Proteoma –definição:
•
“O complemento PROTEico total de um genOMA.”
–
M. Wilkins et al. Electrophoresis 1995, 16, 1090-1094
•
Grupo de proteínas expresso por uma célula em um momento.
•
Proteoma é dinâmico: muda constantemente em resposta a estímulos.
•
Proteomia é o estudo das propriedades proteicas em grande escala, de forma a obter uma
visão mais global e integral dos processos de uma célula.
•
Proteoma: permite identificação de novos genes ainda não identificados em bancos gênicos
de EST ou após o sequênciamento completo do genoma.
2DE com focalização isoelétrica
2DE: 1adim:native elet +SDS-PAGE
Purificação de complexos cromat. afinidade
Crom. Líquida multidimensional
Fracionamento em misturas de solventes (acet, isop,
clorof. E metanol
Rota de uma análise proteômica
2D Gel
Electrophoresis
Coloração
Captura de
imagem
Digestão da
proteína
Planejamento da
excisão
Análise da
imagem
Preparação
Maldi
Preparação
para o MS
Análise pelo
Maldi
Análise pelo
MS
Identificação
da proteína
Identificação
da proteína
Identificacao da proteina
quantificacao da mudanca na expressao
Sistema automatizado “Ettan” da Amersham
1) Filme carlos-cenargem-mov: Espectrometria de massa
2) Filme Maldi-ESI
MALDI-TOF MS
matrix-assisted laser desorption ionisation time of
flight mass spectrometry
Maldi-TOF
ms-ms tutorial.exe
Processes in Proteome
Analysis
C. elegans
Age related
protein differences
• Proteome Expression or Profiling
– identifying which proteins
change levels
of
old
old
expression in response to certain stimuli or
the environment of the cell
•
• Sensitivity
Proteome
Mapping
• Dynamic range
young
young
• Detector linearity
– assigning
the location of a protein (-spot),
quantitation
as defined by is
pI key
and MW, and identification
by mass spectrometry
• Sensitivity of spot detection
• Resolutions and Sensitivity of MS
How to Increase Sensitivity in Proteomics?
•
•
Increasing amounts of low-abundance proteins relative to other proteins by
fractionation
– narrow range pH gradients
• high load
• solubility during separation
– cell compartments
• mitochondria
• peroxisomes
• nuclei
– biochemical pre-fractionation
• solubility
• affinity
Increasing sensitivity by using fluorophores
Profiling the Mitochondrial Proteome
•
•
Acidic proteins left
high molecular weight top
Silver-stained Reference 2D gel
– unfractionated proteins
– average of 1.500 spots per 2D gel
– poor recovery from in-gel digestion
– limited throughput of profiling effort
– 195 (marked) spots excised and processed
– not all could be identified
• low recovery of peptides
• low abundance
• lack of credible hits in databases
CBB-stained Reference 2D gels
– 8-16 times less sensitive than silver
– average of 300 - 500 spots per gel
– good recovery from in-gel digestion
– MS compatibility
CBB = CoomassieTM Brilliant Blue
MALDI-TOF mass spectrum
Profiling the Mitochondrial
Proteome
• Identification of over 100 proteins
• in several days
• high confidence
• based on high mass accuracy (typically 50 ppm
or less
• at least 4 peptides matched
• at least 10% sequence coverage
Pre-fractionation by minispin
columns
• Metal chelate IMAC column
– calcium-charged metal chelate
– enrichment of Calcium binding proteins
• Concanavalin A (Con A) column
– Con A lectin binds high mannose
oligosaccharides
• Phenyl Sepharose column
– hydrophobic protein binding
– much less specific enrichment as above
Calcium binding protein enrichment
•
•
•
•
•
Acidic proteins left
high molecular weight top
CBB-stained 2D gel
819 proteins detected
presumably detected proteins
– calcium binding proteins
– regulated by calcium
identified spots are marked
proof by MS identification
– all proteins are previously
shown to bind calcium or to be
calcium-regulated
Con A binding protein enrichment
•
•
•
•
•
•
Acidic proteins left
high molecular weight top
CBB-stained 2D gel
min. 78 proteins detected
presumably detected proteins
– glycosylated proteins
large amount of protein unresolved
– vertical & horizontal streaking
– possible reasons
• heterogeneity in charge &
mass of putative
glycosylated proteins
clear resolved and identified spots
are marked
little information available on on
glycosylation of mitochondrial
proteins
– e. g. Glutamate DH identified
Hydrophobic protein enrichment
•
•
•
•
Acidic proteins left
high molecular weight top
CBB-stained 2D gel
736 proteins detected
presumably detected proteins
– hydrophobic & membrane
proteins
– less specific
well-resolved 2D gel
– fragment of matrix proteins
– no identification by database
query
• despite excellent spectra and
mass accuracy
– new proteins?
Protein Enrichment by Specific Fractionation
Table 2. Selected proteins identified in affinity enriched 2-D gels of Mitochondrial and ER and peroxisomal proteins.
Affinity ligand
Spot number
Figure
Protein identity
Database
Accession number
calcium
calcium
calcium
calcium
calcium
calcium
calcium
calcium
calcium
Con A
ConA
ConA
ConA
Con A
ConA
ConA
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
7
17
34
36
52
54
66
67
78
11a
11b
11c
22
25
26
30
14
15
16
19
36
39
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
5
GRP 78
Calcium transporting ATPase, ER
ATP synthase beta subunit
Aldehyde DH preprotein
Electron transfer flavoprotein, alpha
Electron transfer flavoprotein alpha
ATP synthase D
ATP synthase alpha
Cytochrome b5
Methylmalonate-semialdehyde DH
Glutamate DH precursor
Aldehyde DH precursor
Acyl-CoA DH precursor
D-beta-hydroxybutyrate precursor
Rhodanese fragment
Pyruvate DH kinase precursor
Mitochondrial matrix P1 precursor
ERP60
Mitochondrial matrix P1 precursor
Aldehyde DH precursor
3-ketoacyl-COA thiolase
Catalase, PX
Swiss Prot
Swiss Prot
NCBInr.32499
NCBInr.32499
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
GenPept.11299
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
Swiss prot
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
Swiss Prot
P06761
P11606
1374715
118505
P13803
P13803
P31399
P15999
AF007107
Q02253
P26443
Q13573
P15651
P29147
P24329
Q15118
P19227
P11598
P19227
P47738
P13437
P00761
ER = Endoplasmic reticulum
PX= peroxisome
Protein Enrichment by Specific Fractionation
• Total Mitochondria
– 300 to 500 proteins
• CBB-stained gels
– 1598 proteins
• silver-stained gel
– 300 to 500 proteins
• Pre-fractionation
– 819 proteins/ CBB stained
• calcium binding protein
enrichment
– min. 78 proteins / CBB
stained
• con A binding protein
enrichment
– resolution
– 736 proteins / CBB stained
• hydrophobic protein
enrichment
– fragmentation
– min. 1633 proteins
More than 3 to 5 times more proteins detected using pre-fractionation!
Overall sensitivity of used
process
• Approximately 125 fmol of protein in the gel spot!!!
– ability to recover sufficient peptides to allow a search and
identification in the databases
– protein dependend
– routine base experiments 250 to 500 fmol in gel spot
– date of experiments 1999
• How to increase this further on?
– Where are we today?
Increase Sensitivity by....
• ... Using fluorophore-staining AND appropriate instrumentation,
because sensitivity is a result of both!
– SYPRO Ruby stain
• performance in comparison to silver and CBB
– new ProXPRESS proteomic imaging system
• exact quantitation of fluorophores
• expression profiling
– new ProPic high-performance protein picker
• imager, analysis software and picker in one
• on-board in-gel fluorophore detection
• proteome mapping
– The PerkinElmer Proteomic product line has been optimised for
fluorophore staining!
Staining Technologies - Comparison PostLabels
Staining Method
Technology
Detection Limit Dynamic End-Point
per 1D band
Range
Stain?
MSComp.
SYPROTM Rubya
Fluorescence
1 ng
3 logs
Yes
Yes
SYPRO™ Redb,e
SYPRO™ Orangec,f
SYPRO™Tangerineg
Fluorescence
2 ngd
3 logs
Yes
Yes
Fluorescence
4-8 ng
3 logs
Yes
Yes
Silver
(destructive)
Silver
(non-destructive)
Colloidal CBB
Absorbance
1 ng
0.8 logs
No
No
Absorbance
4-8 ng
No
Yes
Absorbance
8-16 ng
Yes
Often
CBB
Absorbance
8-16 ng
0.8 logs
(7)
1.3 logs
(20)
2 logs
No
Yes
a
ex 300/480 nm; em 618 nm
b
ex 300/550 nm; em 630 nm
c
ex 300/470 nm; em 570 nm
g
ex 300/490 nm; em 640 nm
d
less sensitive for a 2D gel
Red has a lower background than Orange
f
Orange is slightly brighter than Red
e
SYPROTM Ruby Stain Vs Silver Stain:
Phosphorylase Serial Dilution:
Peptide Matches by MALDI-TOF MS
Phosphorylase
Quantity
Mass
[femtomoles]
[ng]
1546
150
773
75
387
38
193
19
97
9
78
5
38
2
Number of matched peptides
Stained with
Stained with
TM
SYPRO Ruby
Silver
6
8
6
7
8
5
4
2
5
0
2
0
1
0
Conclusion:
 Peptide mass profiling is feasible using either stain, when 40 ng is available.
 Only SYPROTM Ruby stain allows identification with <10 ng of protein.
Aplicações de Microarranjos de Proteínas
* DNA - protein
interaction
* Protein - protein
interactions
* Enzyme-substrate
analysis
* Protein profiling
* Antibody
characterization
* Small molecule
screening
TM
HydroGel
Coated Slides
Protein Penetration Demonstrated by Confocal
Fluorescent Microscope Measurement
~70% penetration of a
160 kD protein
starting
ending
1.9 µm per section in Z axis
Imobilizar a sonda
(anticorpos)
Imobilizar e lavar
Incubar com a amostra alvo
Lavar e detectar
Alvo (target) = sonda
Targets: Cy3- and
Cy5-labeled patient
serum samples
ELISA: Agora em lâminas: múltiplas amostras
Representative commercial ELISA for IFN-g shows detection range of
approximately 10-1000 pg/mL (2 log dynamic range)
Ensaios sanduíche: detecção simultânea de múltiplas
substâncias
Texas Red conjugated Streptavidin
Biotinylated detection antibody
Target (cytokine)
Capture antibody
43 Cytokine Antibody Chip
Each probe is printed in quadruplicate
(350 pL/spot) at 500 um spacing.
Qualitative Screening
A
B
C
Biotin-IgG
IL-1b
IL-8
IL-6
Control
GCSF
Human ER-negative breast cancer cells MDA-MB-231 were screened with a
43 cytokine antibody chip
A: Cell culture media as negative control (left) showing low non-specific binding
B: Conditioned media (center) indicating cells produced IL-8, GCSF and IL –6
C: Cell lysates (right) containing IL-1b, GCSF and IL-8 but lacking IL-6
Exemplos de análise do proteoma em plantas (2001)
 the maritime pine needle (at the organ level) [11];
 the maritime pine xylem(at the tissue level) [11];
 peribacteroid membrane of soybean root nodules (at the subcellular level) [12].
 subproteoma lumenal and peripheral thylakoid proteins. Peltier et al
 descriptive proteomes include the global comparison of green and etiolated rice shoots [8]
 analysis on rice leaf and stem of the effects of jasmonic acid treatment as a model for defence
associated responses [15],
 characterisation of the nodule membrane upon symbiosis with nitrogen-fixating bacteria
 changes in protein synthesis that occur during hypoxic acclimatation using [35S]-methionine
 phloem proteins are differentially distributed in source and sink organs.
Limitações
Difícil extração e separação de proteínas hidrofóbicas em géis 2D (LC-MS)
Número limitado de proteínas (após a maturação: 106proteínas diferentes por
célula)
Bancos de dados: tornando sinérgicos os esforços de uma comunidade de pesquisadores
The maritime pine proteome database
Arabidopsis plasma membrane proteome database