Aplicació de la proteòmica a la cerca de Biomarcadors · PDF filea la cerca de ....
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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
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).
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
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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
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45
50
55
60
65
70
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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