Promises and pitfalls of untargeted metabolomics

Metabolomics is one of the newer omics fields, and has enabled researchers to complement genomic and protein level analysis of disease with both semi-quantitative and quantitative metabolite levels, which are the chemical mediators that constitute a given phenotype. Over more than a decade, methodologies have advanced for both targeted (quantification of specific analytes) as well as untargeted metabolomics (biomarker discovery and global metabolite profiling). Untargeted metabolomics is especially useful when there is no a priori metabolic hypothesis. Liquid chromatography coupled to mass spectrometry (LC-MS) has been the preferred choice for untargeted metabolomics, given the versatility in metabolite coverage and sensitivity of these instruments. Resolving and profiling many hundreds to thousands of metabolites with varying chemical properties in a biological sample presents unique challenges, or pitfalls. In this review, we address the various obstacles and corrective measures available in four major aspects associated with an untargeted metabolomics experiment: (1) experimental design, (2) pre-analytical (sample collection and preparation), (3) analytical (chromatography and detection), and (4) post-analytical (data processing).

[1]  Matej Oresic,et al.  Normalization method for metabolomics data using optimal selection of multiple internal standards , 2007, BMC Bioinformatics.

[2]  Fidele Tugizimana,et al.  A Conversation on Data Mining Strategies in LC-MS Untargeted Metabolomics: Pre-Processing and Pre-Treatment Steps , 2016, Metabolites.

[3]  Age K. Smilde,et al.  UvA-DARE ( Digital Academic Repository ) Assessment of PLSDA cross validation , 2008 .

[4]  Pavel Jandera,et al.  Recent advances in stationary phases and understanding of retention in hydrophilic interaction chromatography. A review. , 2017, Analytica chimica acta.

[5]  Ralf Tautenhahn,et al.  Autonomous Metabolomics for Rapid Metabolite Identification in Global Profiling , 2014, Analytical chemistry.

[6]  Eoin Fahy,et al.  Metabolomics Workbench: An international repository for metabolomics data and metadata, metabolite standards, protocols, tutorials and training, and analysis tools , 2015, Nucleic Acids Res..

[7]  Justin J J van der Hooft,et al.  Metabolite identification using automated comparison of high-resolution multistage mass spectral trees. , 2012, Analytical chemistry.

[8]  C. Junot,et al.  Direct introduction of biological samples into a LTQ-Orbitrap hybrid mass spectrometer as a tool for fast metabolome analysis. , 2008, Analytical chemistry.

[9]  M. Belanger,et al.  Multiple In Vivo Liver Biopsies Using a Freeze-Clamping Technique , 2002, Journal of investigative surgery : the official journal of the Academy of Surgical Research.

[10]  H. G. Rose,et al.  IMPROVED PROCEDURE FOR THE EXTRACTION OF LIPIDS FROM HUMAN ERYTHROCYTES. , 1965, Journal of lipid research.

[11]  Charles R. Evans,et al.  Evaluation of intensity drift correction strategies using MetaboDrift, a normalization tool for multi-batch metabolomics data. , 2017, Journal of chromatography. A.

[12]  Martin T. Wells,et al.  RRmix: A method for simultaneous batch effect correction and analysis of metabolomics data in the absence of internal standards , 2017, PloS one.

[13]  Leo L. Cheng,et al.  Applications of high‐resolution magic angle spinning MRS in biomedical studies II—Human diseases , 2017, NMR in biomedicine.

[14]  Yanli Wang,et al.  PubChem: a public information system for analyzing bioactivities of small molecules , 2009, Nucleic Acids Res..

[15]  J. Heijnen,et al.  Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. , 2009, Analytical chemistry.

[16]  E. Csató,et al.  Preparation and comparison of a pentafluorophenyl stationary phase for reversed-phase liquid chromatography , 1990 .

[17]  Rainer Lehmann,et al.  Effects of pre-analytical processes on blood samples used in metabolomics studies , 2015, Analytical and Bioanalytical Chemistry.

[18]  Andrew N Lane,et al.  Applications of NMR spectroscopy to systems biochemistry. , 2016, Progress in nuclear magnetic resonance spectroscopy.

[19]  D. McCalley,et al.  Understanding and manipulating the separation in hydrophilic interaction liquid chromatography. , 2017, Journal of chromatography. A.

[20]  James N. Hughes,et al.  NAD Deficiency, Congenital Malformations, and Niacin Supplementation , 2017, The New England journal of medicine.

[21]  Polina Golland,et al.  Permutation Tests for Classification: Towards Statistical Significance in Image-Based Studies , 2003, IPMI.

[22]  W. Nyhan,et al.  Perturbations of tyrosine metabolism promote the indolepyruvate pathway via tryptophan in host and microbiome. , 2015, Molecular genetics and metabolism.

[23]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[24]  Jianguo Xia,et al.  Using MetaboAnalyst 3.0 for Comprehensive Metabolomics Data Analysis , 2016, Current protocols in bioinformatics.

[25]  T. Perneger.  What's wrong with Bonferroni adjustments , 1998, BMJ.

[26]  Kristian Fog Nielsen,et al.  Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking , 2016, Nature Biotechnology.

[27]  M. Barker,et al.  Partial least squares for discrimination , 2003 .

[28]  Adam M. Feist,et al.  A model‐driven quantitative metabolomics analysis of aerobic and anaerobic metabolism in E. coli K‐12 MG1655 that is biochemically and thermodynamically consistent , 2014, Biotechnology and bioengineering.

[29]  Jason A. Thomas,et al.  An automated, pressure-driven sampling device for harvesting from liquid cultures for genomic and biochemical analyses. , 2006, Journal of microbiological methods.

[30]  Coral Barbas,et al.  Quality assurance procedures for mass spectrometry untargeted metabolomics. a review , 2018, Journal of pharmaceutical and biomedical analysis.

[31]  O. Fiehn,et al.  Using fragmentation trees and mass spectral trees for identifying unknown compounds in metabolomics. , 2015, Trends in analytical chemistry : TRAC.

[32]  J. Gangoiti,et al.  Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics , 2013, Metabolomics.

[33]  M. Hirai,et al.  MassBank: a public repository for sharing mass spectral data for life sciences. , 2010, Journal of mass spectrometry : JMS.

[34]  Christoph Steinbeck,et al.  The role of reporting standards for metabolite annotation and identification in metabolomic studies , 2013, GigaScience.

[35]  Eoin Fahy,et al.  LIPID MAPS-Nature Lipidomics Gateway: An Online Resource for Students and Educators Interested in Lipids. , 2012, Journal of chemical education.

[36]  R. Goodacre,et al.  Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling , 2011, Nature Protocols.

[37]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[38]  G. Siuzdak,et al.  From exogenous to endogenous: the inevitable imprint of mass spectrometry in metabolomics. , 2007, Journal of proteome research.

[39]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[40]  Agustín Lahoz,et al.  Mammalian cell metabolomics: Experimental design and sample preparation , 2013, Electrophoresis.

[41]  Joshua D. Knowles,et al.  Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry , 2011, Nature Protocols.

[42]  Yang Liu,et al.  Development and Evaluation of a Parallel Reaction Monitoring Strategy for Large-Scale Targeted Metabolomics Quantification. , 2016, Analytical chemistry.

[43]  L. Wasserman,et al.  Operating characteristics and extensions of the false discovery rate procedure , 2002 .

[44]  Jorge Cadima,et al.  Principal component analysis: a review and recent developments , 2016, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences.

[45]  Arjen Lommen,et al.  MetAlign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing. , 2009, Analytical chemistry.

[46]  D. Vuckovic.  Current trends and challenges in sample preparation for global metabolomics using liquid chromatography–mass spectrometry , 2012, Analytical and Bioanalytical Chemistry.

[47]  M. D. Luque de Castro,et al.  Metabolomics analysis I. Selection of biological samples and practical aspects preceding sample preparation , 2010 .

[48]  Antony J. Williams,et al.  The Royal Society of Chemistry and the delivery of chemistry data repositories for the community , 2014, Journal of Computer-Aided Molecular Design.

[49]  B. Warrack,et al.  Normalization strategies for metabonomic analysis of urine samples. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[50]  Ron Wehrens,et al.  Improved batch correction in untargeted MS-based metabolomics , 2016, Metabolomics.

[51]  Ralf Tautenhahn,et al.  An accelerated workflow for untargeted metabolomics using the METLIN database , 2012, Nature Biotechnology.

[52]  D. Watson.  A Rough Guide to Metabolite Identification Using High Resolution Liquid Chromatography Mass Spectrometry in Metabolomic Profiling in Metazoans , 2013, Computational and structural biotechnology journal.

[53]  Nigel W. Hardy,et al.  The metabolomics standards initiative (MSI) , 2007, Metabolomics.

[54]  E. Schleicher,et al.  Simultaneous extraction of metabolome and lipidome with methyl tert-butyl ether from a single small tissue sample for ultra-high performance liquid chromatography/mass spectrometry. , 2013, Journal of chromatography. A.

[55]  S. Panke,et al.  Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data , 2006, Molecular systems biology.

[56]  G. Siuzdak,et al.  Expanding coverage of the metabolome for global metabolite profiling. , 2011, Analytical chemistry.

[57]  L. Heilmeyer.  Cellular Regulation by Protein Phosphorylation , 1991, NATO ASI Series.

[58]  G. Siuzdak,et al.  XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization. , 2008, Analytical chemistry.

[59]  Thaer Barri,et al.  UPLC-ESI-QTOF/MS and multivariate data analysis for blood plasma and serum metabolomics: effect of experimental artefacts and anticoagulant. , 2013, Analytica chimica acta.

[60]  D. E. Atkinson,et al.  Role of the adenylate deaminase reaction in regulation of adenine nucleotide metabolism in Ehrlich ascites tumor cells. , 1976, Cancer research.

[61]  G. Siuzdak,et al.  XCMS Online: a web-based platform to process untargeted metabolomic data. , 2012, Analytical chemistry.

[62]  Robert S Plumb,et al.  Global metabolic profiling of animal and human tissues via UPLC-MS , 2012, Nature Protocols.

[63]  T. Fan,et al.  Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM) , 2009, Molecular Cancer.

[64]  Serge Rudaz,et al.  New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches , 2010, Analytical and bioanalytical chemistry.

[65]  S. K. Jensen.  Improved Bligh and Dyer extraction procedure , 2008 .

[66]  Charles F. Burant,et al.  Impact of Anesthesia and Euthanasia on Metabolomics of Mammalian Tissues: Studies in a C57BL/6J Mouse Model , 2015, PloS one.

[67]  Nigel W. Hardy,et al.  Proposed minimum reporting standards for chemical analysis , 2007, Metabolomics.

[68]  A. Mucci,et al.  MRS study of meningeal hemangiopericytoma and edema: a comparison with meningothelial meningioma. , 2012, Oncology reports.

[69]  Adam D. Kennedy,et al.  Untargeted metabolomic analysis for the clinical screening of inborn errors of metabolism , 2015, Journal of Inherited Metabolic Disease.

[70]  Nicola Zamboni,et al.  High-throughput, accurate mass metabolome profiling of cellular extracts by flow injection-time-of-flight mass spectrometry. , 2011, Analytical chemistry.

[71]  D. Ekman,et al.  A direct cell quenching method for cell-culture based metabolomics , 2009, Metabolomics.

[72]  D. Dunger,et al.  The development and validation of a fast and robust dried blood spot based lipid profiling method to study infant metabolism , 2014, Metabolomics.

[73]  Joshua D Rabinowitz,et al.  Metabolomics in systems microbiology. , 2011, Current opinion in biotechnology.

[74]  D. Bonneau,et al.  A Nontargeted UHPLC-HRMS Metabolomics Pipeline for Metabolite Identification: Application to Cardiac Remote Ischemic Preconditioning. , 2017, Analytical chemistry.

[75]  O. J. Dunn.  Multiple Comparisons among Means , 1961 .

[76]  M. Mcphail,et al.  Magnetic Resonance Spectroscopy: Principles and Techniques: Lessons for Clinicians. , 2015, Journal of clinical and experimental hepatology.

[77]  Tobin J Dickerson,et al.  Characterization of differences between blood sample matrices in untargeted metabolomics. , 2011, Analytical chemistry.

[78]  Åsa M Wheelock,et al.  Trials and tribulations of 'omics data analysis: assessing quality of SIMCA-based multivariate models using examples from pulmonary medicine. , 2013, Molecular bioSystems.

[79]  C. Burant,et al.  Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics. , 2011, Analytical chemistry.

[80]  David S. Wishart,et al.  HMDB: a knowledgebase for the human metabolome , 2008, Nucleic Acids Res..

[81]  Xin Lu,et al.  An alignment algorithm for LC-MS-based metabolomics dataset assisted by MS/MS information. , 2017, Analytica chimica acta.

[82]  M. Rantalainen,et al.  OPLS discriminant analysis: combining the strengths of PLS‐DA and SIMCA classification , 2006 .

[83]  Giuseppe Martano,et al.  Fast sampling method for mammalian cell metabolic analyses using liquid chromatography–mass spectrometry , 2014, Nature Protocols.

[84]  N. Fournier,et al.  A comparison of extraction methods for the isolation of phospholipids from biological sources. , 1986, Analytical biochemistry.

[85]  Jens O Krömer,et al.  Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol. , 2010, Analytical biochemistry.

[86]  W. R. Wikoff,et al.  Metabolomics identifies perturbations in human disorders of propionate metabolism. , 2007, Clinical chemistry.

[87]  John D. Storey,et al.  Statistical significance for genomewide studies , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[88]  U. Sauer,et al.  Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast , 2005, Genome Biology.

[89]  Oliver Fiehn,et al.  Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry , 2007, BMC Bioinformatics.

[90]  G. Siuzdak,et al.  The Expanding Role of Mass Spectrometry in Metabolite Profiling and Characterization , 2005, Chembiochem : a European journal of chemical biology.

[91]  S. Wold,et al.  Orthogonal projections to latent structures (O‐PLS) , 2002 .