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US-12626897-B2 - IROA metabolomics workflow for improved accuracy, identification and quantitation

US12626897B2US 12626897 B2US12626897 B2US 12626897B2US-12626897-B2

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.

Inventors

  • Christopher Beecher
  • Richard A. Yost
  • Robin Hendrikus Johannes Kemperman

Assignees

  • IROA TECHNOLOGIES, LLC

Dates

Publication Date
20260512
Application Date
20251008

Claims (19)

  1. 1 . A method of determining chemical identity of one or more molecules in an experimental sample by performing mass spectral analyses of the experimental sample and concurrently performing quality assurance for an associated mass spectral apparatus, which method comprises: a) preparing an isotopic ratio outlier analysis (IROA) standard sample comprising a known composition of metabolite compounds, which are associated with a library of mass spectral peaks reflecting each of the metabolite compounds, wherein the composition of metabolite compounds comprises a paired set of a first isotopically-labeled compound and a second isotopically-labeled compound, with each isotopically-labeled compound being equally present, wherein about 2% to about 10% by mass of a non-hydrogen/deuterium atom in the first isotopically-labeled compound is a first isotope of the non-hydrogen/deuterium atom and about 90% to about 98% by mass of the non-hydrogen/deuterium atom in the first isotopically-labeled compound is a second isotope of the non-hydrogen/deuterium atom, wherein about 90% to about 98% by mass of the non-hydrogen/deuterium atom in the second isotopically-labeled compound is the first isotope of the non-hydrogen/deuterium atom and about 2% to about 10% by mass of the non-hydrogen/deuterium atom in the second isotopically-labeled compound is the second isotope of the non-hydrogen/deuterium atom, wherein said first isotope atom and said second isotope atom are not the same, wherein the first isotope atom and the second isotope atom are stable to radioactive decay, b) preparing an experimental sample comprising one or more molecules and further comprising an Internal Standard comprising a metabolite compound of the IROA standard sample, said metabolite compound isotopically-labeled with only the heavier isotope of the first isotope and the second isotope of the non-hydrogen/deuterium atom; c) subjecting the IROA standard sample and the experimental sample to liquid chromatography coupled to mass spectrometry (LC/MS) to produce eluted components of the IROA standard sample and the experimental sample; d) separately ionizing the eluted components of the IROA standard sample and the experimental sample via mass spectrometer to produce ions of the IROA standard sample and ions of the experimental sample; e) implementing a windowing scheme comprising variable width and/or variable overlapping fragmentation windows; f) fragmenting the ions using a mass spectrometer to produce fragment ions, and, correspondingly, a pattern of mass spectral peaks for the IROA standard sample and a pattern of mass spectral peaks for the experimental sample; g) comparing the pattern of mass spectral peaks of the heavier isotope from the Internal Standard to the mass spectral pattern of peaks of the first isotopically-labeled compound and the second isotopically labeled compound from the IROA standard sample; h) comparing the pattern of mass spectral peaks of the heavier isotope from the Internal Standard to the mass spectral pattern of peaks of the one or more molecules of the experimental sample, and comparing the LC retention time of the Internal Standard to the LC retention time of the experimental sample, to identify the one or more molecules in the experimental sample; and i) determining whether the mass spectral pattern of peaks found for the IROA standard sample matches the reference library's mass spectral peaks for the IROA standard sample, in order to assess performance of the associated mass spectral apparatus, and if variances are found, correcting for those variances.
  2. 2 . The method of claim 1 , further comprising comparing other data associated with the IROA standard sample to that of the experimental sample wherein the other data comprises ion mobility data, UV/vis data, molecular formula, collision cross section data, and/or IR data.
  3. 3 . The method of claim 2 , wherein the other data comprises collision cross section data.
  4. 4 . The method according to claim 1 , wherein the metabolite compounds comprise a molecular weight of about 2000 AMU or less.
  5. 5 . The method according to claim 1 , wherein said metabolite compounds are biologically-produced or synthetically-produced metabolite compounds.
  6. 6 . The method according to claim 5 , wherein said biologically-produced metabolite compounds are obtained from a cell lysate preparation obtained from culture of single-celled organisms comprising yeast, bacteria, or alga.
  7. 7 . The method according to claim 5 , wherein the biologically-produced metabolite compounds are prepared from a culture of yeast.
  8. 8 . The method according to claim 7 , wherein the yeast is Saccharomyces cerevisiae.
  9. 9 . The method according to claim 7 , wherein the culture of yeast grows on about 2% to about 10% by mass of 13 C U-glucose carbon source and, in a separate run, the culture of yeast grows on about 90% to about 98% by mass 13 C U-glucose carbon source.
  10. 10 . The method according to claim 7 , wherein the culture of yeast grows on a 95% by mass 13 C U-glucose carbon source and, in a separate run, the culture of yeast grows on a 5% by mass 13 C U-glucose carbon source.
  11. 11 . The method according to claim 1 , wherein the metabolite compounds are randomly and universally labeled.
  12. 12 . The method according to claim 1 , wherein the paired set of the first isotopically-labeled compound and the second isotopically-labeled compound comprise isotopes of carbon ( 12 C and 13 C), nitrogen ( 14 N and 15 N), oxygen ( 16 O, 17 O, or 18 O), sulfur ( 32 S, 33 S, 34 S, or 36 S), chlorine ( 35 Cl and 37 Cl), magnesium ( 24 Mg, 25 Mg and 26 Mg), silicon ( 27 Si, 28 Si and 29 Si), calcium ( 40 Ca, 42 Ca, 43 Ca, and 44 Ca), or bromine ( 19 Br and 81 Br).
  13. 13 . The method according to claim 12 , wherein the paired set of the first isotopically-labeled compound and the second isotopically-labeled compound comprises 12 C and 13 C.
  14. 14 . The method according to claim 1 , wherein the first isotopically-labeled compound comprises approximately 5% 13 C by mass as a first isotope of C atom and approximately 95% 12 C by mass as a second isotope of C atom; and wherein the second isotopically labeled compound comprises approximately 95% 13 C by mass as the first isotope of C atom and approximately 5% 12 C by mass as the second isotope of C atom.
  15. 15 . The method according to claim 1 , wherein each of said metabolite compounds has a molecular weight between 60 AMU to 1500 AMU.
  16. 16 . The method according to claim 1 , wherein each of said metabolite compounds has a molecular weight less than 1000 AMU.
  17. 17 . The method according to claim 1 , further comprising storing the pattern of mass spectral peaks for the experimental sample and other data from the IROA standard sample to determine trends in the mass spectral apparatus.
  18. 18 . The method according to claim 1 , wherein the windowing scheme comprises an msms wide-windowing system.
  19. 19 . The method according to claim 18 , wherein the msms wide-windowing system is a SWATH system.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 19/236,271, filed Jun. 12, 2025, which is a continuation of U.S. patent application Ser. No. 19/014,907, filed Jan. 9, 2025, now U.S. Pat. No. 12,354,859, which is a continuation of U.S. patent application Ser. No. 17/321,009, filed May 14, 2021, now U.S. Pat. No. 12,230,488, which is a division of U.S. patent application Ser. No. 15/905,452, filed Feb. 26, 2018, which claims benefit of Provisional Patent application No. 62/463,153, filed on Feb. 24, 2017, whose disclosures are incorporated by reference. BACKGROUND ART Metabolites are small molecular weight compounds (less than about 2000 Da and more usually less than about 1000 Da) that are employed as building blocks or produced as end products in various metabolic pathways and cellular regulatory processes in a biological system. The entire collection of metabolites in a biological system, whether at the cellular, pathway or organism level, is known as a “metabolome”. Levels of these metabolites in a metabolome are either dictated by the genome, proteome, and/or transcriptome of the biological system or imposed by environmental perturbations and results in changes in phenotype. Thus, metabolomics can be applied to map or identify the cause of alteration in phenotype and understand correlations between “omics”. Dwivedi, et al., Int J Mass Spectrom 2010 298:78-90. Isotopic Ratio Outlier Analysis (IROA) has been developed to enable the characterization of carbon information in a given metabolites or a fragment. Unlike other stable isotope labeling methods, rather than utilizing substrates with natural abundance (1.1% of 13C isotopomer seen in carbon atoms in nature) and 98-99% enrichment for the control and experimental populations, respectively, IROA with prototrophic yeast uses randomized 95% 12C glucose (5% 13C), and 95% randomized 13C glucose (5% 12C) as carbon sources. This strategy leads to more predictable and diagnostic patterns for the observable isotopic peaks in the mass spectra. [Qiu et al., Metabolites 2018 8:9]. The promise of IROA for metabolic phenotyping has been demonstrated in model organism studies. Saccharomyces cerevisiae, a prototrophic wild-type strain in the CEN. PK background [Brauer et al., Mol. Biol. Cell 2005, 16:2503-2517] was grown in minimal yeast nitrogen base (YNB) media, containing either randomized 95% 12C, or 95% 13C glucose as the main carbon source, in order that the isotopomer pattern of all metabolites would mirror the labeled glucose [Qiu et al., Anal. Chem. 2016, 88:2747-2754], a protocol that can easily be adapted for microbial species studies. The abundance of the light isotopologues in the 95% 13C samples (Mn-1, Mn-2, etc., the 13C envelope) or the heavy isotopologues in the 95% 12C samples (M0+1, M0+2, etc., the 12C envelope), follows the binomial distribution for 13C, based on the initial substrate enrichment, in the metabolite products generated. The mass difference between the 12C (M0) isotopic peak and the 13C (Mn) isotopic peak indicates the number of carbons (n) in the metabolite's carbon backbone. This narrows possibilities for chemical formula generation (CFG) and for normalization between control (13C) and treated (12C) groups. [Qiu et al., Metabolites 2018 8:9]. It is possible to use metabolomic techniques, such as the IROA basic, or IROA phenotypic protocols (optimally) [de Jong and Beecher, C. Bioanalysis 2012, 4 (18):2303-2314], or standard metabolomic techniques to identify and crudely quantify several hundred or even thousands of compounds in a biological sample. However, to make such measurements and to compare the measurements from any two or more samples, all the samples need to be analyzed in a single batch, ideally during a single day because day-to-day variances are too great to otherwise overcome, and absolute quantitation; i.e., relative to a known standard, cannot be assured. It is currently not quantitatively acceptable to compare samples run on the same instrumentation several days apart, and impossible to compare data generated on different instruments, or based on different methods. Instrument drift, chromatographic drift, and even environmental conditions can alter results sufficiently so that reproducibility is hard to obtain even on the same instrument. In addition to these problems of quantitation, the identification of any compound across many mass spectral techniques alone is unlikely to be successful unless very careful calibrations have been made and authentic standards are run. This is because, not only are there multiple biological compounds that can be confused because they have the same exact mass but, even more problematic, there are often more artefactual or fragmentary compounds that are structurally different from, but can share the correct mass, or even formulae, as biological isobaric equivalents. The invention disclosed hereinafter extends methods describe