Proteomics in biomarker discovery for clinical purposes

(1)

Proteomics in biomarker

discovery for clinical purposes

Deborah Penque, PhD

(2)

Biomarker

NIH-USA official definition:

A characteristic that is objectively measured and evaluated as

indicator of normal or pathogenic biological processes or

pharmacological response to a therapeutic intervention”

Biomarker

Biomarker still needed for

• early detection of diseases to benefit from the potential therapies.

• pharmacodynamic assessment of drug action to help guide dose and schedule

• selection of patients who will benefit from therapy (pharmacoproteomics)

(3)

•

• Abnormalies

Abnormalies in the production or function of

in the production or function of

the proteins are linked to health,

environmental response and many diseases

•

• Protein

Protein is

is the

the main

main target of

target of many

many therapeutic

therapeutic

Why

Why Protein

Protein as

as Biomarker

Biomarker ?

?

To understand

To understand how to control

how to control an

an

environmental response

environmental response and or

and or treat

treat

a particular

a particular disease

disease, it is necessary to

, it is necessary to

identify the proteins

identify the proteins associated with

associated with

•

• Protein

Protein is

is the

the main

main target of

target of many

many therapeutic

therapeutic

drugs

“Proteins are two steps closer than genes to

most biological phenomena and diseases”

Hood et al Proteomics.2012 Sep;12(18):2773-83

identify the proteins

identify the proteins associated with

associated with

these processes and

these processes and understand how

understand how

they

(4)

Clinical

Clinical Proteomics

Proteomics

Clinical

Clinical Proteomics

Proteomics

Dedicated to the study of the PROTEOME

PROFILE associated with the HEALTHY AND

DISEASE STATE, in the search for

DIAGNOSTIC / PROGNOSTIC / MONITORING

BIOMARKERS or as TARGETS for the

(5)

Proteins

Proteins are

are complex

complex

Genes are digital in nature with a 4-letter language, proteins are analog with a 20 letters language; genes operate in a one-dimensional world and proteins in a three-dimensional world

Proteins is extremely complex due to: modifications by gene mutation, RNA editing,

RNA splicing, up to 400 types of covalent changes and protein processing

Proteins are dynamical, changing their 3-dimensional structures, positions in the

cell, concentrations at different cellular sites, sequences, covalent chemistries, and cell, concentrations at different cellular sites, sequences, covalent chemistries, and

interactions with other proteins and molecules of many types in response to

endogenous and exogenous stimuli;

Proteins exhibit a 106 dynamic range in tissues and a 1012 dynamic range in blood,

making quantification essential

Proteins lack the molecular complementarity of DNA and hence cannot be amplified

prior to measurement—thus, higher ultrasensitive techniques to measure and analyze protein molecules is needed

(6)

• Discovery-based approach

Proteomics Technology

(7)

Discovery -based approach

What proteins can be

detected in this sample ?

Discovery Phase Validation/Translation Phase

Penque, Expert Rev Proteomics, 2007, 4:199-209

(8)

Discovery Proteomics approach

Data Aquisition 2D-gel HPLC MALDITOFTOF ESI-MS/MS LC/MS/MS Shotgun MS

Data Base Query

(GPS, Mascot, Sequest, GO, etc

Pathway/Network Analysis

(9)

Discovery-based Proteomics approach

MALDITOFTOF

Data Base Query

(Mascot, Sequest, etc

Imaging MS

protein

protein CHIPCHIP MALDI

SELDI-TOF

Pathway/Network analysis

(10)

Targeted proteomics approach

Is protein X measurable in this sample?

Shotgun proteomics

Biochemical experiments

Antibody /Affinity - based

http://www.proteinatlas.org/

21,500 Abs for 11,000 genes

QQQ-type MS (SRM, SISCAPA) MALDI-type (MISA, iMALDI) Mass spectrometer-based http://www.srmatlas.org/

(170,000SRM assays for human) Literature

(11)

The balance between scope/sensitivity/scalability of discovery and targeted proteomics. Due to the broad-scope nature and sensitivity of discovery proteomics, the ability to

perform a comprehensive analysis of hundreds or thousands of samples is limited. Conversely, targeted proteomic analysis entails the quantitation of discrete subsets of peptides, which allows the ability to analyze these peptides across thousands of samples

(12)

Quantitative Proteomics

Protein abundance and sample complexity are significant factors that

affect the availability of proteins for mass spectrometric quantitation

(13)

Relative

Absolute /

Quantitation

Absolute

(14)

Proteomics Clinical Purposes

(some examples)

(15)

Laboratório de Proteómica

Departamento de Genética Humana Unidade de Tecnologia & Inovação

INSA, I.P. Ricardo Jorge-Lisboa

Missões

Desenvolver uma plataforma I&D inovadora baseada na

proteómica para validação, implementação de

biomarcadores já existentes ou descoberta de novos

Investigação Serviços Outras Actividades

Missão

biomarcadores já existentes ou descoberta de novos

biomarcadores de diagnóstico, prognóstico e

monitorização de doenças ou como alvos a novos

abordagens terapêuticas.

Prestar colaboração e serviços de caracterização de

proteínas pela proteómica

Contribuir para o desenvolvimento da proteómica no nosso

país (promoção/realização de cursos/estágios/conferências,

networking) na área da proteómica

(16)

Running Projects

1. Proteomics of chronic lung diseases leading to biomarkers and therapeutic target discovery. FCT project

POCTI/SAU-MMO/56163/2004. PI: D Penque

2. Environmental Tobacco Smoke Exposure at Portuguese Restaurants, Bars and nightclubs: health effects and early molecular mechanisms underlying respiratory disorders.

Research

molecular mechanisms underlying respiratory disorders.

FCG/ACSS. PI: T Simões & D Penque.

3. MSIA technology development . PI: D Penque & V Torres

4. Obstructive sleep apnea and associated metabolic/cardiovascular disorders: understanding mechanisms towards early diagnosis and

prognosis prediction. HMSP-ICJ/0022/2011- Junior Investigator: A Feliciano,

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Chronic Lung Diseases

Cystic Fibrosis

(18)

Biormaker Discovery of Chronic Lung Diseases

(19)

Basic mechanism responsible for

F508del

-CFTR retention in ER

remains to be elucidated

F508del-CFTR E p it h e li a l c e ll

Investigating by Proteomics the trafficking

defect of F508del-CFTR

Investigating by Proteomics the trafficking

defect of F508del-CFTR

LT (26 ºC) & drugs

Cl

-Trafficking defect

Class II

E p it h e li a l c e ll

LT (26 ºC) & drugs

repair the

trafficking defect of

F508del-CFTR

E p it h e li a l c e ll

Cl

-WT ∆∆∆∆F ∆∆∆∆F26ºC

(20)

Low-temperature

W T -C F T R 37ºC

26ºC

24h _48h 3-10 pH 8 -1 6 % S D S -P A G E

BHK cells stably expressing CFTR

Progenesis PG200v2006 BHK cell line expressing WT or ΔF508del-CFTR ∆∆∆∆ F 5 0 8 -C F T R

3h 35_S-_methionine _{metabolic labelling}

2D Map 2D Map Analysis Analysis

MS

MALDI-TOF-TOF 4700 Gomes-Alves et al 2009

(21)

B pH 3 -10 S D S -P A G E ( 8 -1 6 % ) Hspa5 Hsp90ab1 NI Lamb1-1 NI Psmd2 Vcp Ganab Plec1 Vcl Lonp1 Ogdh NI Pygb Gart Eef2 Snd1 Impdh2 Aco2 Cct4 Qars NI NI Gfm1 Mcm7 Plod3 Pafah1b1 Csde1 Fkbp9 Nasp Ndufs1 NI Vil2 Copd Got1 Eef1g Umps Ccdc105 NI Hspa8 Gdi1 Nap1l1 NI Calu Dpyls3 G3bp1 Atp6v1a Hspa9 Hnrnpk Hspa8 Dlat Rpsa Atp5b NI Actb Txnd4 Des Hnrpf Cct1 Psmc2 NI Enoa Snx6 Adk Pdia3 G6pdx Cct2 Adss A B pH 3 -10 S D S -P A G E ( 8 -1 6 % ) Hspa5 Hsp90ab1 NI Lamb1-1 NI Psmd2 Vcp Ganab Plec1 Vcl Lonp1 Ogdh NI Pygb Gart Eef2 Snd1 Impdh2 Aco2 Cct4 Qars NI NI Gfm1 Mcm7 Plod3 Pafah1b1 Csde1 Fkbp9 Nasp Ndufs1 NI Vil2 Copd Got1 Eef1g Umps Ccdc105 NI Hspa8 Gdi1 Nap1l1 NI Calu Dpyls3 G3bp1 Atp6v1a Hspa9 Hnrnpk Hspa8 Dlat Rpsa Atp5b NI Actb Txnd4 Des Hnrpf Cct1 Psmc2 NI Enoa Snx6 Adk Pdia3 G6pdx Cct2 Adss A A

6 groups (cell types/conditions) 4-5 gels/group

ANOVA, p < 0.05 Normalized volume

Gomes-Alves et al 2009, JOP, in press

Psmd7 Got1 Skp1 NI Snrpf NI NI _S100a10 NI Pfn1 Hist1h4a Ppia Nme1 Fth1 Prdx1 Fabp5 Ube2e Cmpk1 Stmn1 Lgals1 Sh3bgrl Ubc Prdx2 Psmb4 Tmed3 Tpt1 Cbx1 Eif1ya Gnb2l1 Vdac2 Psa2 Pdlim1 Ark72 Psb7 Rexo2 Prdx6 Tpi1 Psa6 Gstm5 Pgam1 Psph Ywhae Pcna Rpsa Anxa5 Cope Psme2 Srm Cdk4 Cops5 Asna1 Npm1 Npm1 Enoa Cnn3 Cnn3 Txnd4 Bdh1 Prps2 Anxa1 Pnp Snx6 Ahcy Ublcp1 Actr2 Eif3a Adk Gdi2 Psmd11 Psmd7 Got1 Skp1 NI Snrpf NI NI _S100a10 NI Pfn1 Hist1h4a Ppia Nme1 Fth1 Prdx1 Fabp5 Ube2e Cmpk1 Stmn1 Lgals1 Sh3bgrl Ubc Prdx2 Psmb4 Tmed3 Tpt1 Cbx1 Eif1ya Gnb2l1 Vdac2 Psa2 Pdlim1 Ark72 Psb7 Rexo2 Prdx6 Tpi1 Psa6 Gstm5 Pgam1 Psph Ywhae Pcna Rpsa Anxa5 Cope Psme2 Srm Cdk4 Cops5 Asna1 Npm1 Npm1 Enoa Cnn3 Cnn3 Txnd4 Bdh1 Prps2 Anxa1 Pnp Snx6 Ahcy Ublcp1 Actr2 Eif3a Adk Gdi2 Psmd11

139 protein spots differentially expressed

125 proteins spots identified Differences over 1.5

(22)

Down-regulation F508del 26ºC 24h 29% 15% 14% 11% 7% 5% 5% Metabolism

Trascription and translation Degradation

Cytoskeleton Folding Antioxidants

Maturation and trafficking Other

Several degradation associated proteins were down-regulated, while BiP and other UPR related proteins were found up-regulated in BHK-F508del cells under the

CFTR-“rescue” treatment at low temperature.

14% Up-regulation F508del 26ºC 24h 25% 19% 15% 13% 13% 9% 6%

Trascription and translation Folding

Metabolism

Maturation and trafficking Degradation

Cytoskeleton Other

(23)

Validation

by

western-blotting

blotting

(24)

(25)

Mutagenic Repair (RXR) motifs Mutagenic Repair (RXR) motifs Low temperature ↑ ↑ BIP, mortalin, Hsp90, Hsp70 ↓ ↓ proteosome (Psme2) Increase folding capacity

UPR

Unfolde protein response

↓

↓ proteosome (Psme2) Increase folding capacity

& diminish degradation

Expression Reversion of some

proteins involved in CFTR

maturation & trafficking

Expression Reversion of some

proteins involved in CFTR

maturation & trafficking

e.g

↑

RACK1

Promote relocation of ∆

∆

∆F-508-CFTR to cell surface

Promote relocation of ∆

∆

∆F-508-CFTR to cell surface

(26)

Freeze in OCT Store at -80ºC Cryodissection naphthalene Challenge Wt F508del Wt F508del

CF

Transgenic

Mice

Cryodissection

PALM Microscope

PALM Microscope _{2000 lung epithelial cells}

2D DIGE & LC/MS/MS

(MALDI-FTICR-MS

The results suggest

the involvement of

prostaglandin and retinoic

acid metabolism in the

abnormal responses of CF

mutant

mice to injury.

Carvalho-Oliveira et al, 2009, JPR, 8:3606-16

(27)

LC/MS/MS

2 D 2 D --g e l g e l Serum

LC/MS/MS

Dysregulated Pathways (~70 p) :

• abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction,

• chronic inflammation, • nutritional imbalance

• P. aeruginosa colonization.

Dysregulated Pathways (~70 p) :

• abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction,

• chronic inflammation, • nutritional imbalance

• P. aeruginosa colonization.

Apolipoproteins family (VDBP, ApoA-I, and ApoB) gradually lower expression from non-CF to CF-carrier individuals and from those to CF patients,

The enzyme NDKB was identified only in the CF, its functions account for ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation and energy production, highlighting its physiological significance in the context of CF.

(28)

(29)

Most enriched Pathways :

• cell-to-cell signaling and interaction • hematological system

• development,

• immune response, • oxidative stress and • cytoskeleton

.

• Chorein (VPS13A) > cell

COPD COPD

• Chorein (VPS13A) > cell

membrane deformation of RBC c

Methemoglobin reductase

• (Cytochrome CYB5R3) > COPD

patients may be at higher risk for

developing methemoglobinemia.

219 proteins dysregulated in COPD RBCm

(30)

2012 Arnaldo Sampaio Award

(31)

Biomarkers for

Obsructive Sleep Apnea is needed

• to distinguish snoring from OSA , facilitating population screening and prevention of OSA-associated outcomes • to provide new insights into pathophysiological aspects • to distinguish snoring from OSA , facilitating population

screening and prevention of OSA-associated outcomes • to provide new insights into pathophysiological aspects • to provide new insights into pathophysiological aspects

of OSA that underlie the increased cardiovascular and metabolic risk in general population

• to provide new insights into pathophysiological aspects of OSA that underlie the increased cardiovascular and metabolic risk in general population

PROTEOMICS

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OSA Patients & Controls Clinical Evaluation & PSG-Laboratory Study Clinical/Sample Database Biobanking

Sample Preparation for PROTEOMICS 2-DIGE tryptic peptides spot excision Protein D IS C O V E R Y P H A S E Shotgun MS Mass spectometer mass spectra Protein identification Protein Networks Bioinformatic Data Analysis

CANDITATES BIOMARKERS OF OSA CANDITATES BIOMARKERS OF OSA

VERIFICATION PHASE

(33)

2DIGE

pH 3-10 1 2 .5 % S D S -P A G E

76 spots identified differentially abundant (Anova p≤ 0.05)

30 spots identified by MALDITOTOF, corresponding

OSA Evening X OSA Morning X Snorers Evening X Snorers Morning

RBC Hemoglobin-depleted cytoplasmic fraction 1 2 .5 % S D S 21 different proteins Existence of Post-translational Modifications

(34)

CATALASE Proteoforms pH-3-10 1 2 .5 % S D S -P A G E 1 2 3 4 5 6 7 8 2DIGE 0,8 1 1,2 1,4 1,6 1,8 N o rm a liz e d V o lu m e Snorers OSA 2 0,6 0,8 1 1,2 1,4 N o rm a liz e d V o lu m e Snorers OSA 4 0,8 1 1,2 1,4 1,6 N o rm a liz e d V o lu m e Snorers OSA 3 0,8 1 1,2 1,4 1,6 N o rm a liz e d V o lu m e Snorers OSA 1 0,6 0,8 1 1,2 1,4 1,6 N o rm a lz ie d V o lu m e Snorers OSA 5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 N o m a liz e d V o lu m e Snorers OSA 6 1,1 1,6 N o m a lz ie d V o lu m e 7 1,1 1,6 N o rm a liz e d V o lu m e 8 -Evening -Morning

Anova p=0.009 Anova p=0.002 Anova 3.8x10-6

Anova p=0.0005 Anova p=0.0009 Anova p=0.01

**

A

D

is

co

ve

ry

P

h

a

se

0,6 N o m a lz ie d V o lu m e Snorers OSA 0,6 N o rm a liz e d V o lu m e Snorers OSA 0 0,2 0,4 0,6 0,8 1 1,2 1,4 Re la tiv e Pr ot ei n Ab un da nc e OSA-CPAP Snorers OSA CATALASE MW (kDa) 80 60 50 -Snorers OSA - Evening - Morning Western Blot B 60000 80000 100000 120000 140000 n m o lC A T/ n m o lC A T/ m in /m g p ro te in * ** 55000 65000 75000 85000 95000 105000 Evening Morning n m o lC A T/ n m o lC A T/ m in /m g p ro te in * CATALASE Activity - Evening - Morning

Snorers OSA OSA-CPAP

C

V

a

li

d

a

ti

o

n

P

h

a

se

(35)

Discovery Phase

P e ro x ir e d o x in 2 P ro te o fo rm s

Validation Phase

(36)

Summary

Proteomics

can provide:

• New

New insights

insights

into the poorly-understood

pathogenetic processes of diseases.

• New

New biomarkers

biomarkers

for diagnosis & prognosis

• New

New targets

targets

for development of novel

therapeutic approaches.

(37)

Team members

INSA

(Proteomics, Mol Biol, Statistic)

Patrícia Alves Bruno Alexandre Nuno Charro Isabel C Oliveira* Deborah Penque Tânia Simões M Fátima Vaz Paula Pacheco Paulo Nogueira ITQB (Mass spectrometry)

Univ Edinburgh(SELDI-TOF consultants)

Margaret Imrie; Robert Gray David Poteous; Chris Boyd

Univ Pittisburgh

HSM/Clinic Pulmonology

(patients recruitment, clinical phenotype)

Pilar Azevedo Carlos Lopes

António Bugalho de Almeida

FCUL

(Bioinformtics)

Francisco Couto, David Santos

Acknowledgements

Ana V Coelho Univ Pittisburgh(MS/consult)

Thomas Conrads & Brian Hood

NCI (MS consultants)

Timothy Veenstra & Josip Blonder

Univ Madrid

(MS consultants)

Juan Pablo Albar

Work partially supported by FCT research grants: PCOTI/ESP/44720/2002,

POCTI/MGI/40848/2001, POCTI/SAU-MMO/56163/2004, Gulbenkian Fondation-ACSS ;

Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011)

•Univ Lund Peter James

(38)

www.redeprocura.com

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