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Proteomics – status i relation til anvendelse i klinikken

Lars Melholt Rasmussen

professor, overlæge

Afd. for Klinisk Biokemi og Farmakologi, KBF

Odense Universitets Hospital, OUH

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Personlig medicin – proteomics status

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-Measurements of proteins are central in clinical medicine

(clin. biochem., microbiol., pathology, clin. immunol)

single markers

diagnosis: troponins, BNP, CRP etc

risk stratification (apolipoproteins, CRP)

monitoring drug effects (INR (factor II, VII, X)

multi markers

double testing (chrom. abberations)

elf-test (liver fibrosis)

MALDI-TOF identif. af bakteriekolonier

-Precision medicine needs more detailed phenotyping

ex: measurement of many proteins simultaneously (proteomics)

-Genomics is already estabished in research and routine

-So what is the status with clinical proteomics?

Proteinerne er her allerede!

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Clinical proteomics - status?

basics – potentials / challenges

methods / types

examples

1: current clin use:

amyloidosis diagnosis

2: recent promising research

athero-diagnostica

3: caution

treatment interference (heparin)

DNA mRNA Protein

Gene

DNA

mRNA1

mRNA2

mRNA3

mRNA4

mRNA5

SplicingProtein1

Protein2

Protein3

Protein4

Protein5

Protein6

....

Proteini

Post-translationalprocessing

Genomics Transcriptomics Proteomics

Nucleic acids: Simple, uniform chemistry

Easy extraction & quantitation, complete sequencing,

Proteins: Heterogenous physical/ chemical properties

Extremely different concentrations

Proteomics

• Large-scale investigations

• Identity & quantity

• Proteins and modifications

Since 1980’s: 2D-gels

Now: LC-MSMS

multiplex binding assays

800 900 1000 1100 1200 1300 1400m/z0

100

%

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Types of plasma proteomics – current state

Multiplex binding assays LC-MSMS (Mass spectrometry)

Types Immunoassays (antibody dependent)

Nucleic acid-binders (aptamers)

Discovery (data-dependent)

Targeted (predetermined, data-independent)

Advantages Easy to run many samples

High sensitivity

Large dynamic range

Not dependent of reagents

Specificity: very high

Identifies posttranslational modifications

Limitations Depends on antibodies or binders

Specificity is variable – often low

Interference: also from other analytes

when multiplexing

Ressource intensive (pre-analyt. and analyt.)

Sensitivity depends on physics of peptides,

sample and method

Which

proteins?

Most often defined by vendor:

OLINK (approx. 100)

Luminex (approx. 10 – 100)

SOMASCAN (approximat. 2000)

Discovery LC-MSMS

100 – 4000 proteins dependent on

method/instrument (not predetermined)

Targeted LC-MSM

1-100 predetermined

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Multiplex immunoassay - example

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Types of plasma proteomics – current state

Multiplex binding assays LC-MSMS (Mass spectrometry)

Types Immunoassays (antibody dependent)

Nucleic acid-binders (aptamers)

Discovery (data-dependent)

Targeted (predetermined, data-independent)

Advantages Easy to run many samples

High sensitivity

Large dynamic range

Not dependent of reagents

Specificity: very high

Identifies posttranslational modifications

Limitations Depends on antibodies or binders

Specificity is variable – often low

Interference: also from other analytes

when multiplexing

Ressource intensive (pre-analyt. and analyt.)

Sensitivity depends on physics of peptides,

sample and method

Which

proteins?

Most often defined by vendor:

OLINK (approx. 100)

Luminex (approx. 10 – 100)

SOMASCAN (approximat. 2000)

Discovery LC-MSMS

100 – 4000 proteins dependent on

method/instrument (not predetermined)

Targeted LC-MSM

1-100 predetermined

Workflow for LC-MSMS proteome analysis

nano-LC-MSMS

Plasma

Tissue

Mass spectra

Peptide sequence

Protein ident.

Bioinformatics

Nano-LC

SeparationIndividual

versus

Pools

Unbiased versus targeted Quantification-spectral counts

-labeled peptides

-isobaric labelling

Fragmentation:

Trypsination

Peptides

Extraction

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Types of plasma proteomics – current state

Multiplex binding assays LC-MSMS (Mass spectrometry)

Types Immunoassays (antibody dependent)

Nucleic acid-binders (aptamers)

Discovery (data-dependent)

Targeted (predetermined, data-independent)

Advantages Easy to run many samples

High sensitivity

Large dynamic range

Not dependent of reagents

Specificity: very high

Identifies posttranslational modifications

Low dynamic range

Limitations Depends on antibodies or binders

Specificity is variable – often low

Interference: also from other analytes

when multiplexing

Ressource intensive (pre-analyt. and analyt.)

Sensitivity depends on physics of peptides,

sample and method

Which

proteins?

Most often defined by vendor:

OLINK (approx. 100)

Luminex (approx. 10 – 100)

SOMASCAN (approximat. 2000)

Discovery LC-MSMS

100 – 4000 proteins dependent on

method/instrument (not predetermined - unbiased)

Targeted LC-MSM

1-100 predetermined

A typical clinical proteomics data output

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Clinical proteomics - status?

basics – potentials / challenges

methods / types

examples

1: current clin use:

amyloidosis diagnosis

2: recent promising research

athero-diagnostica

3: caution

treatment interference (heparin)

Amyloidosis

Misfolded protein deposition in tissue: heart, kidney, skin, tendon, lung, etc

transthyretin

immunglobuliner

serum amyloid protein

many others

Causes MGUS, chron. inflam., genetic basis

Diagnostics

Congo red + Immunohistochem. difficult because of low specificity and no quantitation

New proteome diagnosis

Myokardie-Biopsi

Congo rød farvning + mikrodissektion

Congo rød

Congo rød med filter

Congo rød med filter

Proteomics for the diagnosis and sub type classificationof amyloidosis

Amyloidosis: Extracellular deposition of proteins in an insoluble beta-sheet

physical format

Most important amyloidogenic proteins include:

• ApoE

• ApoA1

• ApoA4

• Serum amyloid P

• Serum amyloid A

• Transthyretin

• IGK (immunoglobulin kappa)

• IGL (lambda light chains)

Management of amyloidosis relys on treatment of the underlying etiology:• High-dose chemotherapy and stem cell transplantation (AL-type amyloidosis)

• Liver transplantation ( hereditary TTR-type amyloidosis)

”Amyloid signature”

Specific subtypes

Immunoelectron microscopy and mass spectrometry for classification of amyloid deposits

Abildgaard et al

Amyloid J, 2019

Amyloidosis

Misfolded protein deposition in tissue: heart, kidney, skin, tendon, lung, etc

transthyretin

immunglobuliner

serum amyloid protein

many others

Causes MGUS, chron. inflam., genetic basis

Diagnostics

Congo red + Immunohistochem. difficult because of low specificity and no quantitation

New proteome diagnosis

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Clinical proteomics - status?

basics – potentials / challenges

methods / types

examples

1: current clin use:

amyloidosis diagnosis

2: recent promising research

athero-diagnostica

3: caution

treatment interference (heparin)

Acute heart attack – acute coronary syndrome – diagnosis & treatment

Other diseasesMuscular pain

Lungproblems

Perforated ulcer etc

Complications-arrythima

-cardiogenic schock

AMBULANCE

Initial diagnosis & treatment:

anticoag: aspirin, heparin, other

HOSPITAL

Diagnosis? ECG, plasma-troponin, CAG

Treatment (stent)

Complication-stratification/treatment?

Acute coronay occlusion

Treatment: balooning/stenting

Ventricular assist

=temporary pump

SurvivalDeath

BIOMARKERS NEEDED:

Better, quicker diagnosis

Complication-stratification

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Myeloperoxidase

sFRP3

Midkine

Syndecan-1

New potential biomarkers for diagnosis

and complication stratification in acute coronary syndrome

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1: 100 autopsy coronary arteries+aortas – atherosclerosis grading + proteomics:

lists of atherosclerosis-abundant proteins (1000 proteiner)

2: Plasma - multiplex LC-MSMS assay for 17 of these proteins

3: 100 individuals having coronary arteriography (CAG): +/- coronary disease

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Associations of proteins to plaque abundancy and type

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1: 100 autopsy coronary arteries+aortas – atherosclerosis grading + proteomics:

lists of atherosclerosis-abundant proteins (1000 proteiner)

2: Plasma - multiplex LC-MSMS assay for 17 of these proteins

3: 100 individuals having coronary arteriography (CAG): +/- coronary disease

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Performance of 15-plex plasma-athero-panel:

+/- angiographically determined coronary atherosclerosis

Acute heart attack – acute coronary syndrome – diagnosis & treatment

Other diseasesMuscular pain

Lungproblems

Perforated ulcer etc

Complications-arrythima

-cardiogenic schock

AMBULANCE

Initial diagnosis & treatment:

anticoag: aspirin, heparin, other

HOSPITAL

Diagnosis? ECG, plasma-troponin, CAG

Treatment (stent)

Complication-stratification/treatment?

Acute coronay occlusion

Treatment: balooning/stenting

Ventricular assist

=temporary pump

SurvivalDeath

BIOMARKERS NEEDED:

Better, quicker diagnosis

Complication-stratification

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Myeloperoxidase

sFRP3

Midkine

Syndecan-1

New potential biomarkers for diagnosis

and complication stratification in acute coronary syndrome

Plasma 17-plex-athero (vitronectin)

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Clinical proteomics - status?

basics – potentials / challenges

methods / types

examples

1: current clin use:

amyloidosis diagnosis

2: recent promising research

athero-diagnostica

3: caution

treatment interference (heparin)

Acute heart attack – acute coronary syndrome – diagnosis & treatment

Other diseasesMuscular pain

Lungproblems

Perforated ulcer etc

Complications-arrythima

-cardiogenic schock

AMBULANCE

Initial diagnosis & treatment:

anticoag: aspirin, heparin, other

HOSPITAL

Diagnosis? ECG, plasma-troponin, CAG

Treatment (stent)

Complication-stratification/treatment?

Acute coronay occlusion

Treatment: balooning/stenting

Ventricular assist

=temporary pump

SurvivalDeath

PREDICT STUDY

(N=500)

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DISCOVERY OF NOVEL BIOMARKERS TO BE USED FOR

ACUTE CORONARY SYNDROME

OVERALL AIM

Discovery plasma proteomics to identify biomarkers for diagnosis and stratification

of patients with acute coronary syndromes

OUR APPROACH

- Plasma proteomics: Pre-analytical enrichment + LC-MSMS (800 proteins)

SUBSTUDY

Influence of heparin on the plasma proteome

A: 9 patients receiving heparin – plasma at 0, 2, 15, 60 min

B: Predict patients +/- heparin in ambulance

Beck et al, Clinical Chemistry, 2018 Oct; 64(10):1474-1484

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Heparin

-Polysaccharide, negatively charged

-Inhibits coagulation: Enhances binding of antithrombin to thrombin

-Is used to prevent further thrombosis development

in acute coronary syndrome and other situations

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0

1

2

3

4

5

0 10 20 30 40 50 60

Re

lati

ve a

bu

nd

ance

Time [min]

Blood sampling followed byHeparin administration

Secreted frizzled-related protein-1

SPARC-related modularcalcium-binding protein 1

Myeloperoxidase

ApoC - I to IV

Effect of heparin administration

on plasma proteins

(PCI procedure, n=9)

>800 protein ID

25 sign. changes

(bonferroni-corrected, p<7x10-5)

FEW PROTEIN

EXAMPLES

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Acute heart attack – acute coronary syndrome – diagnosis & treatment

Other diseasesMuscular pain

Lungproblems

Perforated ulcer etc

Complications-arrythima

-cardiogenic schock

AMBULANCE

Initial diagnosis & treatment:

anticoag: aspirin, heparin, other

HOSPITAL

Diagnosis? ECG, plasma-troponin, CAG

Treatment (stent)

Complication-stratification/treatment?

Acute coronay occlusion

Treatment: balooning/stenting

Ventricular assist

=temporary pump

SurvivalDeath

Heparin ???

75 % had received heparin before hospital entry

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One protein example verified by a

calibrated immunoassay: MIDKINE

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Proposed mechanism

Competition (wash) of proteins bound to endothelial cell surface heparan sulphates

VESSEL WALL

Exoneous

heparin

LUMEN

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Myeloperoxidase

sFRP3

Midkine

Syndecan-1

New potential biomarkers for diagnosis

and complication stratification in acute coronary syndrome

Plasma 17-plex-athero (vitronectin)

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Myeloperoxidase

sFRP3

Midkine

Syndecan-1

New potential biomarkers for diagnosis

and complication stratification in acute coronary syndrome

Plasma 17-plex-athero (vitronectin)Effects of heparin???

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Clinical proteomics - status?

basics – potentials / challenges

methods / types

examples

1: current clin use:

amyloidosis diagnosis

2: recent promising research

athero-diagnostica

3: caution

treatment interference (heparin)

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Clinical proteomics - status?

• proteomics is working !!!!

• technologies are robust, reproducible,

both discovery and targeted approaches

• >1000 protein IDs in discovery modes

• high-throughput: series of

>1000 individual samples

• clinical data needs to be good

• studies should focus on unmet clinical

needs, not technology potentials

• within 5-10 several new specific

applications will appear

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Proteomics – status i relation til anvendelse i klinikken

Thank you for your attention

Lars Melholt Rasmussen

professor, overlæge

Afd. for Klinisk Biokemi og Farmakologi, KBF

Odense Universitets Hospital, OUH

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Personlig medicin

Kendskab til individuelle

egenskaber

Bedre diagnostik

Bedre stratificering

Bedre monitorering

Skræddersyet behandling

Registre

køn, alder, bosted

indlæggelser

lab.data

recepter

Biologiske data

konventionelle

OMICS

Big data

Biostatistik

Art. Intel.

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Myeloperoxidase

sFRP3

Midkine

Syndecan-1

New potential biomarkers are influenced by

heparin !! (in these studies heparin medication in ambulance is not registred)

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Myeloperoxidase

sFRP3

Midkine

Syndecan-1

New potential biomarkers for myocardial damage,

coronary occlusion and heart insufficiency

Effects of heparin???

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New biomarkers for cardiogenic shock

in acute coronary syndrome

Substudy of PREDICT (national study)

Acute coronary syndrome

Pre-hospital Hospital + CAG

Blood sample (BNP, copeptin, TnI/T) + Echo

STEMI

Normal CAG

Cardiogenic shock

(early – late)Plasma proteomics

n=320

+/- heparin (75/25 %) Collab: B: Jacob Eifer Møller, Lisette Okkels Jensen et al

KBF: Hans C Beck, LMR et al

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0

1

2

3

4

5

0 10 20 30 40 50 60

Re

lati

ve a

bu

nd

ance

Time [min]

Blood sampling followed byHeparin administration

Secreted frizzled-related protein-1

SPARC-related modularcalcium-binding protein 1

Myeloperoxidase

ApoC - I to IV

Effect of heparin administration

on plasma proteins

(PCI procedure, n=9)

>1000 protein ID

Ca 75 sign. changes

FEW PROTEIN

EXAMPLES:

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New biomarkers for cardiogenic shock

in acute coronary syndrome

Substudy of PREDICT (national study)

Acute coronary syndrome

Pre-hospital Hospital + CAG

Blood sample (BNP, copeptin, TnI/T) + Echo

STEMI

Normal CAG

Cardiogenic shock

(early – late)Plasma proteomics

n=320

+/- heparin (75/25 %) Collab: B: Jacob Eifer Møller, Lisette Okkels Jensen et al

KBF: Hans C Beck, LMR et al

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*** ** ***

*** ******

** ****

*** p < 10-9 ** 10-6 > p > 10-9 * 10-3 > p > 10-6

***

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-What is clinical proteomics?

methods / types

research example (apoliporot.)

clinical example (amyloidosis)

-Some challenges in plasma proteomics

assays

preanalytical

drug interference

DNA mRNA Protein

Gene

DNA

mRNA1

mRNA2

mRNA3

mRNA4

mRNA5

SplicingProtein1

Protein2

Protein3

Protein4

Protein5

Protein6

....

Proteini

Post-translationalprocessing

Genomics Transcriptomics Proteomics

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OUH maj 2011/MO

Clinical Biochemistry Research Unit, KBF, OUHUniv. of Southern Denmark

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Fra forskning til hverdag med NGS, omics og big data

Lokale Skovbrynet

Mødeleder: Morten Pedersen

10.05-10.30

Fra patient til svar med NGS

Jesper Boulund Kristensen, Lone Andersen

10.30-11.00

Proteomics – status i relation til anvendelse i klinikken

Lars Melholt Rasmussen

11.00-11.30

Big Data er vejen til bedre børnevaccinationer

Tue Bjerg Bennike

8.20 – 9.20

National strategi for personlig medicin - skræddersyet behandling til alle?

Professor Søren Brunak

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Præsentationen vil lægge ud med en diskussion

af begrebet ”proteomics”, og kort

opsummere hvilke teknologier der anvendes, og

hvad de dækker over og hvilke outcomes,

man kan forvente fra forskellige platforme.

Herefter vil eksempler fremdrages som

illustrerer state of the science: status for aktuel

anvendelse af proteomics i klinisk

sammenhæng, områdets potentiale og en

diskussion af, hvad vi kan forvente i fremtiden

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Personalised medicine:

Personalised medicine: a move away from a ‘one size fits all’ approach to the treatment

and care of patients with a particular condition, to one which uses new approaches to

better manage patients’ health and target therapies to achieve the best outcomes in the

management of a patient’s disease or predisposition to disease.

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-Measurements of specific proteins are central

in clinical medicine (clin. biochem., pathology)

single markers

diagnosis: troponins, BNP

risk stratification (apolipoproteins, CRP)

monitoring drug effects (INR (factor II, VII, X)

multi markers

double testing (chrom. abberations)

elf-test (liver fibrosis)

-Precision medicine needs detailed phenotypingmeasurement of many proteins simultaneously (proteomics)

is believed to contain great potentials

-Genomics is already estabished in research and routine

-So what is the status with clinical proteomics?

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-What is clinical proteomics?

methods / types

research example (apoliporot.)

clinical example (amyloidosis)

-Some challenges in plasma proteomics

assays

preanalytical

drug interference

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Crista Cobbaert

All apolipoprotein-multiplex LC-MSMS assay

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INITIAL HEPARIN STUDY

Aim

Effect of heparin administration on plasma proteins?

Patients

PCI procedure, n=9

0, 2, 15, 60 min after 5000 IU of heparin iv

Plasma proteome analysis

>800 protein quantification

Workflow for LC-MSMS proteome analysis

nano-LC-MSMS

Plasma

Tissue

Mass spectra

Peptide sequence

Protein ident.

Bioinformatics

Nano-LC

Separation

(chromat.)

Individual

versus

Pools

Unbiased versus targeted Quantification-spectral counts

-labeled peptides

-isobaric labelling

Fragmentation:

Trypsination

Peptides

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Proteome results