Lars Melholt Rasmussen professor, overlæge Afd. for...
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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)
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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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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
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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
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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)
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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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Myokardie-Biopsi
Congo rød farvning + mikrodissektion
Congo rød
Congo rød med filter
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Congo rød med filter
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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
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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)
![Page 21: Lars Melholt Rasmussen professor, overlæge Afd. for ...deks.dk/wp-content/uploads/2019/09/3.2-LM.pdf1 Proteomics –status i relation til anvendelse i klinikken Lars Melholt Rasmussen](https://reader030.fdocuments.nl/reader030/viewer/2022040210/5e5b6c6af69b09680773e870/html5/thumbnails/21.jpg)
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
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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
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)
![Page 30: Lars Melholt Rasmussen professor, overlæge Afd. for ...deks.dk/wp-content/uploads/2019/09/3.2-LM.pdf1 Proteomics –status i relation til anvendelse i klinikken Lars Melholt Rasmussen](https://reader030.fdocuments.nl/reader030/viewer/2022040210/5e5b6c6af69b09680773e870/html5/thumbnails/30.jpg)
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
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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
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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