Introduction to

Clinical Metabolomics

TransMed Course: Basics in Clinical Proteomics and

Metabolomics. Oct 10-19, 2012

Vidya Velagapudi, Ph.D, Adjunct Professor

Head of the Metabolomics Unit, Tech Centre,
Institute for Molecular Medicine Finland FIMM,
Helsinki, Finland
10/10/12
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© FIMM - Institiute for Molecular Medicine Finland www.fimm.fi

 FIMM Technology Centre
Metabolomics Unit Core Facility
Biomedicum 2U, 2nd Floor

Services: Targeted quantitative high-

throughput metabolomics Analyses
for medical applications

E-mail: vidya.velagapudi@fimm.fi

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© FIMM - Institiute for Molecular Medicine Finland www.fimm.fi

 Metabolomics Unit
Launched in 2011
Waters XEVO-TQS triple quadrupole Hamilton star liquid handling system

Facility up and running in 2012

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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics work flow
 Applications of metabolomics
 Small test

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 What is Metabolomics?

• Metabolites are bio-active small molecules involved in the

biochemical network

• Metabolome represents the collection of all metabolites in a

given biological sample

• Metabolomics is the systematic identification and quantitation

of the metabolites

• Clinical Applications include studies of chronic diseases,

pharmacology, pre-clinical drug trials, toxicology, transplant
monitoring, newborn screening and clinical chemistry.

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 Brief historical note

1500-2000BC 1940s-1970s 21st century

China •Advances in analytics • Advances in analytics
•Ants used to •Pattern recognition • Biostatistics & Bioinformatics
detect  Metabolic profiling  Modern era of metabolomics
patients with and systems biology
diabetes
Metabolomics is not new

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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics work flow
 Applications of metabolomics
 Small test

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 Why Metabolomics ?
- End point of cellular regulation

- Dynamic & time sensitive

- Sensitivity

- High throughput

- Suitable platform for sys bio

- Integrated studies
( Metabolomics + GWAS,
Metabolomics + transcriptomics)

Functional readout
Gate to personalised medicine
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 Why is metabolomics necessary?

 The proteome cannot be completely predicted from the

transcriptome due to some differences in regulatory
mechanisms.

 The metabolome is further down the line from gene

function and so reflects more closely the activities of a
cell at the functional level.

 A metabolite may come from more than one metabolic

pathway and it is only when you conduct a study on the
metabolome as a whole that you can identify which
pathways are involved in it’s metabolism.

 Metabolomics can be viewed as complementary to

transcriptomics and proteomics.

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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics work flow
 Applications of metabolomics
 Small test

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 Structural Diversity of Metabolites

NH2
OH
H
H2N N OH O
P
N OH

O H2 N N O O
NH O
Arginine O P
H2N
OH O OH
O
Glycine OH N N
O
P
O

NH2 OH

HO OH
N
H Adenosine-5'-triphosphate
Tryptophan

O
O

OH OH
HO

Succinic acid
O
O
O
Pyruvic acid

OH
HO

O O
Oxaloacetic acid
Acetyl CoA

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 Metabolome
Homeostasis
‘Housekeeping’
mM (10-3)
Organic Acids
NMR
Lipids
µM (10-6)
Amino Acids
Mass
Nucleotides
Spectrometry nM (10-9)
Steroids

Eicosanoids
pM (10-12)
Neurotransmitters
CUSTOM
Peptides
fM (10-15)
Trace elements

Biofluid metabolic profile = Phenotype 1

2
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Adapted from Sumner, LW, et al., Phytochem, 62, 817,2003 12
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 Challenges in Metabolomics

 Successful extraction of metabolites from cellular

matrices is dependent upon the type extraction procedure.

 When you have a structural diverse group of compounds,

it becomes difficult to develop methods that can separate
them all.

 Ionization/Visualization of all metabolites is also difficult

to achieve, as each compound will vary in its affinity for a
detector.

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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics work flow
 Applications of metabolomics
 Small test

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 Metabolomics platform

High
Concentration
Global profiling NMR profile
(GCx)GCMS-profile LCMS derivatized

Glycerolipids, Bile acids, steroids,

Metabolic Profile
Sphingomyelins, fatty acids,
Ceramides & oxylipids
Cerebrosides
Class-targeted LCMS LC-FTMS

profiling Nucleosides, peptides

Acyl CoA, LCMS
phosphorylated
fatty acids
LCMS
GCMS
Endocannabinoids LCMS

Target (Multiplex)
Biomarker Immunoassays
GC, HPLC
Assays

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 Metabolomics Analyses

 Global analysis
 An analysis of the total detectable content of the
sample (e.g. MS/NMR spectrum of serum/urine)
 Primarily used for the detection of novel entities
• Q-ToF/MS
• GCxGC-ToF/MS

 Targeted analysis
 An analysis focused onto a specific molecule or
molecules (e.g. measurement of a specific m/z)
 Used for the measurement of known variables for a
model
• Triple quadrupole MS

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 Common mass spectrometers
LC-ion trap UPLC-Q-TOF LC-ion trap–FT-ICR

Triple quadrupole GCxGC-TOF GC-TOF

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 Metabolomics_systems biology
 Sensitivity
Small changes in activities of individual enzymes lead to small
changes in metabolic fluxes, but can lead to large changes in
metabolite concentrations
 Throughput
The metabolomics platforms afford higher throughput than
current transcriptomics or proteomics technologies

Cells Cellular Organisms Clinical

pathway studies
models

SYSTEMS
BIOLOGY
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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics work flow
 Applications of metabolomics
 Small test

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 Metabolomics work flow
Experiment design + Analytical chemistry + Chemometrics + Bioinformatics

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 Metabolite extraction

Extraction protocols differ based on the type of sample

• Serum/plasma
• Tissues
• Cells
• Urine
• CSF
• Saliva
• Dried blood spots

And the type of analysis

• Polar metabolites
• Non-polar metabolites

There is NO single extraction method that

could extract all the metabolites
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 Metabolite Separation

Different techniques are available based on the type of analysis

 Liquid chromatography
Suitable for compounds that are non-volatile in nature
• HPLC
• UPLC
• Nano-LC
• 2D-LC

 Gas chromatography
Suitable for compounds that are volatile in nature
• GC
• 2D-GC

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 Metabolite analysis

Ionization Mass Sorting (filtering) Detection

Ion Ion
Source Mass Analyzer Detector
Form ions
Sort Ions by Mass (m/z) Detect ions
(charged molecules)

Acquiring a Mass Spectrum

100

75

Inlet • Liquid 50

• Vapor 25

The separation of ions based on 0

1330 1340 1350

their mass / charge ratio’

Mass Spectrum
MS types: quadrupole, triple quad, TOF, quad-TOF, ion trap, ICR

All compounds must be ionized prior to MS detection!

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 LC/MS metabolomics platform

Sample
Separation Mass
extract
spectrometer
ISs RPLC/CapLC +ESI (Qtof)
Metabolites

MS/MS fragmentation pattern

MS survey scan MS-only ion chromatogram
y9 100
1047.5
100 100
864.43

95 100
90 Selected
864.92
85
80 Ion
865.41

75
70 %

65
60
%
55 865.92

y8
b14 866.44
%
50 863.04 867.42
862.70
1683.6 868.34 869.45
45 960.4
40 0 m/z
862 863 864 865 866 867 868 869
35 y10
30 1160.5 y14
y7 y11 b13
25 1659.5

20
y5 873.4 1273.6
y12 y131570.4
646.4 y6 b9 b15
b10 b111402.6 1531.6 b16 b17
15 745.5 b7 b12 1771.3
826.3 1071.3 1199.5 1328.6 y15 1857.9 1985.5
10
5 425.9 545.3 610.1 647.3 y16 0 m/z 0 Time
0
400 600 800 1000 1200 1400 10.00 15.00 20.00 25.00
400 600 800 1000 1200 1400 1600 1800 2000

Mass / Charge m/z

Mass /charge Retention time

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 Data processing
MZmine 2
1. Platform independent (e.g. Windows, Linux, Mac)
2. Modular software design (easy to join development effort)

dd.mm.yyyy (http://mzmine.sourceforge.n25et/)
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 Data Analysis

Pre-processing & Normalization & QC

Exploratory Analysis Univariate Analysis Correlation Analysis

Analysis of Variance
PCA and (ANOVA) Correlation Networks
Selection of peaks displaying significant changes Linear and Non-Linear approach
DiscriminantAnalysis to profile association calculation
between Wild Type and Transgenic, separately from
gender or age specific effects

Parametric Non-parametric Select peaks with high

Study general trends
Tests Tests Level of correlations to
In data
(t-test) (Kolmogorov-Smirnov) Strongest outliers

Prioritization of Important Peaks for Identification

Verification of Metabolite IDs. Databases Extensions

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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics Work flow
 Applications of metabolomics
 Small test

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Clinical Metabolomics Applications

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 Clinical Metabolomics Applications

 > 95% of all diagnostic clinical assays look for small molecules
 89% of known drugs are small molecules
 50% of all drugs are derived from pre-existing metabolites
 30% of identified genetic disorders involve diseases of small
molecule metabolism
 Monitor/measure metabolite flux, consequences from gene
KOs, and enzyme or pathway kinetics

Goldsmith, P., et al., J Surg Res, 2010. 160(1), 122-32.

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 Clinical Metabolomics Applications

 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 and functions of unknown genes
 Monitor gene/environment interactions
 Rapidly track effects from toxins/drugs/surgery

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 Metabolomics : Future?

› Metabolomics is one out of 14 fields

› Other fields in Nature‘s 2020 vision in which
metabolomics plays/can play a major role
’Personalized Medicine‘,
’Microbiome‘,

’Drug Discovery‘,
’Mental Health‘.
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 Outline

 What is metabolomics?
 Why metabolomics?
 Challenges in metabolomics
 Metabolomics platforms
 Metabolomics Work flow
 Applications of metabolomics
 Small test

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 Small assignment

1. What is metabolomics?

2. What is the biggest challenge in metabolomics?

3. What are different metabolomics platforms?

4. What is the typical metabolomics work flow?

5. What are the clinical applications of metabolomics?

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