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Nmr-based metabolite screening platform

a metabolite and platform technology, applied in the field of nmr-based metabolite screening platforms, can solve the problems of limiting analysis, not all metabolites, including nucleotide analogs and lipids, are easily ionizable, and cannot be detected via mass spectrometry, so as to improve the dynamic range data reconstruction, simplify statistical analysis, and improve the effect of nmr efficiency

Inactive Publication Date: 2018-06-14
PRESIDENT & FELLOWS OF HARVARD COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a new method for screening metabolites in cells using a combination of techniques such as stable isotope labeling, spectral width folding, random phase sampling, non-uniform sampling, and data extension for enhanced dynamic range data reconstruction. This platform allows for global, unbiased, ultra-high resolution analysis of both water-soluble and lipid-based metabolites. The method can identify differentially expressed metabolites in cells, making it useful for identifying therapeutic targets and diagnostic markers. The patent also describes how the method can be used to determine the dosage, toxicity, and therapeutic efficacy of therapeutic compounds.

Problems solved by technology

Untargeted mass spectrometry approaches are possible but require several rounds of purification and further identification methods.
In addition, not all metabolites, including nucleotide analogs and lipids, are easily ionizable and thus cannot be detected via mass spectrometry.
Further, the fragmentation pattern resulting from mass spectrometry is not always suitable to distinguish between molecules such as sugars that have equal mass, but different structures, hence limiting the analysis.

Method used

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Examples

Experimental program
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example 1

gy for the Screening Platform Utilizing NMR

[0097]Preparation of Biological Sample:

[0098]FIG. 1 provides an overall description of the platform. Approximately 20 million cells were used for each sample (however it is possible to use as few as 2 million). Before harvesting, 13C-labeled precursors (glucose and glutamine in these examples) were added directly to the media and allowed to incubate for a user-defined amount of a time and in this case 4 hrs. After aspirating the media and washing two times with phosphate-buffered saline (PBS), the cells were again counted and collected. An equal number of cells with no label were also harvested to serve as a 13C background control. Cells were lysed by the addition of ice-cold methanol, and an aqueous extraction was performed by adding equal parts water and chloroform. After centrifugation the water soluble and organic metabolites were separately collected, dried, and stored until ready to be further analyzed. No additional purification was ...

example 2

ra High Resolution NMR Data Acquisition

[0102]Folding the Spectra:

[0103]While the aforementioned sample preparation alleviated the physical demands on the amount sample, the long acquisition time required to record high resolution 2D NMR spectroscopy was still a concern. To combat this, a multi-prong approach was taken: “folding” the spectra width, using random phase sampling (RPS), implementing non-uniform sampling (NUS) techniques and data extension in the analysis.

[0104]As described above, the spectral width (sw) is the range of frequencies over which NMR signals are to be detected. Metabolite mixtures contain diverse molecules, and the spectral width necessary to cover all potential carbon chemical shifts spans over −220 ppm. In FIG. 2A, the HSQC spectra for all water soluble metabolites in KRAS mutant pancreatic cancer cells is shown. In this instance the 13C-sw spans 220 ppm and a total 1024 points were collected. By solving the equation, resolution=SW / TD (where TD is the total...

example 3

Metabolite NMR Data

[0112]NMR Analysis: As summarized in FIG. 4, a custom “NMR Metabolite Array” program was created to automate the NMR analysis process using the method herein described. First, the spectra are phased, aligned, and scaled with an internal control. 1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was added into each sample, and its concentration was used as a reference to allow relative quantification comparisons between samples to be made. We created an automatic peak picking program to generate peak lists for each sample, where each resonance peak was converted into an X, Y coordinate with an intensity value. Next we created a “MASTER PEAK LIST” program that generates a “master” look up table for all the resonances in the spectra under investigation and was subsequently run.

[0113]In short, this program reads all X, Y points from the individual peak list files, removes duplicates within defined tolerances and writes the resulting set of peaks to a standard outp...

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Abstract

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 13C, 15N, or 31P). 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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of and claims priority to U.S. patent application Ser. No. 14 / 377,257, filed on Aug. 7, 2014, which claims priority to International Patent Application Serial No. PCT / US2013 / 025628, filed on Feb. 11, 2013, which claims benefit of prior U.S. Provisional Application Ser. No. 61 / 597,298, filed on Feb. 10, 2012. The above applications are incorporated herein by reference in their entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH[0002]The inventions were made with Government support under R21 AI087431 awarded by the National Institutes of Health. The government has certain rights in the inventions.FIELD OF THE INVENTION[0003]The invention relates to NMR-based screening platforms.BACKGROUND OF THE INVENTION[0004]The metabolic output of a cell is the summation of the functional genomic, transcriptomic and proteomic networks that define that cell type. Metabolomics is the comprehensive and simultaneous syste...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/50G01R33/465G01R33/46G01N24/08
CPCG01N33/5038A61B5/055G01N24/08G01R33/4633G01R33/465G01N33/5005G01N2458/15G01N2570/00A61P35/00A61P43/00G01N33/5011G01N2500/10
Inventor O'DAY, ELIZABETH M.LIEBERMAN, JUDYWAGNER, GERHARD
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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