Aplicació de la proteòmicaa la cerca de

Biomarcadors proteics

Barcelona, 08 de Juny 2010

Eliandre de OliveiraPlataforma de Proteòmica – Parc Científic de Barcelona

Protein ChemistryProteomics

Hypothesis-freeHigh-scale

To identify as many components of the proteome as possible

The proteome is the set of proteins expressed from a determined genome.

What we can do?

Protein Mining: Identification of different proteins in a sample.

Protein Expression Profiling: Comparison of protein expressionunder determined conditions.

Protein Network Mapping: Approach to look at the interactionbetween proteins from different systems.

Mapping of Protein Modifications: Identification of proteinmodifications and site mutation localization.

Applications

Systems biology - understand cell-pathways, network, and complex interacting.

Biological processes - characterize sub-proteomes such as protein complexes, cellular machines, organelles

Biomarkers - discovery of disease (serological, urine, other biological fluids) -diagnostics, treat patients, monitor therapies

Drug targets - evaluate toxicity & other biological or pharmaceutical parameters associated with drug treatment

Protein Profiling

• 2-DE gel electrophoresis

• Difference gel electrophoresis (DIGE)

• LC-MS/MS using stable isotopic labeling(ICAT, iTRAQ, SILAC..)

• ProteinChip (SELDI analysis)

• Antibody arrays

Measure the expression of a set of proteins in two samples and compare them - Comparative proteomics

2-DE gels, DIGE

- High resolving power

- Absolute / relative quantity

- Easily archived for further comparison

- Detects some PTMs and alternatives splices

- Low troughput

- Poor detection of large, acidic, basic and membrane proteins

- Only high abundance proteins

Coomassie blue stained gels

Silver stained

Mix labeled extractsInternal standard

SELDISurface Enhanced Laser Desorption Ionization

- High throughput

- Small amounts of sample

- More reproducible than 2DE, but lower resolving power

- Applied for the analysis of crude samples

- Process is not standardized

Ionized proteins are detected and their mass accurately determined by Time-of-Flight Mass Spectrometry

Chemical Surfaces

(Hydrophobic) (Anionic) (Metal Ion) (Normal Phase)(Cationic)

(Antibody - Antigen) (Receptor - Ligand) (DNA - Protein)(PS10 or PS20)

Biological Surfaces

Laser

Antibody array

Forward phaseSandwich assay Direct assay

Analyte

Detection with Labeled Analyte

Detection with 2nd

Antibody

Antibody immobilized on glass substrate

Reverse phase

Analytes immobilized on glass substrate

Detection with Labeled Antibody

Modified from slide; FullMoonBiosystemsInc. (http://www.fullmoonbio.com/Doc/Overview.pdf)

- Not discovery based

- Must have 1 or 2 specific high affinity antibodies

- Very high throughput

- Can be highly quantitative - relative and absolute

- Can design reagents to detect PTMs, splice forms

Protein Identification by LC-MS/MS

Protein mixture

Digestion

Peptides

400 800 1200 1600m/z

MS

MS/MS

10 20 30 min0

HPLC

DatabaseSearching

LLTTIADAAK

SAGGNYVVFGEAK

EDDVEEAVQAADR

All peptide sequences Identification of many proteins

Aebersold and Mann, Nature, 2004

Stable-isotope labeling

Isobaric tag reagent (iTRAQ)

Isobaric Tag (Total mass =145 Da)

Peptide reactive group

Reportermass=114 to 117

Gives strong signature ion in MS/MS

Balancemass 31 to 28

Balances the mass change of reporter to maintain a total mass of 145

Amine specific

Isobaric tags for relative and absolute quantificationAllows us to compare the relative abundance of proteins from four different samples in a single mass spectrometry experiment

Quantitative proteomics by using isobaric tag reagent-iTRAQ

31

1347.0 1349.6 1352.2 1354.8 1357.4 1360.0Mass (m/z)

1352.84111.0 112.8 114.6 116.4 118.2 120.0

Mass (m/z)

114

116

115

117

9.0 292.8 576.6 860.4 1144.2 1428.0

Mass (m/z)

8396.7

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity y8

P

A b2 y10

q,H

72.1 b4

b145.1 b7LT 11

2.1

y3 b10

y5 b9y2 b674.1 13

52.8

y6142.

1

39.0 y4 b8 y9 y1

1 PP PP

PP PP

EE EE

EE EE

TT TT

DD DD

…………

P E P T I D E…

P E P T I D E…

P E P T I D E…

+

+

+

+

P E P T I D E…

-PRG114 31

-PRG115 30

-PRG116 29

-PRG117 28

-Reporter-Balance-Peptide INTACT- 4 samples identical m/z

MS

Mix -NH

P E P T I D E…114-NH

P E P T I D E…-NH

P E P T I D E…-NH

P E P T I D E…

114

115

116

117- Reporter ions DIFFERENT

MS/MSP E P T I D E…

- Peptide fragments EQUAL

3011529116

28117

Approaches to Protein Biomarkers

Proteomics(Discovery)

Candidate- Single - Panel

Validation Value Added

Single/Few Protein Assay(s)(Hypothesis)

Multiplex Protein Assay(s)(Hypothesis/ Discovery)

Detection Limits in Clinical Diagnosticsfor a 50 kdal protein analyte

BiomarkerConcentration pg/ml amol/ml Molecules/ml

50 mg/ml 50,000,000,000 1,000,000,000 6.02E+1710 mg/ml 10,000,000,000 200,000,000 1.20E+171 mg/ml 1,000,000,000 20,000,000 1.20E+16100 ug/ml 100,000,000 2,000,000 1.20E+1510 ug/ml 10,000,000 200,000 1.20E+141 ug/ml 1,000,000 20,000 1.20E+13100 ng/ml 100,000 2,000 1.20E+1210 ng/ml 10,000 200 1.20E+111 ng/ml 1,000 20 1.20E+10100 pg/ml 100 2 1.20E+0910 pg/ml 10 0 . 0.2 1.20E+081 pg/ml 1 0 .02 1.20E+07

Immunoassays QqQMS given 1000-fold enrichment

Biofluid Proteomics

Human plasma - Biomarkers of Sepsis (in collaboration with Dr. Ricard Ferrer and Dr. Antoni Artigas, Hospital de Sabadell)

Human CSF - Biomarkers of ALS, Amyotrophic lateral sclerosis (in collaboration with Dra. Marta Vilaseca, Dr. Claudio Dilema and Dr. Joan Guinovart of the IRB, and Dr. Jacques Borg of Saint Étienne University Hospital)

(Anderson, N. L., and Anderson, N. G. (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, 845 –867).

Am J Respir Crit Care Med Vol 178. pp 701–709, 2008

admissionplasma sample

dischargeplasma sample

Pipeline

Protein Identification LC-MS/MS

Sample Preparation

Patient Selection

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400m

/z

05

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

690.81

1027.87

570.33 1156

.84599.13

635.85

1138.861122

.831251.79

371.25

799.93 1010

.89242.26

727.23258

.19881.99

389.22

561.21

958.89

276.24

832.76

1269.83

286.28

1234.85

1107.00

1346.63

1252.9

579.3

643.8Fr

agm

ent I

on in

tens

ity

Mass / Charge Ratio

Ion

inte

nsity

Retention Time

LC-MS/MS Proteomics

Clinical Plasma Samples

Peptides

Liquid Chromatography

Preparation& Digestion

Mass Spectrometry

MS/MS

Separation By Mass/ChargeMeasurement of Intensity

ProteinIdentification

Separation By Retention Time

- Major plasma proteins

Immunodepletion-Digestion

-iTRAQ labelling

- 4 sepsis patients

- samples analyzed in two groups: (A1D1A2D2) + (A3D3A4D4)

- Protein expression level is represented as a ratio: admission/discharge

- ( abundance ratio)

⌫

Pre-treatmentPlasma sample 1

Pos-treatmentPlasma sample 2,3

Pipeline

Protein Identification LC-MS/MS

Sample Preparation

Patient Selection

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400m

/z

05

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

690.81

1027.87

570.33 1156

.84599.13

635.85

1138.861122

.831251.79

371.25

799.93 1010

.89242.26

727.23258

.19881.99

389.22

561.21

958.89

276.24

832.76

1269.83

286.28

1234.85

1107.00

1346.63

1252.9

579.3

643.8Fr

agm

ent I

on in

tens

ity

Mass / Charge Ratio

Ion

inte

nsity

Retention Time

LC-MS/MS Proteomics

Clinical Plasma Samples

Peptides

Liquid Chromatography

Preparation& Digestion

Mass Spectrometry

MS/MS

Separation By Mass/ChargeMeasurement of Intensity

ProteinIdentification

Separation By Retention Time

- Major plasma proteins

Immunodepletion-Digestion

-iTRAQ labelling

Parc Científic de Barcelona

Alex CamposMª Antonia OdenaKostadin Evgeniev Atanasov

Hospital de SabadellCorporació Sanitària Parc Taulí:

Dr. Antoni ArtigasDr. Ricard FerrerDra. Gemma FusterGemma GomàGisela Gili

Thank you !