http://creativecommons.org/licenses/by-sa/2.0/

Large Scale Approaches to the Study of Metabolite Levels

Prof:Rui Alvesralves@cmb.udl.es

973702406Dept Ciencies Mediques Basiques,

1st Floor, Room 1.08Website of the

Course:http://web.udl.es/usuaris/pg193845/Courses/Bioinformatics_2007/ Course: http://10.100.14.36/Student_Server/

Genome, Proteome, now what?

Metabolites!!!

Why Studying Metabolites Directly?

•Just because a protein is changing its activity do levels of product/substrate change?• What happens with non-covalently bound regulators?•What about the levels of the different metabolites?•Which metabolites do cell regulate for each response?•How can we know what to change in the cell for biotechnological purposes of producing some metabolite (e.g. antibiotics) if we don’t know how the levels of these metabolites change?

From metabolites to metabolomics

• Metabolite is an intermediate of metabolism

• Metabolome is the metabolic complement (metabolite pool) of a cell, tissue or organism under a given set of conditions

• Metabolomics is the study of the metabolome

The Metabolome

• The Metabolome– Metabolite complement of a proteome

• Variable– In different cell and tissue types in same organism– In different growth and developmental stages of organism

• Dynamic– Depends on response of genome & proteome to environmental

factors» Disease state» Drug challenge» Growth conditions» Stress

What can we do with Metabolomics

• Metabolomics enables:

• Qualitative and quantitative display of metabolite concentration patterns

• Assessment of global changes• Comparative analysis of samples

• Provides information from which biological hypotheses may be developed

Tissue or biofluid sample

Measure the metabolite profile

Treat profile as ‘fingerprint’ for

classification purposes

Explore profile to gain mechanistic insight into the biological response

Statistical bioinformatic tools

Bioanalytical tools

(applied/clinical) (basic research)

1. Mass spectrometry2. 1H NMR spectroscopy

The procedure

Low molecular weight organic metabolites:

Amino acids

Organic acids and bases

Nucleotides

Carbohydrates

Osmolytes

Lipids (broad non-specific

resonances)

Which metabolites can be observed by NMR?

NMR is possible because of Nuclear Spin

• All nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum

• Nuclear spin angular momentum is a quantized property of the nucleus in each atom, which arises from the sub-atomic properties of neutrons and protons

• The nuclear spin angular momentum of each atom is represented by a nuclear spin quantum number (I)

• All nuclei with even mass numbers have I=0,1,2…

• All nuclei with odd mass numbers have I=1/2,3/2...

• NMR is possible with all nuclei except I=0, but I=1/2 has simplest physics

Biomolecular NMR primarily 1H, 13C, 15N (31P)

Organism

The experiment

Markedmetabolite

Organism

Magnetic field generator

(frequency: what compound)

(how much)

Chemical shift is how much the spectrum changes with respect to a specific well known ground state

What the hell is chemical shift?

• All nuclei have a specific resonance

spectrum

• This spectrum changes depending on the environment of an atom

• Thus the 1H spectrum in CH4 is different from that in 1H2

A few simple 1H spectra

What about more complicated molecules?

1H NMR Spectrum of Ubiquitin

• Things get very messy

• Subspectra become entangled

What to do about this?• Use a different magnetic pulse to measure another spectrum!

Magnetic field

generator

Magnetic field

generator

Cs ppm (pulse 1)

Cs ppm (pulse 2)

2D NMR!!!!

Use 2D NMR to Resolve Overlapping Signals

1D

2D

Sub-spectraoverlapped

Coupled spins

Crosspeaksresolved!

ppm (pulse 1)

ppm (pulse 1)

ppm (pulse 2)

ppm (pulse 2)

Concept can be extended to N-dimensional NMR!!!

Multi-Dimensional NMR

If 2D cross peaks overlap go to 3D or 4D …..

HN

H

H

Rule of thumb• If two groups are different then you can

always resolve the spectrum by applying a sufficiently high magnetic field

Data Analysis• Fitting 5-10 rounded peaks is trivial, fitting 1000+

sharp peaks is not, i.e. dense matrix problem with very high probability of cumulative rounding errors and singularities(LLSOL - Stanford)

• Peak positions & shapes dependent on salt, pH, temperature, ligands, ligand/ion interactions, shimming, signal-to-noise digital resolution, phasing, field strength, etc. etc.

Metabolome Pipeline

• Multi-disciplinary teams required• Meta-data (data about data) extremely important• Data storage (database) important for large datasets• Brown et al, Metabolomics, 2005, 1, 39-51

•Spectrum identification can be made using for example Fourier Transforms

•Problems similar to those for “ID”ing mass spec spectra for proteins

Example

• Metabolomic changes due to polution in fresh water japanese fish

PC1 score

PC

2 s

co

re

Day 1

2

6

54

3

7

8Developmental

trajectory

PCA scores plot: Summarizes changes in NMR-visible metabolome throughout embryogenesis in Japanese

medaka

Fertilization HatchChemical shift (ppm)12345678910

PC

1 lo

adin

gs

-0.4

-0.2

0.0

0.2

0.4

Tyros

ine

ATPHist

idin

e

Creat

ine

Alanin

e

Lacta

telate stage embryos

early stage embryos

Developmental toxicity of trichloroethylene (TCE) in Japanese medaka

Expose medaka embryos to TCE throughout embryogenesis.

Preserved replicates of ~100 eggs on day 7 of development.

PCA scores plot: Dose-dependent effects of TCE on medaka metabolome

PC1 score

PC

2 sc

ore

2

6

54

3

7

8

Day 1

46 ppm TCE

Day 7 controls

3 ppm TCETrajectory?

PC1 score

PC

2 s

co

re

Permanent toxicant-induced

perturbation

stage specific toxicity identified for targeted gene

expression studies

Perturbations to normal developmental trajectory

Normal development

C. A. Pincetich, et al, Comp. Biochem. Physiol. C 140, 103-113 (2005).

Advantages of metabolomics

• Changes in the levels of individual enzymes:• expected to have little effect on measured

metabolic fluxes• do have significant effects on the concn of

metabolites

• ‘Downstream’ result of gene expression• changes in metabolome are amplified relative

to changes in the transcriptome and the proteome.

Advantages of metabolomics• Metabolomics is complementary to

transcriptomics and proteomics, but closer to the phenotype

• Number of metabolites expected to be smaller than number of genes or proteins (S. cerevisiae 6000 genes and 600 metabolites)

• Metabolomic analyses can cost up to two thirds less than other ‘omic’ analyses (more appropriate for high-throughput/large sample number studies than proteomics and transcriptomics)

Plants have hundreds of thousands different chemical compounds, many still unknown!!!

Disadvantages• Sometimes chemistry changes, depending

on isotope composition

• Less sensitive than Mass spec

Central repositories• No central repository that i know of.

Molecular Phenotype

Post-genomic Era of Biology

Genome

Gene expression

Proteins

Metabolism

Metabolomics

Proteomics

Transcriptomics

Genomics

Genotype

Environmental stressors