Mass Spectrometry in Metabolomics pp 147-167

Part of the Methods in Molecular Biology book series (MIMB, volume 1198) | Cite as

Stable Isotope-Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

  • Richard M. Higashi
  • Teresa W.-M. Fan
  • Pawel K. Lorkiewicz
  • Hunter N. B. Moseley
  • Andrew N. Lane
Protocol

Abstract

Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics are poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, stable isotope-resolved metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks in a wide range of experimental systems, including human subjects. MS offers a wide range of instrumental capabilities involving different levels of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS that is affordable by many individual laboratories to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter focuses on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM.

Key words

Metabolomics Stable isotope Mass spectrometry FT-MS GC-MS 

References

  1. 1.
    Schlotterbeck G, Ross A, Dieterle F, Senn H (2006) Metabolic profiling technologies for biomarker discovery in biomedicine and drug development. Pharmacogenomics 7:1055–1075PubMedCrossRefGoogle Scholar
  2. 2.
    Oresic M, Vidal-Puig A, Hanninen V (2006) Metabolomic approaches to phenotype characterization and applications to complex diseases. Expert Rev Mol Diagn 6:575–585PubMedCrossRefGoogle Scholar
  3. 3.
    Dunn W, Broadhurst D, Deepak S, Buch M, McDowell G, Spasic I, Ellis D, Brooks N, Kell D, Neyses L (2007) Serum metabolomics reveals many novel metabolic markers of heart failure, including pseudouridine and 2-oxoglutarate. Metabolomics 3:413–426CrossRefGoogle Scholar
  4. 4.
    Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu J, Laxman B, Mehra R, Lonigro RJ, Li Y, Nyati MK, Ahsan A, Kalyana-Sundaram S, Han B, Cao X, Byun J, Omenn GS, Ghosh D, Pennathur S, Alexander DC, Berger A, Shuster JR, Wei JT, Varambally S, Beecher C, Chinnaiyan AM (2009) Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 457:910–914PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Griffiths WJ, YuqinWang TK, Kohl M, Enot DP, Deigner H-P (2010) Targeted metabolomics for biomarker discovery. Angew Chem Int Ed 49:5426–5446CrossRefGoogle Scholar
  6. 6.
    Theodoridis G, Gika HG, Wilson ID (2011) Mass spectrometry-based holistic analytical approaches for metabolite profiling in systems biology studies. Mass Spectrom Rev 30:884–906PubMedGoogle Scholar
  7. 7.
    Want EJ, Smith CA, Qin CA, VanHorne KC, Siuzdak G (2006) Phospholipid capture combined with non-linear chromatographic correction for improved serum metabolite profiling. Metabolomics 2:145–154CrossRefGoogle Scholar
  8. 8.
    Clayton TA, Lindon JC, Cloarec O, Antti H, Charuel C, Hanton G, Provost J-P, Le Net J-LØ, Baker D, Walley RJ, Everett JR, Nicholson JK (2006) Pharmaco-metabonomic phenotyping and personalized drug treatment. Nature 440:1073–1077PubMedCrossRefGoogle Scholar
  9. 9.
    Harrigan GG, Brackett DJ, Boros LG (2005) Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics. Mini Rev Med Chem 5:13–20PubMedCrossRefGoogle Scholar
  10. 10.
    Boros LG (2005) Metabolic targeted therapy of cancer: current tracer technologies and future drug design strategies in the old metabolic network. Metabolomics 1:11–15CrossRefGoogle Scholar
  11. 11.
    Fan TWM, Higashi RM, Lane AN (2006) Integrating metabolomics and transcriptomics for probing Se anticancer mechanisms. Drug Metab Rev 38:707–732PubMedCrossRefGoogle Scholar
  12. 12.
    Robertson DG (2005) Metabonomics in toxicology: a review. Toxicol Sci 85:809–822PubMedCrossRefGoogle Scholar
  13. 13.
    Griffin JL, Bollard ME (2004) Metabonomics: its potential as a tool in toxicology for safety assessment and data integration. Curr Drug Metab 5:389–398PubMedCrossRefGoogle Scholar
  14. 14.
    Chen C, Gonzalez FJ, Idle JR (2007) LC-MS-based metabolomics in drug metabolism. Drug Metab Rev 39:581–597PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Fan TWM, Lorkiewicz P, Sellers K, Moseley HNB, Higashi RM, Lane AN (2012) Stable isotope-resolved metabolomics and applications for drug development. Pharmacol Ther 133:366–391PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Lane AN, Fan TW, Higashi RM (2008) Isotopomer-based metabolomic analysis by NMR and mass spectrometry. Methods Cell Biol 84:541–588PubMedCrossRefGoogle Scholar
  17. 17.
    Fan TW, Lane AN, Higashi RM, Farag MA, Gao H, Bousamra M, Miller DM (2009) Altered regulation of metabolic pathways in human lung cancer discerned by (13)C stable isotope-resolved metabolomics (SIRM). Mol Cancer 8:41PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Fan TW-M, Yuan P, Lane AN, Higashi RM, Wang Y, Hamidi A, Zhou R, Guitart X, Chen G, Manji HKM, Kaddurah-Daouk R (2010) Stable isotope-resolved metabolomic analysis of lithium effects on glial-neuronal metabolism and interactions. Metabolomics 6:165–179PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) (13)C-based metabolic flux analysis. Nat Protoc 4:878–892PubMedCrossRefGoogle Scholar
  20. 20.
    Fan T, Lane A, Higashi R, Yan J (2011) Stable isotope resolved metabolomics of lung cancer in a SCID mouse model. Metabolomics 7:257–269PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Moseley H, Lane A, Belshoff A, Higashi R, Fan T (2011) A novel deconvolution method for modeling UDP-GlcNAc biosynthetic pathways based on 13C mass isotopologue profiles under non steady-state conditions. BMC Biol 9:37PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Hiller K, Metallo C, Stephanopoulos G (2011) Elucidation of cellular metabolism via metabolomics and stable-isotope assisted metabolomics. Curr Pharm Biotechnol 12:1075–1086PubMedCrossRefGoogle Scholar
  23. 23.
    Fan TW, Lane AN (2011) NMR-based stable isotope resolved metabolomics in systems biochemistry. J Biomol NMR 49:267–280PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Lorkiewicz PK, Higashii RM, Lane AN, Fan TW-M (2011) High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS. Metabolomics 8:930–939PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Le A, Lane AN, Hamaker M, Bose S, Gouw A, Barbi J, Tsukamoto T, Rojas CJ, Slusher BS, Zhang H, Zimmerman LJ, Liebler DC, Slebos RJC, Lorkiewicz PK, Higashi RM, Fan TWM, Dang CV (2012) Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metab 15:110–121PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Boros LG, Lerner MR, Morgan DL, Taylor SL, Smith BJ, Postier RG, Brackett DJ (2005) [1,2-13C2]-D-glucose profiles of the serum, liver, pancreas, and DMBA-induced pancreatic tumors of rats. Pancreas 31:337–343PubMedCrossRefGoogle Scholar
  27. 27.
    Bian F, Kasumov T, Thomas KR, Jobbins KA, David F, Minkler PE, Hoppel CL, Brunengraber H (2005) Peroxisomal and mitochondrial oxidation of fatty acids in the heart, assessed from the C-13 labeling of malonyl-CoA and the acetyl moiety of citrate. J Biol Chem 280:9265–9271PubMedCrossRefGoogle Scholar
  28. 28.
    Olszewski KL, Mather MW, Morrisey JM, Garcia BA, Vaidya AB, Rabinowitz JD, Llinas M (2010) Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature 466:774–778PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Fan TW-M, Lane AN, Higashi RM (2004) The promise of metabolomics in cancer molecular therapeutics. Curr Opin Mol Ther 6:584–592PubMedGoogle Scholar
  30. 30.
    Arita M. Personal Communication from metabolome database downloadGoogle Scholar
  31. 31.
    Moseley HNB (2013) Error analysis and propagation in metabolomics data analysis. Comp Struct Biotechnol J 4:e201301006Google Scholar
  32. 32.
    Fan TWM, Higashi RM, Lane AN, Jardetzky O (1986) Combined use of proton NMR and gas chromatography-mass spectra for metabolite monitoring and in vivo proton NMR assignments. Biochim Biophys Acta 882:154–167PubMedCrossRefGoogle Scholar
  33. 33.
    Fiehn O, Kopka J, Dormann P, Altmann T, Trethewey RN, Willmitzer L (2000) Metabolite profiling for plant functional genomics. Nat Biotechnol 18:1157–1161PubMedCrossRefGoogle Scholar
  34. 34.
    Kind T, Meissen JK, Yang D, Nocito F, Vaniya A, Cheng YS, Vandergheynst JS, Fiehn O (2012) Qualitative analysis of algal secretions with multiple mass spectrometric platforms. J Chromatogr A 1244:139–147PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Budczies J, Denkert C, Muller BM, Brockmoller SF, Klauschen F, Gyorffy B, Dietel M, Richter-Ehrenstein C, Marten U, Salek RM, Griffin JL, Hilvo M, Oresic M, Wohlgemuth G, Fiehn O (2012) Remodeling of central metabolism in invasive breast cancer compared to normal breast tissue—a GC-TOFMS based metabolomics study. BMC Genomics 13:334PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Liu W, Le A, Hancock C, Lane AN, Dang CV, Fan TW, Phang JM (2012) Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC. Proc Natl Acad Sci U S A 109:8983–8988PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Dong C, Yuan T, Wu Y, Wang Y, Fan TW, Miriyala S, Lin Y, Yao J, Shi J, Kang T, Lorkiewicz P, St Clair D, Hung MC, Evers BM, Zhou BP (2013) Loss of FBP1 by Snail-mediated repression provides metabolic advantages in basal-like breast cancer. Cancer Cell 23:316–331PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Yoo H, Antoniewicz MR, Stephanopoulos G, Kelleher JK (2008) Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line. J Biol Chem 283:20621–20627PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Vizan P, Boros LG, Figueras A, Capella G, Mangues R, Bassilian S, Lim S, Lee WN, Cascante M (2005) K-ras codon-specific mutations produce distinctive metabolic phenotypes in NIH3T3 mice [corrected] fibroblasts. Cancer Res 65:5512–5515PubMedCrossRefGoogle Scholar
  40. 40.
    Lee WN, Boros LG, Puigjaner J, Bassilian S, Lim S, Cascante M (1998) Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose. Am J Physiol 274:E843–E851PubMedGoogle Scholar
  41. 41.
    Lee WN, Go VL (2005) Nutrient-gene interaction: tracer-based metabolomics. J Nutr 135:3027S–3032SPubMedGoogle Scholar
  42. 42.
    Yang TH, Wittmann C, Heinzle E (2006) Respirometric 13C flux analysis—Part II: in vivo flux estimation of lysine-producing Corynebacterium glutamicum. Metab Eng 8:432–446CrossRefGoogle Scholar
  43. 43.
    Lee PNW, Wahjudi PN, Xu J, Go VL (2010) Tracer-based metabolomics: concepts and practices. Clin Biochem 43:1269–1277CrossRefPubMedCentralGoogle Scholar
  44. 44.
    Fan TWM, Higashi RM, Frenkiel TA, Lane AN (1997) Anaerobic nitrate and ammonium metabolism in flood-tolerant rice coleoptiles. J Exp Bot 48:1655–1666Google Scholar
  45. 45.
    Fan TW-M, Lane AN, Higashi RM (2012) The handbook of metabolomics, vol 17. Springer, New YorkCrossRefGoogle Scholar
  46. 46.
    Moseley H (2010) Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinform 11:139CrossRefGoogle Scholar
  47. 47.
    Fan TWM, Colmer TD, Lane AN, Higashi RM (1993) Determination of metabolites by proton NMR and GC analysis for organic osmolytes in crude tissue extracts. Anal Biochem 214:260–271PubMedCrossRefGoogle Scholar
  48. 48.
    Gradwell MJ, Fan TWM, Lane AN (1998) Analysis of phosphorylated metabolites in crayfish extracts by two-dimensional 1H-31P NMR heteronuclear total correlation spectroscopy (hetero TOCSY). Anal Biochem 263:139–149PubMedCrossRefGoogle Scholar
  49. 49.
    Fan T, Bandura L, Higashi R, Lane A (2005) Metabolomics-edited transcriptomics analysis of Se anticancer action in human lung cancer cells. Metabolomics 1:325–339CrossRefGoogle Scholar
  50. 50.
    Lane AN, Fan TW-M (2007) Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY. Metabolomics 3:79–86CrossRefGoogle Scholar
  51. 51.
    Fan TW-M (ed) (2010) Metabolomics-edited transcriptomics analysis (meta), vol 2. Academic, OxfordGoogle Scholar
  52. 52.
    Fan TW-M, Tan JL, McKinney MM, Lane AN (2012) Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells. Metabolomics 8:517–527CrossRefGoogle Scholar
  53. 53.
    Yuneva MO, Fan TW-M, Higashi RM, Allen TA, Balakrishnan A, Goga A, Ferraris DV, Tsukamoto T, Wang C, Seo Y, Chen X, Bishop JM (2012) The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type. Cell Metab 15:157–170PubMedCrossRefPubMedCentralGoogle Scholar
  54. 54.
    Fan TW-M (2012) Sample preparation for metabolomics investigation. In: Fan TW-M, Lane AN, Higashi RM (eds) The handbook of metabolomics: pathway and flux analysis, Methods in pharmacology and toxicology. Springer Science, New York, pp 7–27CrossRefGoogle Scholar
  55. 55.
    Mattingly SJ, Xu T, Nantz MH, Higashi RM, Fan TW-M (2012) A carbonyl capture approach for profiling oxidized metabolites in cell extracts. Metabolomics 8:989–996PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Lane AN, Fan TW-M, Xie X, Moseley HN, Higashi RM (2009) Stable isotope analysis of lipid biosynthesis by high resolution mass spectrometry and NMR. Anal Chim Acta 651:201–208PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Stadtman TC (1996) Selenocysteine. Annu Rev Biochem 65:83–100PubMedCrossRefGoogle Scholar
  58. 58.
    Stadtman TC (2000) Selenium biochemistry—mammalian selenoenzymes. In: Reactive oxygen species: from radiation to molecular biology. New York Acad. Sci, New York, pp 399–402Google Scholar
  59. 59.
    Duarte NC, Becker SA, Jamshidi N, Thiele I, Mo ML, Vo TD, Srivas R, Palsson BO (2007) Global reconstruction of the human metabolic network based on genomic and bibliomic data. Proc Natl Acad Sci U S A 104:1777–1782PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Allwood JW, Erban A, de Koning S, Dunn WB, Luedemann A, Lommen A, Kay L, Loscher R, Kopka J, Goodacre R (2009) Inter-laboratory reproducibility of fast gas chromatography-electron impact-time of flight mass spectrometry (GC-EI-TOF/MS) based plant metabolomics. Metabolomics 5:479–496PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Fan TW-M (2010) Metabolomics-edited transcriptomics analysis (meta). In: McQueen CA (ed) Comprehensive toxicology. Academic, Oxford, pp 685–706CrossRefGoogle Scholar
  62. 62.
    Want EJ, O’Maille G, Smith CA, Brandon TR, Uritboonthai W, Qin C, Trauger SA, Siuzdak G (2006) Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry. Anal Chem 78:743–752PubMedCrossRefGoogle Scholar
  63. 63.
    Villas-Boas SG, Hojer-Pedersen J, Akesson M, Smedsgaard J, Nielsen J (2005) Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast 22:1155–1169PubMedCrossRefGoogle Scholar
  64. 64.
    Winder CL, Dunn WB, Schuler S, Broadhurst D, Jarvis R, Stephens GM, Goodacre R (2008) Global metabolic profiling of Escherichia coli cultures: an evaluation of methods for quenching and extraction of intracellular metabolites. Anal Chem 80:2939–2948PubMedCrossRefGoogle Scholar
  65. 65.
    Sellick CA, Hansen R, Maqsood AR, Dunn WB, Stephens GM, Goodacre R, Dickson AJ (2009) Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells. Anal Chem 81:174–183PubMedCrossRefGoogle Scholar
  66. 66.
    Bolten CJ, Kiefer P, Letisse F, Portais JC, Wittmann C (2007) Sampling for metabolome analysis of microorganisms. Anal Chem 79:3843–3849PubMedCrossRefGoogle Scholar
  67. 67.
    de Koning W, van Dam K (1992) A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Anal Biochem 204:118–123PubMedCrossRefGoogle Scholar
  68. 68.
    Buchholz A, Hurlebaus J, Wandrey C, Takors R (2002) Metabolomics: quantification of intracellular metabolite dynamics. Biomol Eng 19:5–15PubMedCrossRefGoogle Scholar
  69. 69.
    Daykin CA, Foxall PJD, Connor SC, Lindon JC, Nicholson JK (2002) The comparison of plasma deproteinization methods for the detection of low-molecular-weight metabolites by H-1 nuclear magnetic resonance spectroscopy. Anal Biochem 304:220–230PubMedCrossRefGoogle Scholar
  70. 70.
    Rabinowitz JD, Kimball E (2007) Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem 79:6167–6173PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Richard M. Higashi
    • 1
  • Teresa W.-M. Fan
    • 1
  • Pawel K. Lorkiewicz
    • 2
  • Hunter N. B. Moseley
    • 3
  • Andrew N. Lane
    • 1
  1. 1.Graduate Center of ToxicologyUniversity of KentuckyLexingtonUSA
  2. 2.Diabetes and Obesity CenterUniversity of LouisvilleLouisvilleUSA
  3. 3.Molecular and Cellular BiochemistryUniversity of KentuckyLexingtonUSA

Personalised recommendations