IN SILICO ANALYSIS TO

METABOLOMICS
Toral Joshi
M.Phil (Bioinformatics)
Disha Life Sciences
 INTRODUCTION
Metabolism:- It is the set of chemical reactions that happen in
living organisms to maintain life.
Catabolism :- Breaks down organic matter.
Anabolism :- Uses energy to construct components of cells
such as proteins and mucliec acid
Metabolite :- Metabolites are the intermediates and products of metabolism.
Usually metabolites refers to small molecules.
Metabolic Pathway :- Series of Chemical reaction occuring within the
cell.
Metabolic Network :- Collection of Metabolic pathways is called a
metabolic Network.
 INTRODUCTION
• Metabolome :- Metabolome refers to the complete set of small-
molecule metabolites
• Metabolomics:-Investigation of metabolic regulation and fluxes in
individual cells or cell types.Metabolomics is the "systematic study of
the unique chemical fingerprints that specific cellular processes leave
behind" - specifically, the study of their small-molecule metabolite
profiles.
• Metabonomics:- the determination of systemic biochemical profiles
and regulation of function in whole organisms by analysing biofluids
and tissues
 The History of Metabolomics
Linus Pauling hypothesised on the predictive capacity of
chromatographic profiling of bodily fluids for detection and
diagnosis of human disease.

Chromatographic separation techniques were

developed in the late 1960's.
Robinson and Pauling published “Quantitative Analysis of Urine Vapor and Breath by Gas-
Liquid Partition Chromatography” in 1971.

The Metabolome and Metabolomics were coined in the 1990s.

In January 2007 the Human Metabolome Project, completed the first draft of the
human metabolome, consisting of 2,500 metabolites, 1,200 drugs and 3,500 food
components.
 WHAT IS A METABOLITE?

• Any organic molecule detectable in the body with a MW <

1000 Da
• Includes peptides, oligonucleotides, sugars, nucelosides,
organic acids, ketones, aldehydes, amines, amino acids,
lipids, steroids, alkaloids and drugs (xenobiotics)
• Includes human & microbial products
• Concentration > 1mM
 SIZE OF METABOLOMES VARIES
GREATLY
• Saccharomyces cereviciae ~ 600 metabolites (compared to over
~6,000 genes)
• Plants: ~ 200,000 primary & secondary metabolites
• Human metabolome: Much larger

• Degree of diversity encompasses:

• Molecular weights (wide range of mwt)
• Polar (carbohydrates)
• Non-polar (terpenoids & lipids)
• Volatile vs. non-volatile organic compounds
 METABOLITES
Some common metabolites include:
• cholesterol
• glucose, sucrose, fructose
• amino acids
• lactic acid, uric acid
• ATP, ADP
• drug metabolites, legal and illegal

These are produced in metabolic pathways, such as the Krebs

(citrate) cycle for oxidation of glucose.
 METABOLITES & FUNCTION

• Serum Creatinine
• Late stage organ stress and tissue breakdown
• TMAO
• Early stage buffering response
• Creatine, methyl-histidine, taurine, glycine
• Tissue damage, muscle breakdown, remodelling
• Citrate, lactate, acetate, acetone
• Oxidative stress, apoptosis, anoxia, ischemia
• Histamine, chlorotyrosine, thromoxane, NO3
• Immune response, inflammation
THE PYRAMID OF LIFE

Metabolomics
1400
Chemicals

Proteomics
2500 Enzymes

Genomics
25,000 Genes
Metabolomics

Primary Molecules

Secondary Molecules

Chemical Fingerprint
 METABONOMICS

• Evaluation of tissues & biological fluids for changes in

endogeneous metabolite levels resulting from disease,
genetic changes or (particularly important for
pharmaceuticals) from therapeutic treatments.
 METABOLITE PROFILING

Both Metabolomics and Metabonomics involve nonselective or

non bias analysis.

In contrast ‘Metabolite profiling’ involves the identification

and quantitation by a particular analytical procedure of a
predefined set of metabolites of known or unknown identity
and belonging to a selected metabolic pathway.
 Metabolome analysis

Metabolite Metabolite Metabolite

Metabolomics
target analysis profiling fingerprinting

The intention is not

Group of All metabolites, to identify each
Specific metabolites, present
metabolites e.g. a class of observed
in a cell or sample.
e.g. particular compounds such compound but to
Comprehensive
enzyme system that as compare patterns
analysis of the or
would be directly carbohydrates, whole
amino fingerprints of
affected by abiotic metabolome under
acids or those a metabolites that
or biotic
associated with a given set of change in response
perturbation.
specific pathway. conditions. to disease or toxin
exposure.
 IMPORTANT TERMINOLGIES

• Some important terminology that may be confusing:

• Target metabolite analysis: Focussed approach, few metabolites
• Metabolite profiling: Metabolic networks and compound classes
• Metabolomics: Analysis of “all” metabolites in a specific living
organism
• Metabonomics = Metabolomics in clinical disease
• Metabolic fingerprinting: Rapid classification of metabolite groups
• Metabolic pathway – metabolic network
• Pleiotropic effects – rather a rule than an exception
 METABOLOMICS
Integration of genomics, transcriptomics, proteomics and
metabolomics is a goal of systems biology.
THE ”OMICS”

• Term Investigates Role

Genomics DNA sequences Information
Transcriptomics mRNA sequences Messenger
Proteomics Protein sequences Factory
Metabolomics Metabolites Function
Phenomics Phenotype Form
 HUMAN METABOLOME PROJECT
• $7.5 million Genome Canada Project launched in Jan. 2005
• Mandate to quantify (normal and abnormal ranges) and
identify all metabolites in urine, CSF, plasma and WBC’s
• Make all data freely and electronically accessible (HMDB)
• Make all cmpds publicly available (HML)
 HUMAN METABOLOME PROJECT
• Purpose is to facilitate Metabolomics
• Objective is to improve
• Disease identification
• Disease prognosis & prediction
• Disease monitoring
• Drug metabolism and toxicology
• Linkage between metabolome & genome
• Development of software for metabolomics
 BIOCHEMICAL PROFILE MAP TO
METABOLIC PATHWAYS

Biochemical Profile

20
 ANALYTICAL TECHNOLOGIES

GC

Separation
HPLC
Methods

Capillary
Metabolomics Electrophoresis

MS
Detection
Methods
NMR
 ANALYTICAL TECHNOLOGIES: SEPARATION

Gas chromatography
It offers high resolution, but requires chemical derivatization for many
biomolecules and only volatile chemicals can be analysed without derivatization.
Gas-liquid chromatography - involves
a sample being vapourised and
injected onto the head of the
chromatographic column. The sample
is transported through the column by
the flow of inert, gaseous mobile
phase. The column itself contains a
liquid stationary phase which is
adsorbed onto the surface of an inert
solid.
 ANALYTICAL TECHNOLOGIES: SEPARATION

High performance liquid chromatography

HPLC has lower resolution than GC, but it does have the advantage that a much
wider range of analytes can potentially be measured.
 ANALYTICAL TECHNOLOGIES: SEPARATION

Capillary electrophoresis
It has a higher theoretical separation efficiency than HPLC and is suitable for use
with a wider range of metabolite classes than is GC. As for all electrophoretic
techniques, it is most appropriate for charged analytes.
 ANALYTICAL TECHNOLOGIES: DETECTION

Mass spectrometry
Used to identify and to quantify metabolites after separation by GC, HPLC, or
CE. In addition, mass spectral fingerprint libraries exist that allow
identification of a metabolite according to its fragmentation pattern.

There are many types of mass

Sector instrument
spectrometers that not only analyze
the ions differently but produce
different types of ions; however they
all use electric and magnetic fields to
change the path of ions in some way.
 ANALYTICAL TECHNOLOGIES: DETECTION

Nuclear magnetic resonance (NMR) spectroscopy

NMR is almost the only detection technique which
does not rely on extraction and separation of the
analytes, and the sample can thus be analysed in vivo
and recovered for further analyses.

Any molecule containing one or more atoms with a

non-zero magnetic moment can potentially be
detected. In practice metabolites are labelled by
feeding substrates containing 1H, 13C, 14N, 15N or 31P
isotopes.

NMR is close to being a universal detector. However,

it possesses one major disadvantage, which is that it is
relatively insensitive compared to mass spectrometry-
based techniques.
 POST-GENOMIC ERA OF BIOLOGY

Genome

Gene expression
(mRNA)
Metabolism

Proteins
 POST-GENOMIC ERA OF BIOLOGY

Genome
Transcriptomics (Microarrays)

Metabolomics
Gene expression
Genomics
(mRNA)
Metabolism

Proteins

Proteomics
 POST-GENOMIC ERA OF BIOLOGY
Genotype
Genome
Transcriptomics (Microarrays)

Metabolomics
Gene expression
Genomics
(mRNA)
Metabolism

Proteins
FunctionalM
olecular
Proteomics Phenotype
 POST-GENOMIC ERA OF BIOLOGY
Genotype
Genome
Transcriptomics (Microarrays)

Metabolomics
Gene expression
Genomics
(mRNA)
Metabolism

Environmental Proteins
stressors FunctionalM
olecular
Proteomics Phenotype
 "METABOLOMICS: HOW AND WHAT FOR ? "

6 steps:
1- sampling (storage)
2- metabolite extraction (standardisation, reproducibility)
3- biochemical analysis (GC-MS, LC-MS, NMR)
4- data pre-processing (base line correction….)
5- data visualisation and mining (PCA, data bases)
6- integration of data (metabolic pathways, genome..)
Identify metabolites and pathways that influence drug response
Nature Reviews Genetics 5; 669-676 (2004);
 To monitor in parallel hundreds or even
thousands of metabolites, high-
throughput techniques are required that
enable screening for relative changes
rather than absolute concentrations of
compounds
METABOLOME ANALYSIS

• Samples (complex tissues, cells, etc.)

• Extract metabolites from sample.

• Separate metabolites (chromatography).

• Detect and characterize individual metabolites

• Quantify and perform data analysis.

METABOLOME DATA ANALYSIS

Data is collected from instruments

(GCMS,LCMS,NMR,CEMS,FTM
S,etc.) in high a throughput manner.

Data is deconvoluted and stored

automatically in appropriate format
and database

Computer based applications

automatically transform analyse
data .

Statistically significant differences and/or

similarities are reported to researcher in an
easy to understand format.
Gene Protein
Transcript
Metabolite
APPLICATIONS
 METABOLIC PROFILING: THE
POSSIBILITIES
• Toxicology Testing
• Clinical Trial Testing
• Fermentation Monitoring
• Food & Beverage Tests
• Nutraceutical Analysis
• Drug Phenotyping
• Water Quality Testing
• Organ Transplantation
• Genetic Disease Tests
• Nutritional Analysis
• Clinical Blood Analysis
• Clinical Urinalysis
• Cholesterol Testing
• Drug Compliance
• Dialysis Monitoring
• MRS and fMRI
 MEDICAL METABOLOMICS
• Generate metabolic “signatures” for disease states or host
responses
• Obtain a more “holistic” view of metabolism (and treatment)
• Accelerate assessment & diagnosis
• More rapidly and accurately (and cheaply) assess/identify disease
phenotypes
• Monitor gene/environment interactions
• Rapidly track effects from drugs/surgery
APPLICATIONS IN
METABOLITE IMAGING
Lactate N-acetyl-aspartate

Glutamate

Citrate
Alanine
 Absent
Normal
Below Normal
Above Norrmal

Patient 9
Patient 8
Patient 7
Patient 6
Patient 5
Patient 4
Patient 3
Patient 2
Patient 1

Patient 11

Patient 15
Patient 14
Patient 13
Patient 12
Patient 10
Acetic Acid
Betaine
Carnitine
Citric Acid
Creatinine
Dimethylglycine
Dimethylamine
Hippulric Acid
Lactic Acid
METABOLIC MICROARRAYS

Succinic Acid
Trimethylamine
Trimn-N-Oxide
Urea
Lactose
Suberic Acid
Sebacic Acid
Homovanillic Acid
Threonine
Alanine
Glycine
Glucose
 The “Omics” of Nutrition
N
u DNA
t Nutrigenetics
r
i Nutritional
g
Epigenetics
e
n RNA
o
Nutritional
Bioactive Food m
Transcriptomics Phenotype
Component i
c
s
Protein

Proteomics

Metabolomics
Metabolite
 Nutritional Metabolomics
N
u DNA
t Nutrigenetics
r
i Nutritional
g
Epigenetics Metabolomics
e
n RNA
o
Nutritional
Bioactive Food m Metabolite
Transcriptomics
Component i
c
s
Protein
Proteomics

Phenotype
 Can Metabolomics Shed Light
on these 3 Nutrition Related Biomarkers
Inactive Metabolite
Absorbed
Dose Biologically
Effective
Dose

Intake of Susceptibility Early

Dietary (Genetic/ Biologic
Constituent Environment) Effect

Health Effects Altered

Structure/
Structure
+ and - Function
CAN METABOLOMICS PROVIDE CLUES
ABOUT THE PROGRESSION OF DISEASE
Natural History of Disease Treatment History
Quality
Of Life
Outcomes
Environment
Treatment
+ Lifestyle Options
Disease
Staging
Patient
Stratification

Early
Detection
Genetic Biomarkers
Risk
 METABOLOMICS: APPLICATIONS
• Identification of metabolic biomarkers that change
as an indicator of the presence of disease or in
response to drug-based intervention.

• Determination of the effect of biochemical or

environmental stresses on plants or microbes
APPLICATIONS (CONT’D)

• Bacterial characterizations

• Human health assessments (potential for

“translational research”?)

• Metabolic engineering
 Metabolomics Applications

Diagnosis

Disease (e.g. coronary heart disease).

Toxicology

Functional genomics

Ascribing functions to genes

Systems biology

Integration with data sets from other omics.

 OTHER APPLICATIONS

• Genetic Disease Tests • Food & Beverage Tests

• Nutritional Analysis • Nutraceutical Analysis
• Clinical Blood Analysis • Drug Phenotyping
• Clinical Urinalysis • Water Quality Testing
• Cholesterol Testing • Petrochemical Analysis
• Drug Compliance • Fermentation Monitorin
• Toxicology Testing
• Transplant Monitoring
• Clinical Trial Testing
• MRS and fMRI
Metabolomics approaches have been widely used to
provide a phenotypic description of a cell as a function
of time and/or condition by a set of metabolites.

Changes in levels of metabolic intermediates of a

sequential series of reactions are often more
pronounced than the changes in enzymatic kinetics or
individual fluxes. For this reason, metabolomics is
considered a sensitive tool for the study of genotype-
phenotype correlations as well as the pharmacological
and toxicological effects of drugs.
DATABASES FOR METABOLOMICS
 KEGG
• KEGG is a suite of databases.
• PATHWAY
• holds the current knowledge on molecular interaction networks,
• including metabolic pathways, regulatory pathways,and molecular complexes.
• GENES
• is a collection of gene catalogs for all the complete genomes and some
• partial genomes. Each gene catalog is computationally derived from public
• resources, and is manually reannotated for reconstruction of KEGG pathways.
• KEGG GENES is associated with KEGG GENOME containing chromosome maps,
• KO for manually curated ortholog groups, and KEGG SSDB for computationally
• generated ortholog/paralog clusters and gene clusters.
• COMPOUND/
• GLYCAN/REACTION contains information about chemical
• compounds and reactions.
 KEGG:- LIGAND DATABASE
• LIGAND Database of Chemical Compounds and Reactions in
Biological Pathways
• provide the linkage between chemical and biological aspects of life in
the
• light of enzymatic reactions.
• The database consists of four sections:
• COMPOUND, GLYCAN, REACTION, and ENZYME.
FOOD COMPONENT DATABASE
SOFTWARES FOR METABOLOMICS
LIST OF OTHER SOFTWARES
• mzmine and mzmine2 (http://mzmine.sourceforge.net/) - mzxml, mzdata,
netCDF and XCalibur data (LC-MS, GC-MS, MS data)
• metAlign (RIKILT-WUR Institute of Food Safety) - LC-MS and GC-MS
data
• BinBase (fiehnlab.ucdavis.edu)
• xcms and xcms2 (Scripps) - netCDF data (LC-MS, GC-MS, MS and
MS2 data)
• MarkerLynx (Waters) (LC-MS data)
• BluFuse (BlueGnome) - for MS and NMR data
• SpecAlign University of Oxford (Jason Wong) - Alignment of SELDI,
MALDI, NMR, RAMAN, IR (via TXT import)
• HiRes (Columbia University Medical Center) - for NMR data
• msInspect (Proteomics Fred Hutchinson Cancer Center)
• Progenesis PG600 (Nonlinear) - for MALDI and SELDI mass spectra
• caMassClass (NCBI) - for SELDI protein mass spectra
SOFTWARES
Xalign - for LC-MS data [DOI] - request here

msalign from Matlab Bioinformatics Toolbox - for MS data (example using msalign)
pairseqsim - (Bioconductor - Witold Wolski) - for mass spectra [DOI]
Randolph Yasui code - [DOI] download Matlab code and WMTSA wavelet toolbox
RTAlign algorithm of MSFACTs (noble.org) - GC-MS and LC-MS data
Genedata Expressionist (genedata.com) - for LC-MS and infusion MS data.
MS Align (David Grant - Uconn.edu) - for high resolution mass spectral data [DOI]
LCMSWARP (PNNL) - for proteomics and metabolomics LC-MS data (http://ncrr.pnl.gov/software)
ChromAlign (Thermo) - included in Sieve and Biosieve package for LC-MS and LC-MS-MS data
PETAL - Peptide Element Alignment for LC-MS data (http://peiwang.fhcrc.org/research-project.html)
MarkerView (ABI/Sciex) - for LC-MS and MALDI data peak picking and alignment and statistics
SOFTWARES
• MathDAMP (Keio University) - for GC-MS, LC-MS, CE-MS data with Mathematica source code [DOI]
• NameLess - for MALDI MS and FT-MS data with JAVA source code [DOI]
• CPM MatLab toolbox (J Listgarten) - for LC-MS, proteomics, metabolomics and time series data + source
code.
• GASP (genedrift.org) - for GC-MS alignment
• AnalyzerPro (SpectralWorks) - for alignment of GC-MS data
• meta-b (Vladimir Likic) - for alignment of LC-MS data with python source code (go SVN)
• spectconnect (MIT) - for alignment of GC-MS data using AMDIS for deconvolution
• ChenomX Profiler (Chenomx) - for binning and alignment of NMR signals (+ DB search)

• KnowItAll Metabolomics Editions (BioRad) - with IntelliBucket bucketing and binning of NMR data (+ DB
search)
• MS-Xelerator (MSMETRIX) - Advanced Algorithms for LC/MS Data Processing (Marco Ruijken)
• OBI-Warp (U Texas) - Ordered Bijective Interpolated Warping for LC-MS data [PDF]
THANK YOU FOR YOUR PATIENCE