabolites can present unique druggable targets to which small molecule therapeutics can be designed.
1. A method for monitoring metabolism of a substrate within a given type of cell in a sample, the method comprising:
a. culturing a given type of cell of a first sample with a substrate for a sufficient period of time to allow metabolic breakdown of the substrate into substrate metabolites, wherein at least a portion of the substrate is labeled with a nuclear magnetic resonance (NMR) stable isotope;
b. harvesting the substrate metabolites from the cells of step (a) to obtain a second sample of substrate metabolites; and
c. performing multi-dimensional NMR on the second sample of step (b) to determine a resonance spectrum of the metabolized substrate, wherein the resonance spectrum represents the metabolites of the substrate, and
wherein the multi-dimensional NMR comprises any one of the following techniques:
spectral width folding, random phase sampling in which a phase of a signal detector is randomly alternated between two values at different time points in an NMR signal, non-uniform sampling in which a parameter of an indirect domain of the multi-dimensional NMR is varied by an amount that changes with each experiment during performance of the multi-dimensional NMR, and data extension for enhanced dynamic range data reconstruction in which a number of data points in the indirect domain is increased after performing the multi-dimensional NMR.
2. The method of claim 1 , wherein the multi-dimensional NMR comprises any two, in any combination, of the following techniques: spectral width folding, random phase sampling, non-uniform sampling, and data extension for enhanced dynamic range data reconstruction.
3. The method of claim 1 , wherein the multi-dimensional NMR comprises all four of the following techniques: spectral width folding, random phase sampling, non-uniform sampling, and data extension for enhanced dynamic range data reconstruction.
4. The method of claim 1 , wherein the substrate is labeled with a stable isotope and the multi-dimensional NMR comprises random phase sampling, non-uniform sampling, and data extension for enhanced dynamic range data reconstruction.
5. The method of claim 1 , wherein the substrate metabolites that are present in the sample are not purified away from the other molecules in the sample.
6. The method of claim 1 , wherein the substrate concentration within the population of cells is reduced for a period of time prior to loading the cells with the NMR-labeled substrate.
7. The method of claim 1 , wherein the resonances of the metabolites of the labeled substrate are determined using NMR pulse programs or filtering techniques, or both, customized to the substrate.
8. The method of claim 1 , wherein the number of cells within the population of cells is less than 2×10 6 .
9. A method for identifying differentially expressed substrate metabolites between a first population of cells and a second population of cells, the method comprising:
a. loading a first and a second population of cells with a nuclear magnetic resonance (NMR) stable isotope-labeled substrate;
b. culturing the first and the second population of cells of step (a) for a sufficient period of time to allow metabolic breakdown of the substrate into substrate metabolites;
c. harvesting the substrate metabolites from the first and the second population cells of step (b) to obtain a sample of substrate metabolites from each of the first and the second cell populations;
d. performing multi-dimensional NMR on the sample of step (c) for each of the first and the second cell populations to determine a resonance spectrum of the metabolized substrate of the first population of cells and of the second population of cells, wherein the resonance spectrum represents the metabolites of the substrate, wherein the multi-dimensional NMR comprises any one of the following techniques: spectral width folding, random phase sampling in which a phase of a signal detector is randomly alternated between two values at different time points in an NMR signal, non-uniform sampling in which a parameter of an indirect domain of the multi-dimensional NMR is varied by an amount that changes with each experiment during performance of the multi-dimensional NMR, and data extension for enhanced dynamic range data reconstruction in which a number of data points in the indirect domain is increased after performing the multi-dimensional NMR; and
e. comparing the resonance spectrum of the first population of cells with the resonance spectrum of the second population of cells to determine which resonances are differentially expressed, wherein the differentially expressed resonances provide a resonance signature that represents differentially expressed metabolites.
10. The method of claim 9 , wherein the substrate is labeled with a stable isotope and the multi-dimensional NMR comprises any two, in any combination, of the following techniques: spectral width folding, random phase sampling, non-uniform sampling, and data extension for enhanced dynamic range data reconstruction.
11. The method of claim 9 , wherein the substrate metabolites that are present in the sample are not purified away from the other molecules in the sample.
12. The method of claim 9 , wherein the population of cells in each of the first cell population and second cell population is a heterogeneous population of cells, or is a homogeneous population of cells.
13. The method of claim 9 , further comprising comparing the resonance signature of step (e) with a database of known resonance signatures to determine the molecular structure that the resonance signature represents, and thereby determine the substrate metabolites that are differentially expressed between the first and the second population of cells.
14. The method of claim 9 , further comprising identifying a biosynthetic pathway involved in generation of the substrate metabolites and identifying proteins/enzymes of the pathway that may be targeted to modulate the differential expression of the metabolite, to thereby modulate the phenotype of the cells.
15. The method of claim 9 , wherein the first population of cells and the second population of cells have different phenotypes.
16. The method of claim 9 , wherein the first population of cells is a control population of cells and the second population of cells has been contacted with a test compound or agent.