quantitative proteomics research: breast and prostate cancer biology spiros d. garbis, phd...
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Quantitative Proteomics Research: Breast and Prostate
Cancer Biology
Spiros D. Garbis, PhDInvestigator, Faculty MemberCenter for Basic Research IIDivision of Biotechnology
OOutlineutline
• Background and OverviewBackground and Overview• Clinical Tissue ProcurementClinical Tissue Procurement• Mass Spectrometry EssentialsMass Spectrometry Essentials• Summary of Current Findings Summary of Current Findings
Breast and Prostate Cancer
• Poor cancer diagnosis and prognosis
• Higher prevalence in elderly populations
• Complex process
• Synergic behavior of pathways
• Combinatorial Signaling aspects
• Related to the metabolic syndrome (epidemiology)
Albini et al. 2007
Considerations for Experimental Design
The accurate study of Carcinogenesis must account for:
1. Multi-parameter nature of cancer initiation, promotion, progression
2. Crucial role of tumor microenvironment
3. Biological variance of tissue specimens
Fully accounted for in our LC-MS based proteomics infrastructure
To provide a window in gaining a better insight to cancer biology
Perspective and Perspective and ConsiderationsConsiderations
• The current state-of-the-art in the molecular characterization require prior knowledge of the proteins involved
• Promising pre-clinical data is essential (for confirmation and justification for the pharmacoproteomic study)
• What is frequently missed is all the systemic effects
• The global (discovery or hypothesis generating science) targeted (hypothesis driven science)
• Long term implications (i.e xenografted or mutated mouse models, pilot scale human clinical trials, etc)
• NOT about generating lists of proteins. It’s about de-coding quantitative profiles into canonical and novel pathways
Selection of Clinical Material
“Garbage in…Garbage out”
Poorly characterized specimens are usually poor of
information
Well characterized specimens can minimize variability
and facilitate analytical methodology Iterative process process involving a perpetual
feedback loop between tissue procurement/preparation &
analytical method development in order to generate a
reproducible and accurate quantitative protein profile
PROTEOMICS of PROTEOMICS of •TISSUE,TISSUE,•CELL CULTURE,CELL CULTURE,•SERUM SERUM
Protein MAPPING:The ID of a maximum number of proteins constituting a sample
Protein MAPPING:The ID of a maximum number of proteins constituting a sample
Protein EXPRESSION:The ID of altered proteins as a response to a stimulus
Protein EXPRESSION:The ID of altered proteins as a response to a stimulus
Protein MODIFICATION:Determine how and where a protein is post-translationally modified
Protein MODIFICATION:Determine how and where a protein is post-translationally modified
Ligand-enzyme INTERACTION:Determine how proteins interact with medicinal agents
Ligand-enzyme INTERACTION:Determine how proteins interact with medicinal agents
-80 OC within 5 min
Proteomics Lab and analysis after histochemical evaluation of the mirror part
Well defined inclusion/exclusion criteria were used for both clinical groups (staging, Gleason Score 6, no androgen intervention, etc.) Adherance to GCP/ICH guidelines
10 Patients BPH [mean age ± S.D of 70 ± 9 years, mean serum PSA ± S.D of 4.1 ± 1.9 ng/mL]
10 PCa Cases [mean age ± S.D of 66 ± 4 years, mean serum PSA ± S.D of 9.3 ± 4.6 ng/mL]
Cancer specimens histochemically confirmed (>70%)
PCa
Tissue Procurement and Patient Characteristics
5 mm
Proteomics as an INTERDISCIPLINARY science should Proteomics as an INTERDISCIPLINARY science should reliably provide greater insight:reliably provide greater insight:
•Proteins mediate Proteins mediate molecular and biochemical molecular and biochemical functionsfunctions(signalling, cell division, metabolism, cell migration)(signalling, cell division, metabolism, cell migration)
•Alteration of proteinsAlteration of proteins denote disease states denote disease states
•Proteins serve as Proteins serve as viable biomarkersviable biomarkers of disease of disease
•Proteins can serve as Proteins can serve as drug/therapeutic targetsdrug/therapeutic targets
ProteomicsProteomics
BIOLOGICAL SAMPLE (CELLS, TISSUE, FLUID)
SUBFRACTIONATIONSUBFRACTIONATION
EXCISE SPOTS, IN-GEL DIGEST
EXCISE SPOTS, IN-GEL DIGEST
2DGE OR 2D DIGE2DGE OR 2D DIGE
MALDI-TOF MS ANALYSISMALDI-TOF MS ANALYSIS
DATA ANALYSISDATA ANALYSIS
PROTEIN ID & PROTEIN EXPRESSION ANALYSISPROTEIN ID & PROTEIN EXPRESSION ANALYSIS
STABLE ISOTOPE LABELINGSTABLE ISOTOPE LABELING
MULTI-DIMENSIONAL CHROMATOGRAPHY
MULTI-DIMENSIONAL CHROMATOGRAPHY
ON-LINE LC-MS-MS ANALYSIS
ON-LINE LC-MS-MS ANALYSIS
Garbis SD, et al. J. of Chromatogr. A, 2005, 1077, 1-18.
HPLC-ESI-QIT-MSn (6330 MSD) HPLCHPLC--ESIESI--QITQIT--MSMSnn (6330 MSD) (6330 MSD)
DIAGNOSTIC PEPTIDE ABSOLUTE
QUANTIFICATION
DIAGNOSTIC PEPTIDE DIAGNOSTIC PEPTIDE ABSOLUTE ABSOLUTE
QUANTIFICATION QUANTIFICATION
-LC-MALDI-TOF-MS
•2DGE-MALDI-MS
--LCLC--MALDIMALDI--TOFTOF--MSMS
••2DGE2DGE--MALDIMALDI--MSMS
TISSUETISSUETISSUE PLASMAPLASMAPLASMA
PROTEIN I.D. & RELATIVE QUANTIFICATIONPROTEIN I.D. & RELATIVE QUANTIFICATIONPROTEIN I.D. & RELATIVE QUANTIFICATION
CATALOGUE DIAGNOSTIC TRYPTIC PEPTIDESCATALOGUE DIAGNOSTIC TRYPTIC PEPTIDESCATALOGUE DIAGNOSTIC TRYPTIC PEPTIDES
EXTRACT PROTEIN & GENERATE TRYPTIC PEPTIDES EXTRACT PROTEIN & GENERATE TRYPTIC PEPTIDES EXTRACT PROTEIN & GENERATE TRYPTIC PEPTIDES
2DLC-nESI-QqTOF-MS-MS (QSTAR XL)2DLC2DLC--nESInESI--QqTOFQqTOF--MSMS--MS (QSTAR XL)MS (QSTAR XL)
CROSS-PLATFORM VALIDATIONCROSSCROSS--PLATFORM VALIDATIONPLATFORM VALIDATION
STRATEGY FOR MULTISTRATEGY FOR MULTI--PLATFORM PROTEOMICSPLATFORM PROTEOMICS
CELL CULTURECELL CULTURECELL CULTURE
Chromatography Directed Protein Chromatography Directed Protein Isolation and EnrichmentIsolation and Enrichment
PROTEIN CATEGORY PEPTIDE CHROMATOGRAPHIC CHEMISTRY
OPTIMUM pH
Hydrophilic Proteins (proteases, kinases, secreted, phosphate
modified)
Ion Exchange, Biphasic Ion Exchange- Hydrophilic Interaction, Zwitterion Ion
Chromatography, Graphite, TiO2
2.5 - 11
Extrinsic Membrane Proteins)
(non-covalently associated)
Hydrophobic Interaction, Reverse Phase – Polymer phase
11
Intrinsic Membrane Proteins (covalently bound)
Hydrophilic Interaction, Normal Phase
6.5 – 7.5
PRINCIPLE:PRINCIPLE:
1. Isolate proteins in concordance to chromatographic chemistry to be used,
2. Separate and enrich these tryptic peptides with HPLC (high selectivity, reproducibility and capacity)
m/z, Da
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
Inten
sity
0
100
200
300
400
500
600
700
800
900
1000
114.11
145.11
291.22
339.18174.13420.25251.15 701.42
452.28 533.34380.17
Multidimensional Liquid Chromatography
RP-nanoHPLC
SCX
MS/MS
Peptide
mixture
1st Dimension 2nd Dimension
Separation according to i.e. charge state –
polarity index
Separation according to hydrophobicity index ORTHOGONAL
CHEMISTRIES
Tandem Mass Spectrometry (MS/MS)
Collision induced dissociation, CID
MS fragmentation MS
Precursor ion Product ions
MS-ΜS spectrum
MS spectrum
Reveals SEQUENCE and PTM information about the peptides
Mixture of Peptide ions (LC-
ESI)PEPTID
EPEPTIDEPE
PEP
PEPTPEPTIPEPTID
P
CID m/z seperatio
n
PEPTIDEPEPTIDE
PEPTIDE P E P T
P E P T I D E
P E P
P
P E PT IP E P T
I D
P E
Protein Identification
…is based on peptide sequence determination
m/z, Da
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
Intensity
0
100
200
300
400
500
600
700
800
900
1000
114.11
145.11
291.22
339.18174.13420.25251.15 701.42
452.28 533.34380.17
EGETITEVIHGEPIIK
TITEVIH
GR
m/z, Da
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
Intensity
0
100
200
300
400
500
600
700
800
900
1000
114.11
145.11
291.22
339.18174.13420.25251.15 701.42
452.28 533.34380.17
Protein
confidencePeptide
confidence
Quality of MS/MS spectra
•Peptide length
•Number of peptides
•Uniquely occurring peptides
•Peptide abundance
•Peptide ionization
•Peptide volatility
•Instrument status
•Preparation process
m/z
mix
115
114
114
114 11
4115
11511
5 115 11
6116 11
6117
MS/MS spectrum
+
+
+
+
Different reporter ions
Protein with differential abundance
114
115
116
117
31
30
29
28
iTRAQPeptide with differential abundance
The same mass in MS The
same peptide fragmen
ts
2D-LC
A
B
C
D
A
B
CD
114 115 116 117
114-117
145
31-28
Isobaric Tags for Relative and Absolute Quantification (iTRAQTM)
Methodology
Tandem Mass Spectrometry (MS/MS)
2-Dimentional Liquid Chromatography (2D-LC)
Differential peptide labeling with isotopic reagents (iTRAQ)
Algorithmic Data processing & Evaluation
Mix Peptides
Trypsin digestion of protein extracts
Tissue1 PCa
Tissue2
PCa
Tissue3
BPH
Tissue4
BPH
iTRAQ 115
iTRAQ 117
iTRAQ 114
iTRAQ 116
Summary of BrCa & PCa Proteomics QSTAR XL MS-MS Resolution ~ 12,000 @ 500 – 1500 Da
MS Accuracy 3-ppm; MS-MS Accuracy 15-ppm
Reproducible identification of ~ 1000 proteins ( ≥ 95% confidence)
spanning the high mol – low amol on-column amounts
Wide range of physico-chemical properties
100s of PTM variants identified of which > 60% are novel
Differentially expressed proteins are traceable to canonical and
novel mechanistic pathways of carcinogenesis
Independent biological confirmation
Extensive commonality of proteins with respective human cell lines
and sera specimens (secreted proteins)
Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158. Bouchal P, Roumeliotis T, et al. J Proteome Res. 2008, In Press.
Putative NPutative NFFB B AActivating ctivating PProtein rotein
Effective multidimensional
chromatography is essential to the quality
of the product-ion spectra
Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158. Bouchal P, Roumeliotis T, et al. J Proteome Res. 2008, In Press.
Proteomic Feature Maps: A new visualization approach in proteomic
analysis
Giannopoulou EG, et al. J Biomed Inf 2008, In Press.
Novel & Quantitative Phosphoproteome
Supplementary Information:Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158. Bouchal P, Roumeliotis T, et al. J Proteome Res. 2008, In Press.
200 300 400 500 600 700 800 900
232.141
175.119
30
60
309.683
619.364
366.230 544.269
347.168 706.396447.316
696.361
562.343
850.450
y1y2
y4 y5
y12++
y15++
b2
b4++ b7
++
b9++
SDK[IT4]LPEEMGLLQGSSGDK[IT4]RPrec. Mass: 2478.3386, z: +4
BPHPCa
BPHPCa
b1
y3 y6793.428
y7
873.938
b3++
637.800
INT
EN
SIT
Y
m/z
BPH PCa
Cell surface glycoprotein (CD146 Antigen)
Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158.
Voltage-dependent N-type calcium channel subunit α-1B: A Quantitative and Temporal
Proteomics Appoach
m/z, Da
200 400 600 800 1000 1200 1400 1600 1800
Inte
nsity
0
5
10
15
20
25
30
35
40
45b7b7-H3PO486.10
120.08
219.19 699.39642.85201.18
451.31
238.12507.29 756.45467.22 810.39
1066.48 1260.46 1451.78 1617.63 1801.34
m/z, Da
670 680 690 700 710 720 730 740 750 760 770 780
Inte
nsi
ty
5
10
15
20
25
30
35
40b7b7-H3PO4
699.393
756.451700.387735.461
669.355 713.390684.388758.333755.461 772.361
694.379 723.336
m/z, Da
113.0 114.0 115.0 116.0
Intens
ity
114.1100
116.1111
115.1075
113.1025
CTL 6h 12h 36h•Novel Phosphopeptide
•Time-dependent phosphorylation
•Novel apoptosis mechanism
•Physiologic Target
Activity-Based Proteomic Profiling
Covalently boundpurine
Cell Extract
Specific binding of purine target to the
affi nity matrix
N
NN
N
NH
OH
NHNH
ONH
O( )
5
N
NN
N
NH
OH
NHNH
ONH
O( )
5
45-
31-
66.2-97.4-
MW(kDa)
SDS-PAGE, microsequencing,mass spectrometry
Covalently boundpurine
Cell Extract
Specific binding of purine target to the
affi nity matrix
N
NN
N
NH
OH
NHNH
ONH
O( )
5
N
NN
N
NH
OH
NHNH
ONH
O( )
5
45-
31-
66.2-97.4-
MW(kDa)
SDS-PAGE, microsequencing,mass spectrometry
Bach, et al 2005
Cellular structure and growth
Tumor microenvironment and stroma-epithelium interactions (inflammation
factors, angiogenesis factors)
Detoxification
Breast & Prostate physiology
Targets for pharmaceutical or chemopreventive intervention
Differentially expressed proteins are relative to:
The majority of differentially expressed proteins is relative to established carcinogenesis pathways
The methodology used made possible the simultaneous detection of multiple molecular alterations
Novel findings amenable to further investigation as novel pathways of BrCa & PrCa
Detection of proteins which have also been detected in human plasma proteome
MOLECULAR EVIDENCE FOR NUTRITIONAL AND IMMUNOLOGIC FACTORS IN THE MANIFESTATION OF BREAST AND PROSTATE CANCER
Overall Overall ConclusionsConclusions
ALTERNATIVE QSAR INTERPRETATION:ALTERNATIVE QSAR INTERPRETATION:Pharmaco-PROTEOMIC & Toxico-Pharmaco-PROTEOMIC & Toxico-
PROTEOMICSPROTEOMICS
OO
XX
OO
OO
OO HH
OO
XX == SS ee ,,
O
O
HO
OH
O
OHHO
OH
TOCOTRIENOL-X TOCOTRIENOL-X CONJUGATESCONJUGATES
BENZOFURANBENZOFURAN
DEOXYBENZOINDEOXYBENZOIN SOY ISOFLAVONE SOY ISOFLAVONE DERIVATIVESDERIVATIVES
CollaboratorsUniversity of Cyprus• Prof. Andreas Constantinou• Prof. Anastasios Keramidas• Dr. Andreani Odysseos
University of Athens• Prof. Leandros Skaltsounis• Prof. Evi Lianidou• Prof. Elias Manolakos
University of Toronto• Prof. Eleftherios Diamandis
CNRS Cell-Cycle Biology Lab• Prof. Laurent Meijer
Hellenic National Research Institute• Dr. Mixalis Alexis• Dr. Xanthipi Alexi
Demokritos• Dr. Evangelos Gogolides• Dr. Aggeliki Tserepi
Masaryk Memorial Cancer Institute, Brno, The Check Republic
• Dr. Pavel Bouchal• Dr. Roman Hrstka• Dr. Rudolf Nenutil• Dr. Borivoj Vojtesek
University of Adelaide, Australia• Prof. Andreas Evdokiou
Biomedical Research Foundation-Academy of Athens
• Dr. Antonia Vlahou• Dr. Sophia Kossida• Dr. Constantin Tamvakopoulos
Laiko University Hospital• Prof. Konstantinos Constantinides• Prof. Kitty Pavlaki• Dr. Stavros Tyritzis
Biosolutions-Applied Biosystems• Konstantina Tasiouka• George Magkaniotis
BRFAA Relevant References
Garbis SD, et al. J. of Chromatogr. A, 2005, 1077, 1-18.
Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158.
Bouchal P, Roumeliotis T, et alJ Proteome Res. 2008, In Press.
Giannopoulou EG, et al. J Biomed Inf. 2008, In Press.
Delehouz C, et al., J. Biol. Chem. 2008, In Press.
Manadas B, et al. Mol. & Cell. Proteomics 2008, In Press
7 more manuscripts in preparation!