Introduction to Clinical Metabolomicsresearch.med.helsinki.fi/corefacilities/proteinchem/TransMed... · Metabolomics can be viewed as complementary to ... Data Analysis Analysis of

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

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Introduction to

Clinical Metabolomics

Vidya Velagapudi, Ph.D, Adjunct Professor

Head of the Metabolomics Unit, Tech Centre,

Institute for Molecular Medicine Finland FIMM,

Helsinki, Finland

10/10/12

TransMed Course: Basics in Clinical Proteomics and

Metabolomics. Oct 10-19, 2012

© 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: [email protected]

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Metabolomics Unit

Waters XEVO-TQS triple quadrupole Hamilton star liquid handling system

Launched in 2011

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


• 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.

What is Metabolomics?


6

Brief historical note

1500-2000BC

China

•Ants used to

detect patients

with diabetes

1940s-1970s

•Advances in analytics

•Pattern recognition

Metabolic profiling

21st century

•Advances in analytics

•Biostatistics & Bioinformatics

Modern era of metabolomics

and systems biology

Metabolomics is not new


Outline


Why metabolomics?





Small test

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Functional readout

Why Metabolomics ?

- End point of cellular regulation

- Dynamic & time sensitive

- Sensitivity

- High throughput

- Suitable platform for sys bio

- Integrated studies

( Metabolomics + GWAS,

Metabolomics + transcriptomics)

Gate to personalised medicine


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.


Outline


Why metabolomics?





Small test


H2N

O

OH

Glycine

NH2

NH

O

OH

Tryptophan

NH2

HN

NH

H2N

O

OH

Arginine

OHHO

ONN

H2N

N

N

PO

O

OH

O

P

O

OH

O

PO

OH

OH

Adenosine-5'-triphosphate

O

O

OH

Pyruvic acid

O

OH

O

HO

Succinic acid

O

O

HO

O

OH

Oxaloacetic acidAcetyl CoA

Structural Diversity of Metabolites


12

Metabolome

Homeostasis

‘Housekeeping’

Organic Acids

Lipids

Amino Acids

Nucleotides

Steroids

Eicosanoids

Neurotransmitters

Peptides

Trace elements

nM (10-9)

pM (10-12)

mM (10-3)

µM (10-6)

NMR

Mass

Spectrometry

CUSTOM

Biofluid metabolic profile = Phenotype Adapted from Sumner, LW, et al., Phytochem, 62, 817,2003

fM (10-15)


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.

Challenges in Metabolomics


Outline


Why metabolomics?





Small test


Metabolomics platform

Meta

bolic

Pro

file

Global profiling

High Concentration

NMR profile

LCMS derivatized

(GCx)GCMS-profile

Target Biomarker

Assays (Multiplex) Immunoassays

GC, HPLC

Class-targeted

profiling

Bile acids, steroids,

fatty acids,

oxylipids

LC-FTMS

peptides

LCMS

Nucleosides,

Acyl CoA,

phosphorylated

LCMS

Glycerolipids,

Sphingomyelins,

Ceramides &

Cerebrosides

LCMS

fatty acids

GCMS

Endocannabinoids LCMS


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

Metabolomics Analyses


Common mass spectrometers

GC-TOF

LC-ion trap–FT-ICR LC-ion trap

GCxGC-TOF

UPLC-Q-TOF

Triple quadrupole

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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

Cellular

pathway

models

SYSTEMS

BIOLOGY

Clinical

studies Cells Organisms


Outline


Why metabolomics?





Small test



Experiment design + Analytical chemistry + Chemometrics + Bioinformatics


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


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


Acquiring a Mass Spectrum

Inlet

Ionization

Mass Analyzer

Mass Sorting (filtering)

Ion

Detector

Detection

Ion

Source

• Liquid

• Vapor

Detect ions Form ions

(charged molecules) Sort Ions by Mass (m/z)

1330 1340 1350

100

75

50

25

0

Mass Spectrum

The separation of ions based on

their mass / charge ratio’

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

All compounds must be ionized prior to MS detection!

Metabolite analysis


Separation

RPLC/CapLC +ESI

Mass

spectrometer

(Qtof)

10.00 15.00 20.00 25.00

Time 0

100

%

Retention time

MS-only ion chromatogram

400 600 800 1000 1200 1400 m/z 0

100

%

Mass /charge

862 863 864 865 866 867 868 869 m/z 0

100

%

864.43

863.04 862.70

864.92

865.41

865.92

866.44 867.42

869.45 868.34

Selected

Ion

MS survey scan

400 600 800 1000 1200 1400 1600 1800 2000 m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100 1047.5

1683.6 960.4

1160.5

1659.5

873.4 1273.6 1570.4

1531.6 646.4 1402.6 745.5 1771.3 1071.3 1199.5

1985.5 826.3 1857.9 610.1 647.3 545.3 425.9

b 14

b 7 b 9 b 10 b 11

b 12

b 13

b 15 b 17 b 16

y 8

y 15

y 14

y 13 y 12

y 11

y 10

y 9

y 7

y 6 y 5

1328.6

y 16

Mass / Charge

MS/MS fragmentation pattern

Sample

extract

ISs

Metabolites

LC/MS metabolomics platform


Data processing

25

MZmine 2

1. Platform independent (e.g. Windows, Linux, Mac)

2. Modular software design (easy to join development effort)

(http://mzmine.sourceforge.net/)


Data Analysis

Analysis of Variance

(ANOVA) Selection of peaks displaying significant changes

between Wild Type and Transgenic, separately from

gender or age specific effects

Univariate Analysis

Parametric

Tests (t-test)

Non-parametric

Tests (Kolmogorov-Smirnov)

Pre-processing & Normalization & QC

Prioritization of Important Peaks for Identification

Correlation Analysis

Correlation Networks Linear and Non-Linear approach

to profile association calculation

Select peaks with high

Level of correlations to

Strongest outliers

Exploratory Analysis

PCA and

Discriminant Analysis

Study general trends

In data

Verification of Metabolite IDs. Databases Extensions


Outline


Why metabolomics?



Metabolomics Work flow


Small test


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.



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


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‘.


Outline


Why metabolomics?



Metabolomics Work flow


Small test


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?

Documents

Introduction to Clinical Metabolomicsresearch.med.helsinki.fi/corefacilities/proteinchem/TransMed... · Metabolomics can be viewed as complementary to ... Data Analysis Analysis of