Iroa metabolomics workflow for improved accuracy, identification and quantitation

Abstract

An IROA Matrix of metabolite compounds is disclosed. Each of whose compounds has a molecular weight of 2000 AMU or less, and is present as first and second isotopomers that are equally present at two predetermined isotopomeric balances, and contain 2 to 10% of a first isotope, and 90 to 98% of a second isotope, respectively. A reagent pair for transforming a natural abundance mass spectral analysis metabolite sample into an IROA sample is also disclosed and comprises two reactively identical reagents that constitute first and second isotopomers containing 2 to 10% of a first isotope, and 90 to 98% of a second isotope, respectively. Each of the reagent pair contains the same reactive group that reacts with and bonds to a functional group of one or more compounds present in a composition of biologically-produced metabolite compounds. Methods of making and using the above and related materials are also disclosed.

What is claimed: 
     
         1 . An IROA Matrix composition of biologically-produced metabolite compounds, each of said metabolite compounds having a molecular weight of about 2000 AMU or less, and each of said metabolite compounds being present as first and second isotopomers that are equally present at two predetermined isotopomeric balances, said first isotopomers containing about 2 to about 10% of a first isotope, and said second isotopomers containing about 90 to about 98% of a second isotope of the same atom, said first and second isotopes being stable to radioactive decay and other than hydrogen and deuterium. 
     
     
         2 . The IROA Matrix composition according to  claim 1 , wherein said biologically-produced metabolite compounds are obtained from a cell lysate preparation obtained from culture of single-celled or multi-celled organisms, and the molecules are randomly and universally labeled. 
     
     
         3 . The IROA Matrix composition according to  claim 2 , wherein the cells of said cell lysate preparation are obtained from culture of single-celled organisms and the molecules are randomly and universally labeled. 
     
     
         4 . The IROA Matrix composition according to  claim 1 , wherein said first and second isotopes of the same atom are one or more elements selected from the group consisting of isotopes of carbon (12C and 13C), nitrogen (14N and 15N), oxygen (16O, 17O, or 18O), sulfur (32S, 33S, 34S, or 36S), chlorine (35Cl and 37Cl), magnesium (24Mg, 25Mg and 26Mg), silicon (27Si, 28Si and 29Si), calcium (400a, 42Ca, 43Ca, and 44Ca), and bromine (79Br and 81Br). 
     
     
         5 . The IROA Matrix composition according to  claim 1 , wherein said first and second isotopes of the same atom are 12C and 13C. 
     
     
         6 . The IROA Matrix composition according to  claim 1 , wherein each of said metabolite compounds has a molecular weight of about 1500 AMU or less. 
     
     
         7 . A method of creating a reference library of identity data of compounds in an IROA Matrix of  claim 1  that comprises the steps of
 1) mass spectrally determining the identity of the compounds of said IROA Matrix that are within the resolution and sensitivity of the apparatus to provide its symmetrical IROA peak pattern, and additionally determining one or more of:
 a) the gas and/or liquid chromatographic properties of the compounds present, 
 b) the ion mobility of the compounds present, and 
 c) the IROA fragmentation pattern of the compounds present, and 
 
 2) maintaining the compound identity data so determined for use in identifying one or more of the same compounds in a later-analyzed sample. 
 
     
     
         8 . The method according to  claim 7 , wherein the identity of compounds of said IROA Matrix are additionally determined by collisional cross sections of the compounds. 
     
     
         9 . The method according to  claim 7 , wherein the identity of compounds of said IROA Matrix are additionally determined by the IROA fragmentation pattern of the compounds present. 
     
     
         10 . The reference library of identity data of compounds in an IROA Matrix of  claim 7 . 
     
     
         11 . A method of quantifying and identifying compounds in a natural abundance sample using an Internal Standard that is of the same chemical composition as isotopomers containing the about 90 to about 98% of the heavier molecular weight isotope-containing compounds of an IROA Matrix composition are inserted into said natural abundance sample and the so combined sample is analyzed at least by mass spectral analysis, wherein each compound in said Internal Standard is itself identified in a reference library of identity data of  claim 10 . 
     
     
         12 . The method according to  claim 11 , wherein the IROA characteristics of each compound in the Internal Standard are used to support the identity of each compound. 
     
     
         13 . The method according to  claim 11 , wherein the quantity of each natural abundance sample compound is determined. 
     
     
         14 . The method according to  claim 11 , wherein the quantity of each natural abundance sample compound is determined relative to said Internal Standard. 
     
     
         15 . A method of measuring quality assurance and/or a quality control on the operational constancy of a mass spectral apparatus and associated ion mobility channel and chromatographic apparatus, when present, that comprises the steps of assaying the sample of an IROA Matrix composition of  claim 1 , and determining whether the same sets and amplitudes of symmetric IROA mass spectral peaks are present in each analysis. 
     
     
         16 . The method of determining quality assurance and/or a quality control according to  claim 15 , wherein said biologically-produced metabolite compounds are obtained from a cell lysate preparation. 
     
     
         17 . The method of determining quality assurance and/or a quality control according to  claim 15 , wherein the cells of said cell lysate preparation are obtained from culture of single-celled or multi-celled organisms. 
     
     
         18 . The method of determining quality assurance and/or a quality control according to  claim 15 , wherein said first and second isotopes of the same atom are selected from the group consisting of isotopes of carbon (12C and 13C), nitrogen (14N and 15N), oxygen (16O, 17O, or 18O), sulfur (32S, 33S, 34S, or 36S), chlorine (35Cl and 37Cl), magnesium (24Mg, 25Mg and 26Mg), silicon (27Si, 28Si and 29Si), calcium (40Ca, 42Ca, 43Ca, and 44Ca), and bromine (79Br and 81Br). 
     
     
         19 . The method of determining quality assurance and/or a quality control according to  claim 15 , wherein said first and second isotopes of the same atom are 12C and 13C. 
     
     
         20 . The method of determining quality assurance and/or a quality control according to  claim 15 , wherein each of said metabolite compounds has a molecular weight of about 1500 AMU or less. 
     
     
         21 . A reagent pair capable of transforming the biologically-produced metabolite compounds of a natural abundance mass spectral analysis sample into an IROA sample that comprises two reactively identical reagents that constitute first and second isotopomers, said first isotopomers containing about 2 to about 10% of a first isotope, and said second isotopomers containing about 90 to about 98% of a second isotope of the same atom, said first and second isotopes being stable to radioactive decay and being other than hydrogen and deuterium, each of said reagent pair containing the same reactive group that reacts with and bonds to a functional group of one or more compounds present in a composition of biologically-produced metabolite compounds, each of said metabolite compounds having a molecular weight of about 2000 AMU or less. 
     
     
         22 . The reagent pair according to  claim 21 , wherein said reactive group reacts with and bonds to a functional group selected from the group consisting of one or more of an amine, aldehyde or ketone, hydroxyl, thiol and carboxylic acid. 
     
     
         23 . The reagent pair according to  claim 22 , wherein said reactive group reacts with and bonds to an amine functional group. 
     
     
         24 . The reagent pair according to  claim 23 , wherein said reactive group is an isothiocyanate. 
     
     
         25 . The reagent pair according to  claim 24 , wherein said isothiocyanate is phenylisothiocyanate. 
     
     
         26 . The reagent pair according to  claim 22 , wherein said reactive group reacts with and bonds to a ketone or aldehyde group. 
     
     
         27 . The reagent pair according to  claim 26 , wherein said reactive group is a hydrazine or a semicarbazide.