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 in 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.

1 - 23 . (canceled) 
     
     
         24 . A method of 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, said method comprising:
 a. administering one or more isotope-labeled organic metabolites or organic metabolite precursors to said cell, tissue or organism for a period of time sufficient for one or more isotope labels from said one or more isotope-labeled organic metabolites to be incorporated into the a plurality of organic metabolites in the organeome or portion thereof of the cell, tissue or organism;   b. obtaining the organeome or portion thereof from the cell, tissue, or organism;   c. identifying a plurality of the mass isotopomeric envelopes of ions representing individual organic metabolites in the organeome or portion thereof by mass spectrometry;   d. quantifying relative and absolute mass isotopomer abundances of ions within the mass isotopomeric envelope corresponding to each organic metabolite identified in step (c) by mass spectrometry; and   e. calculating the rates of synthesis or removal of the identified organic metabolites to determine the rates of synthesis or removal of said plurality of organic metabolites.   
     
     
         25 . The method of  claim 24 , wherein the one or more organic metabolites or organic metabolite precursors are selected from the group consisting of H 2 O, CO 2 , NH 3 , HCO 3 , amino acids, monosaccharides, carbohydrates, lipids, fatty acids, nucleic acids, glycolytic intermediates, acetic acid, and tricarboxylic acid cycle intermediates. 
     
     
         26 . The method of  claim 24 , wherein the isotope label is selected from the group consisting of  2 H,  13 C,  15 N,  18 O,  33 S or  34 S. 
     
     
         27 . The method of  claim 24 , wherein said administering step (a) is continuous. 
     
     
         28 . The method of  claim 24 , wherein said administering step (a) comprises administering said precursor at regular measured intervals. 
     
     
         29 . The method of  claim 24 , wherein the one or more organic metabolites or organic metabolite precursors are administered orally. 
     
     
         30 . The method of  claim 24 , comprising the additional step of discontinuing said administering step (a). 
     
     
         31 . The method of  claim 24 , further comprising modifying said organic metabolites prior to said measuring step. 
     
     
         32 . The method of  claim 24 , wherein said identifying step (c) further comprises identifying individual organic metabolites in the organeome or portion thereof by chromatography. 
     
     
         33 . The method of  claim 24 , wherein said plurality of organic metabolites comprises the organeome of said cell, tissue, or organism. 
     
     
         34 . The method of  claim 24 , further comprising the step of displaying the rates of synthesis or removal of said plurality of organic metabolites. 
     
     
         35 . The method of  claim 24 , wherein the organic metabolite precursor is selected from the group consisting of H 2 O, CO 2 , NH 3 , and HCO 3 . 
     
     
         36 . The method of  claim 26 , wherein said isotope is  2 H. 
     
     
         37 . The method of  claim 35 , wherein said organic metabolic precursor is H 2 O. 
     
     
         38 . The method of  claim 24 , wherein said isotope-labeled organic metabolite precursor is selected from the group consisting of  2 H 2 O,  3 H 2 O, and H 2   18 O. 
     
     
         39 . The method of  claim 38 , wherein said isotope-labeled organic metabolite precursor is  2 H 2 O. 
     
     
         40 . The method of  claim 24 , wherein the organism is a human. 
     
     
         41 . The method of  claim 24 , further comprising administering a diagnostic or therapeutic agent to said cell, tissue, or organism prior to said administering step (a). 
     
     
         42 - 47 . (canceled)