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Proteomics in biomarker
discovery for clinical purposesdiscovery for clinical purposes
Deborah Penque, [email protected]
BiomarkerBiomarker
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”
BiomarkerBiomarker 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)
impact on patient well being and financial viability of healthcare systems
•• AbnormaliesAbnormalies in the production or function of in the production or function of the proteins are linked to health, the proteins are linked to health, environmental response and many diseasesenvironmental response and many diseases
•• ProteinProtein isis thethe mainmain target of target of manymany therapeutictherapeutic
WhyWhy ProteinProtein as as BiomarkerBiomarker ? ?
To understand To understand how to control how to control an an
environmental response environmental response and or and or treattreat
a particular a particular diseasedisease, it is necessary to , it is necessary to
identify the proteins identify the proteins associated with associated with •• ProteinProtein isis thethe mainmain target of target of manymany therapeutictherapeuticdrugsdrugs
“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 they functionfunction..
ClinicalClinical ProteomicsProteomicsClinicalClinical ProteomicsProteomics
Dedicated to the study of the PROTEOME
PROFILE associated with the HEALTHY AND
DISEASE STATE, in the search for DISEASE STATE, in the search for
DIAGNOSTIC / PROGNOSTIC / MONITORING
BIOMARKERS or as TARGETS for the
development of new therapeutic approaches
ProteinsProteins are are complexcomplex
�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, andcell, 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
Hood et al Proteomics. 2012 Sep;12(18):2773-83
• Discovery-based approach
Proteomics Technology
• Targeted –based approach
Discovery -based approach
What proteins can be
detected in this sample ?
Discovery Phase Validation/Translation Phase
Penque, Expert Rev Proteomics, 2007, 4:199-209
Torre et al, 2015. Book chapter in Methods in Molecular&Biology, in press
Discovery Proteomics approach
Data Aquisition
2D-gel
HPLC
MALDITOFTOF
ESI-MS/MSLC/MS/MS
Shotgun MS
Data Base Query(GPS, Mascot, Sequest,
GO, etc
Pathway/Network
Analysis
Discovery-based Proteomics approach
MALDITOFTOF
Data Base Query(Mascot, Sequest, etc
Imaging MS
proteinprotein CHIPCHIPMALDI
SELDI-TOF
Pathway/Network
analysis
Targeted proteomics approach
Is protein X measurable in this sample?
Shotgun proteomics
Biochemical experiments
Antibody /Affinity - basedhttp://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
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
with the highest level of sensitivity.
Quantitative Proteomics
Protein abundance and sample
complexity are significant factors that
affect the availability of proteins for
mass spectrometric quantitation
RelativeAbsolute /
Quantitation
Absolute
Proteomics
Proteomics Clinical Purposes
(some examples)
Laboratório de Proteómica Departamento de Genética Humana
Unidade de Tecnologia & Inovação
INSA, I.P. Ricardo Jorge-Lisboa
MissõesMissões
�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
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,
HPV
Chronic Lung DiseasesChronic Lung Diseases
Cystic Fibrosis
Asthma COPD
Biormaker Discovery of Chronic Lung Diseases
D Penque Expert Rev. Proteomics 4 (2) 2007
� Basic mechanism responsible for
F508delF508del-CFTR retention in ER
remains to be elucidated
F508del-CFTR
Ep
ith
eli
al c
ell
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 IIClass II
Ep
ith
eli
al c
ell
LT (26 ºC) & drugs
repair the
trafficking defect of
F508del-CFTR
Ep
ith
eli
al c
ell
Cl-
WT ∆∆∆∆F ∆∆∆∆F26ºC
Low-temperature
WT
-CF
TR
37ºC 26ºC24h 48h
3-10 pH
8-1
6%
SD
S-P
AG
E
BHK cells stably expressing CFTR
Progenesis PG200v2006
BHK cell line
expressing WT or
ΔF508del-CFTR
∆∆ ∆∆F
50
8-C
FT
R
3h 35S-methionine metabolic labelling
2D Map 2D Map
AnalysisAnalysis
MS
MALDI-TOF-TOF 4700
Gomes-Alves et al 2009
BpH 3 -10
SD
S-P
AG
E (
8-1
6%
)
Hspa5
Hsp90ab1
NI
Lamb1-1
NI
Psmd2Vcp
Ganab
Plec1
Vcl
Lonp1
Ogdh
NI
PygbGart
Eef2
Snd1
Impdh2
Aco2
Cct4
Qars
NI
NI
Gfm1
Mcm7
Plod3
Pafah1b1
Csde1
Fkbp9Nasp
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
Snx6Adk
Pdia3
G6pdx
Cct2
Adss
A
BpH 3 -10
SD
S-P
AG
E (
8-1
6%
)
Hspa5
Hsp90ab1
NI
Lamb1-1
NI
Psmd2Vcp
Ganab
Plec1
Vcl
Lonp1
Ogdh
NI
PygbGart
Eef2
Snd1
Impdh2
Aco2
Cct4
Qars
NI
NI
Gfm1
Mcm7
Plod3
Pafah1b1
Csde1
Fkbp9Nasp
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
Snx6Adk
Pdia3
G6pdx
Cct2
Adss
AA
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
NINI
S100a10
NI
Pfn1Hist1h4a
PpiaNme1
Fth1
Prdx1
Fabp5
Ube2e
Cmpk1
Stmn1
Lgals1
Sh3bgrl
Ubc
Prdx2
Psmb4
Tmed3
Tpt1
Cbx1
Eif1ya
Gnb2l1
Vdac2
Psa2
Pdlim1
Ark72
Psb7
Rexo2Prdx6 Tpi1
Psa6
Gstm5
Pgam1
Psph
Ywhae
Pcna
Rpsa
Anxa5
Cope Psme2
Srm
Cdk4
Cops5
Asna1
Npm1
Npm1 Cnn3Enoa
Cnn3
Txnd4
Bdh1
Prps2
Anxa1
Pnp
Snx6
Ahcy
Ublcp1
Actr2
Eif3a
Adk
Gdi2 Psmd11Psmd7
Got1
Skp1
NI
Snrpf
NINI
S100a10
NI
Pfn1Hist1h4a
PpiaNme1
Fth1
Prdx1
Fabp5
Ube2e
Cmpk1
Stmn1
Lgals1
Sh3bgrl
Ubc
Prdx2
Psmb4
Tmed3
Tpt1
Cbx1
Eif1ya
Gnb2l1
Vdac2
Psa2
Pdlim1
Ark72
Psb7
Rexo2Prdx6 Tpi1
Psa6
Gstm5
Pgam1
Psph
Ywhae
Pcna
Rpsa
Anxa5
Cope Psme2
Srm
Cdk4
Cops5
Asna1
Npm1
Npm1 Cnn3Enoa
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
Down-regulation F508del 26ºC 24h
29%
15%14%
11%
7%
5%5%
Metabolism
Trascription and translation
Degradation
Cytoskeleton
Folding
Antioxidants
Maturation and traff icking
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
Gomes-Alves et al 2010
Validation
by western-
blottingblotting
Mutagenic Repair(RXR) motifs
Mutagenic Repair(RXR) motifs
Low temperature
↑↑ BIP, mortalin, Hsp90, Hsp70
↓↓ proteosome (Psme2)Increase folding capacity
UPRUnfolde 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
Freeze in OCT
Store at -80ºC
Cryodissection
naphthalene
Challenge
Wt
F508del
Wt
F508del
CF CF TransgenicTransgenic MiceMice
Cryodissection
PALM MicroscopePALM 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
Carvalho-Oliveira et al, 2007, Expert Rev Mol Diag, 7:407-417
LC/MS/MSLC/MS/MS
2D
2D
--ge
lg
el
Serum
LC/MS/MSLC/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.
Most enriched Pathways : • cell-to-cell signaling and interaction
• hematological system
• development,
• immune response,
• oxidative stress and
• cytoskeleton.
• Chorein (VPS13A) > cell
COPDCOPD
• 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
2012 Arnaldo
Sampaio Award
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
PROTEOMICSPROTEOMICS
OSA Patients& Controls
Clinical Evaluation
& PSG-LaboratoryStudy
Clinical/Sample Database
Biobanking
Sample Preparation for
PROTEOMICS
2-DIGE
tryptic peptidesspot
excisionProtein
DIS
CO
VE
RY
P
HA
SE
Shotgun MS
Mass spectometer
mass spectra
Proteinidentification
Protein Networks
Bioinformatic Data
Analysis
CANDITATES BIOMARKERS OF OSA CANDITATES BIOMARKERS OF OSA
VERIFICATION PHASE
Feliciano et al Sleep Medicine 2015
2DIGE
pH 3-10
12.5
% S
DS
-PA
GE
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
12.5
% S
DS
21 different proteins
Existence of Post-translational
Modifications
CATALASE Proteoforms
pH-3-10
12.5
%S
DS
-PA
GE
1 2 3 4 5 6 7 8
2DIGE
0,8
1
1,2
1,4
1,6
1,8
Norm
aliz
ed V
olu
me
Snorers OSA
2
0,6
0,8
1
1,2
1,4
Norm
aliz
ed V
olu
me
Snorers OSA
4
0,8
1
1,2
1,4
1,6
Norm
aliz
ed V
olu
me
Snorers OSA
3
0,8
1
1,2
1,4
1,6
Norm
aliz
ed V
olu
me
Snorers OSA
1
0,6
0,8
1
1,2
1,4
1,6
Norm
alz
ied V
olu
me
Snorers OSA
5
0,7
0,9
1,1
1,3
1,5
1,7
1,9
Nom
aliz
ed V
olu
me
Snorers OSA
6
1,1
1,6
Nom
alz
ied V
olu
me 7
1,1
1,6
Norm
aliz
ed V
olu
me
8
-Evening
-Morning
Anova p=0.009 Anova p=0.002 Anova 3.8x10-6
Anova p=0.0005 Anova p=0.01Anova p=0.0009
**
A
Dis
cove
ryP
ha
se
0,6
Nom
alz
ied V
olu
me
Snorers OSA0,6
Norm
aliz
ed V
olu
me
Snorers OSA
0
0,2
0,4
0,6
0,8
1
1,2
1,4
Rela
tive
Prot
ein
Abun
danc
e
OSA-CPAPSnorers OSA
CATALASE
MW (kDa)
80 -
60 -50 -
Snorers OSA
- Evening
- Morning
Western Blot
B
60000
80000
100000
120000
140000
nm
olC
AT/
nm
olC
AT/
min
/mg
pro
tein
*
**
55000
65000
75000
85000
95000
105000
Evening Morningnm
olC
AT/
nm
olC
AT/
min
/mg
pro
tein
*
CATALASE Activity
- Evening
- Morning
Snorers OSA OSA-CPAP
C
Va
lid
ati
on
Ph
ase
continuous positive airway pressure (CPAP),
Discovery Phase
Pe
rox
ired
ox
in2
Pro
teo
form
s
Validation Phase
SummarySummary
Proteomics can provide:
• NewNew insightsinsights into the poorly-understood
pathogenetic processes of diseases.
• NewNew biomarkersbiomarkers for diagnosis & prognosis
• NewNew targetstargets for development of novel
therapeutic approaches.
Team membersTeam 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 AzevedoCarlos LopesAntónio 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
www.redeprocura.com
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