Method for automated, large-scale measurement of the molecular flux rates of the proteome or the organeome using mass spectrometry

Abstract

Disclosed here is a method for measuring the kinetics (i.e., the molecular flux rates—synthesis and breakdown or removal rates) of a plurality of proteins or organic metabolites inn living systems. The methods may be accomplished in a high-throughput, large-scale automated manner, by using existing mass spectrometric profiling techniques and art well known in the fields of static proteomics and static organeomics, without the need for additional biochemical preparative steps or analytic / instrumental devices.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. patent application 60 / 399,950 filed on Jul. 30, 2002 which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION [0002] The invention relates to methods for measuring molecular flux rates (synthesis and breakdown or input and removal rates from pools of molecules) in the proteome and the organeome (dynamic proteomics and dynamic organeomics, respectively) using mass spectrometry. The methods disclosed are capable of high-throughput, large-scale, automated applications. The methods are applicable to studies in genetics, functional genomics, drug discovery and development, drug toxicity, clinical diagnostics and patient management. BACKGROUND OF THE INVENTION [0003] Recent advances in the Human Genome project have, paradoxically, led to the wide-spread recognition of the inadequacy of gene sequence information by itself. Sequence information (i.e. structural genomics) is unlike...

AI Technical Summary

Benefits of technology

[0010] In order to meet these needs, the present invention is directed to a method of determining the molecular flux rates (i.e., the rates of synthesis or breakdown of a plurality of proteins in all or a portion of the proteome of a cell, tissue or organism). One or more isotope-labeled protein precursors are administered to a cell, tissue or organism for a period of time sufficient for one or more isotope labels to be incorporated into a plurality of proteins in the proteome or portion thereof of the cell, tissue or organism. The proteome or portion thereof are then obtained from the cell, tissue, or organism. A plurality of mass isotopomeric envelopes representing individual proteins in the proteome or portion of the proteome are then identified by mass spectrometry. In addition, the relative and absolute mass isotopomer abundances of the ions within the isotopomeric envelope corresponding to each identified protein are quantified by mass spectrometry. These relative and absolute mass isotopomer abundances allow the molecular flux rates of each identified protein to be calculated and the molecular flux rates of the plurality of proteins thereby to be determined.

[0018] In another aspect, the invention is drawn to determining the molecular flux rates of a plurality of organic metabolites in all or a portion of the organeome of a cell, tissue or organism. One or more isotope-labeled organic metabolites or organic metabolite precursors are administered to the cell, tissue or organism for a period of time sufficient for one or more isotope labels to be incorporated into a plurality of organic metabolites in the organeome or portion thereof of the cell, tissue or organism. The organeome or portion thereof is obtained from the cell, tissue, or organism. A plurality of mass isotopomeric envelopes of ions representing individual organic metabolites in the organeome or portion thereof are identified by mass spectrometry. In addition, the relative and absolute mass isotopomer abundances of the ions within the isotopic envelopes corresponding to each identified organic metabolite are quantified by mass spectrometry. These relative and absolute mass isotopomer abundances allow the rates of synthesis or removal of each identified organic metabolite to be calculated, and the molecular flux rates of the plurality of organic metabolites thereby to be determined.

[0023] In another aspect, the invention is drawn to methods of administering a diagnostic or therapeutic agent to the cell, tissue, or organism prior to administering the precursor. In one embodiment, the invention is drawn to a method of determining the effect of a diagnostic or therapeutic agent on a cell, tissue, or organism by determining the rates of synthesis or removal of a plurality of organic metabolites in the cell, tissue, or organism, administering an agent, and determining the rates of synthesis or removal on the plurality of organic metabolites in the cell, tissue or organism. By this means, drug discovery and development may be facilitated or achieved.

Problems solved by technology

Enumerating the expressed genome (i.e. the complement of mRNA species), even in its entirety, however, does not ultimately provide information about biochemical function (phenotype) in a living system.

Although impressive as a technology, gene expression chips do not solve the central problems of phenotype and function in biochemistry, which relate to the flow of molecules through the complex interactive network of proteins that comprise fully assembled living systems.

This log-jam arises from bottle-necks in the testing of phenotypic consequences of inhibiting particular targets—i.e. the “tail-end” of drug development.

One of the central problems in this area relates to the absence of routine, high-throughput dynamic measurements in biology and medicine.

Yet rarely, if ever, are rates of biochemical processes measured in medical diagnostics.

Static markers of dynamic processes are often helpful and may be better than nothing, but they are not the true measure of disease activity or disease risk.

Nor do static measures allow for personalized biochemical monitoring.

Thus, the current art in mass spectrometric proteomics and organeomics is characterized by a shared and fundamental limitation: the information is static, not dynamic.

Although the current techniques of static proteome and organeome characterization can provide a snapshot of what is present, these techniques cannot provide information concerning flows of molecules through the system (kinetics).

Images