Metabolomics, part 1 PCB 5530 Antje Thamm Fall 2016

Day 2 (Guillaume Beaudoin) Metabolomics Day 1 (Antje Thamm) Introduction to Metabolomics: Basics & limitations of metabolomics Sample preparation Chromatography Mass Spectrometry in metabolomics Day 2 (Guillaume Beaudoin) Metabolomics data analysis Day 3 (Thomas Niehaus)

Definitions and Background Metabolome = the total metabolite pool • All low molecular weight (< 2000 Da) organic molecules in a sample such as a leaf, fruit, seedling, etc. Sugars Nucleosides Organic acids Ketones Aldehydes Amines Amino acids Small peptides Lipids Steroids Terpenes Alkaloids Drugs (xenobiotics)

Definitions and Background Metabolomics = high-throughput analysis of metabolites Metabolomics is the simultaneous measurement of the levels of a large number of cellular metabolites (typically several hundred). Many of these are not identified (i.e. are just peaks in a profile). Not hypothesis driven snapshot

Definitions and Background

Metabolites Are the Canaries of the Genome A single base change can lead to a major change in metabolite levels. BUT: metabolites are the building blocks of genetic information other examples? Bioinformatics.ca

Definitions and Background Scope Accuracy Metabolomics -measures many compounds (ratios) Metabolic profiling -measures a set of related compounds (e.g. phosphate esters) more quantitative Targeted analysis -measures a specific compound; quantification possible

How old is the field of metabolomics? Profiling of blood and urine for clinical detection of human disease has been carried out for Centuries. Ulrich Pinder, 1506: Epiphanie Medicorum Urine wheel describes possible colors, smells and tastes of urine Nicholson, J. K. & Lindon, J. C. Nature 455, 1054–1056 (2008).

How old is the field of metabolomics? Advanced chromatographic separation techniques developed in late 1960’s. Linus Pauling published “Quantitative Analysis of Urine Vapor and Breath by Gas-Liquid Partition Chromatography” in 1971 Chuck Sweeley at MSU helped pioneer metabolic profiling using gas chromatography/ mass spectrometry (GC-MS) Plant metabolic biochemists (e.g. Lothar Willmitzer) were among other early leaders in the field. # of metabolomics publications  Metabolomics is expanding to catch up with other multiparallel analytical techniques (transcriptomics, proteomics) but remains far less developed and less accessible.

The metabolome: Size and Concentrations 200,000 Chemicals 20,000 8000 All Mammals All Microbes All Plants The Pyramid of Life All plant species combined contain 90,000 - 200,000 compounds. Individual plant species contain 5,000 (Arabidopsis) – 30,000 compounds Ratio of metabolites/genes much smaller than in microbes Metabolic profiling much harder than in other organisms

Power of Metabolomics Silent Knockout Mutations. ~90% of Arabidopsis knockout mutations are silent (no visible phenotype) ~85% of yeast genes are not needed for survival Metabolic Control Analysis: Growth rate (sum of all metabolic fluxes) is unchanged in silent knockout mutations Pool sizes of metabolites have to change to compensate for effect of mutation, metabolic fluxes are unchanged  this can be measured!

Silent knockout mutations Power of Metabolomics Silent knockout mutations Example. • Chloroplast 2010 project (phenotype analysis of knockouts of Arabidopsis genes encoding predicted chloroplast proteins): • Various knockouts showed essentially normal growth and color but highly abnormal free amino acid profiles, e.g. At1g50770 (‘Aminotransferase-like’)

Why Metabolomics is Difficult 5 Bases 20 Amino acids 2x105 Chemicals Metabolomics Proteomics Genomics Chemical Diversity The Pyramid of Life Bioinformatics.ca

Why Metabolomics is Difficult Proteomics Genomics Response Time Concentrations of cellular metabolites vary over several orders of magnitude (mM to pM) Differences in molecular weight (20-2000 Da) Concentration High turnover rates Some metabolites are labile Concentration Concentration Bioinformatics.ca

Metabolomics Steps in metabolomics sample preparation sample extraction chromatography detection data analysis

Sample Preparation Growth/Sample Size Grow organisms (e.g. plants or bacteria) under identical conditions Randomize the treatment groups (Make sure effects are measured due to variability in samples, not in experimental set up • number of replicates… depends on what you want to find Large differences = small replication needed Small differences = large replication needed • In general, a minimum of six replicates for each treatment are needed (due to high biological variability) Grow more than you think you’ll need!

Sample Preparation Sample bias Grow organisms (e.g. plants or bacteria) under identical conditions Randomize the treatment groups (Make sure effects are measured due to variability in samples, not in experimental set up

Sample Preparation Biological replicates: High variability, but “this is life” Technical replicates: “Is your sampling/ extraction method robust?”

Sample Preparation Sample collection • Uniform sample sizes (e.g. hole punches in leaves) • Be consistent - similar tissue - time of day • Quickly freeze sample in liquid nitrogen, store samples at -80°C • Fast-harvesting method for bacteria (~30 sec)

Choosing an extraction method Sample Extraction Choosing an extraction method • No universal extraction method exists • Some solvents may degrade certain compounds • Its good to have some idea of what metabolites you want to extract: Untargeted metabolomics / Metabolic profiling / Targeted analysis

Choosing an extraction method Sample Extraction Choosing an extraction method Physical disruption: Grind by hand? Mechanical? Extraction time Extraction efficiency How do you check for sufficient extraction?

Sample Extraction Sample extraction • The method should be consistent and reproducible • Further workup may be required; esp. for targeted analysis (e.g. solid phase extraction)

Chromatography introduction Invented in 1900 by Mikhail Tsvet (used to separate plant pigments) • Types include: - TLC (thin-layer chromatography) - GC (gas chromatography) - LC (liquid chromatography) Y GC and LC are routinely used in metabolomics

Principle of chromatography Principle: Separation of compounds based on differential partitioning between solid and mobile phases. https://www.khanacademy.org/test-prep/mcat/chemical-processes/separations-purifications/a/principles-of-chromatography

Chromatography Gas Chromatography • GC = ‘good chromatography’ Separation according to difference in volatility & structure For compounds with sufficient volatility thermostable

Chromatography Gas Chromatography Mostly used for untargeted metabolomics optimized over several decades High reproducibility Easy to use Good software ‘standardized’ GC method with very good databases for compounds identification Very universal for compounds <600 Da Great coverage for polar compounds (for same coverage, 3+ LC-MS methods are needed Only tool for volatiles Limitations: - high temperatures can destroy labile compounds - polar compounds cannot ‘fly’ on GC columns and must first be derivatized - impossible to collect fractions - heat may cause degradation

Sample derivatization Chromatography Sample derivatization Step 1) Methoximation Step 2) Silylation Z/E isomer have same mass spectrum but differ 2 seconds in retention time Gas chromatography requires volatile compounds (two step derivatization in vial) 1) Methoximation of aldehyde and keto groups (primarily for opening reducing ring sugars) 2) Silylation of polar hydroxy, thiol, carboxy and amino groups with silylation agent MSTFA A single compound with multiple active groups will result in multiple peaks (1TMS, 2TMS, 3TMS) GC-MS can distinguish between stereoisomers Anal Chem. 2009 Dec 15;81(24):10038-48. doi: 10.1021/ac9019522. FiehnLib: mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Kind T, Wohlgemuth G, Lee do Y, Lu Y, Palazoglu M, Shahbaz S, Fiehn O.

Liquid Chromatography LC = ‘Lousy chromatography’ Mobile phase: Liquid Analyte separation based on difference in interaction with column & mobile phase For small and macro-molecules, ionic compounds (not volatiles) Thermostable & thermolabile compounds

Liquid Chromatography LC = ‘Lousy chromatography’ Relatively new, recent advantages Thousand of columns available… normal phase, reverse phase, ion exchange, HILIC New columns constantly being developed to improve resolution, sensitivity and run time Infinite solvent systems possible: Selection of chromatographic configuration depends on physicochemical properties: solubility, polarity, weight Separation is based on complex interaction of analytes with column and mobile phase Advantages: compound can be collected after separation derivatization not necessary a separation protocol can be optimized for nearly any compound BUT: Low reproducibility  no massive databases

Liquid Chromatography LC = ‘Lousy chromatography’ Normal phase chromatography HILIC Reversed Phase Chromatography Stationary Phase Polar (silica) Polar (silica, modified silica) Non-polar modified silica (C18, C8, phenyl) Mobile phase Non-polar (hexane, Ethyl acetate, chloroform) Polar (Water, Acetonitrile) Polar (water, Acetonitrile, Methanol) Analytes Non-polar and water insoluble (Lipids) Very polar metabolites Polar metabolites Polar compounds are retained Polar compounds elute first Not MS compatible MS compatible

What to use for what compounds? Chromatography What to use for what compounds? (Reversed Phase High Performance Liquid Chromatography) RP-HPLC GC (Gas Chromatography) HILIC (Hydrophilic Interaction Chromatography) Non-polar vitamins Amino/organic acids Volatile organic compounds Sterols Fatty acids Sugars Triglycerides Very polar Polar Medium-polar Non-polar