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.

«Bottom-Up Proteomics» «Bottom-Up Proteomics» WorkflowWorkflow

Comparative Proteomics

Beecher, 1999

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.

PCA

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.

Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158.

Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158.

Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158.

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.

Prostate Specific Antigen (PSA)

Garbis SD, et al. J Proteome Res. 2008;7(8):3146-3158.

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.

p22HBP

GST -1

Bouchal P, Roumeliotis T, et al. J Proteome Res. 2008, In Press.

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

Glycogen Debranching Enzyme

Woo, et al. 2008

Roscovitine-Affinity Site

CANONICAL PATHWAY : Apoptosis (p < 0.001)

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

From Proteomics to Proteonomics:

The New Biology

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!