1. PCB 5530 Antje Thamm & Tom Niehaus Fall 2015
Metabolomics
PCB 5530
Antje Thamm & Tom Niehaus
Fall 2015

2. Learning Outcomes Day 1 (Antje Thamm) Day 2 (Tom Niehaus)
• Lecture
- Learn the basics of metabolomics
- Understand the limitations of metabolomics
- Things to consider when using metabolomics for your own research
Day 2 (Tom Niehaus)
• Activity 1: Identifying an unknown peak
• Activity 2: Analyzing a metabolomics dataset

3. Definitions and Background
Metabolome = the collection of all metabolites in a sample
• All low molecular weight (< 2000 Da) organic molecules in a sample such as a leaf, fruit, seedling, serum, urine, etc.
Sugars
Nucleosides
Organic acids
Ketones
Aldehydes
Amines
Amino acids
Small peptides
Fatty acids
Isoprenoids
Phenols
Alkaloids
Drugs and pesticides

4. 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

5. Definitions and Background

6. Definitions and Background
Scope
Accuracy
Metabolomics
-measure many compounds
- relative measurements
Metabolic profiling
-measure a set of related compounds (e.g. phosphate esters)
Targeted analysis
-measure a specific compound
-absolute quantification is possible

7. Definitions and Background History and Development
• Metabolic profiling is not new. Profiling for clinical detection of human disease using urine samples has been carried out for Centuries.
This urine wheel was published in 1506 by Ullrich Pinder, in his book Epiphanie Medicorum. The wheel describes the possible colors, smells and tastes of urine, and uses them to diagnose disease.
eg maple syrup urine disease: metabolism of branched chain amino acids; ketones are detected in urine; diabetics secrete sugars into urine
Nicholson, J. K. & Lindon, J. C. Nature 455, 1054–1056 (2008).

8. Definitions and Background History and Development
• Advanced chromatographic separation techniques were developed in the 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.
• Metabolomics is expanding to catch up with other multiparallel analytical techniques (transcriptomics, proteomics) but remains less developed and less accessible.

9. Definitions and Background
Plant Metabolome Size
• It is estimated that all plant species combined contain 90, ,000 compounds.
• Each individual plant species contains about 5,000 – 30,000 compounds.
e.g. ~ 5,000 in Arabidopsis
The plant metabolome is much larger than that of yeast, where there are far fewer metabolites than genes or proteins (<600 metabolites vs genes). The size of the plant metabolome reflects the vast array of plant secondary compounds. This makes metabolic profiling in plants much harder than in other organisms.

10. Definitions and Background The Power of Metabolomics
Silent Knockout Mutations.
~90% of Arabidopsis knockout mutations are silent – i.e. have no visible phenotype
no direct clues to gene function. (The search for some sort of visible phenotype therefore often becomes desperate.)
Similar in yeast, where up to 85% of genes are non-essential
Even if there is no change in growth rate (visible phenotype; the sum of all fluxes), pool sizes of metabolites may have changed  Can be measured by metabolomics

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

12. Definitions and Background Limitations of metabolomics
• High biological variance in metabolite levels (i.e., high variation between genetically identical plants grown in the same conditions)
• Unlike nucleic acids and proteins, metabolites have a vast range of chemical structures and properties. Their molecular weights span two orders of magnitude (20–2000 Da). Therefore no single extraction or analysis method works for all metabolites. (Unlike DNA sequencing, microarrays, MS analysis of proteins – all are general methods.)
• The concentrations of various metabolites can vary dramatically from mM to pM
• Metabolite pools can be very dynamic – may change within seconds
• Some metabolites are labile and won’t survive extraction and analysis
• Issues with chromatography, detection, and data analysis
• one gene can influence many metabolites; the concentration of one metabolite can regulate expression of many genes

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

14. Sample Preparation Growth/Sample Size
• Grow organisms (e.g. plants or bacteria) under identical conditions
• Randomize the treatment groups
(Make sure the effects you measure are due to the variable being tested, not variables in experiment set up)
• number of replicates… depends on what you want to find
Large differences = small replication needed
Small differences = large replication needed
• In general, six replicates for each treatment are needed (due to high biological variability)

15. 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)

16. Choosing an extraction method
Sample Extraction
Choosing an extraction method
• No universal extraction method exists – always compromise
• Some solvents may degrade certain compounds
• It’s good to have some idea of what metabolites you want to extract

17. Chromatography Introduction
• Invented in 1900 by Mikhail Tsvet (used to separate plant pigments)
• There are several types of chromatography, but all consist of a stationary phase and a mobile phase. Compounds are separated based on differential partitioning between the two phases.
• Types include:
- TLC (thin-layer chromatography)
- GC (gas chromatography)
- LC (liquid chromatography) Y
GC and LC are routinely used in metabolomics

18. Chromatography Gas Chromatography GC = ‘good chromatography’
optimized over several decades, high reproducibility
Identification of compounds based on ‘conventional GC’ method
Suitable for
compounds with sufficient volatility
Thermostable compounds
Limitations:
- high temperatures can destroy labile compounds
- polar compounds cannot ‘fly’ on GC columns and must first be derivatized
by blocking reactive groups

19. Liquid Chromatography
• LC = ‘Lousy chromatography’
• fairly new, recent advances
• thousands of columns available
- normal phase -ion exchange
- reverse phase -HILIC
• infinite solvent systems possible
• low reproducibility
Advantages:
- compound can be collected after separation
- derivatization not necessary
- a separation protocol can be optimized for nearly any compound