Download Proteomics pathway Most common properties of proteins

Proteomics pathway
Sample
Separation
Data Analysis
Selection of spot(s)
G Q K NR E
E M T
Post-separation
analysis
Databases
Data Processing
... NRTKGG ...
Most common properties of
proteins
Protein
properties
Measured
parameters
Range
Abundance
Level of expression
Volume/Size
Molecular weight
50<level<106 copies
per cell
8-20000 kD
Hydrophobicity
More than 20 scales
of hydrophobicity
Charge
pI
Kyte-Doolitlle:
[-4.5, 2.8]
2-14
How to determine these parameters?
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Protein analysis
Samples => Complex proteins mixtures
Protein separation before identification procedure
Liquid
chromatography
Hydrophobicity Ionic strength
Mr
But also:
Immuno-selectivity
Affinity capture
Multidimensional chromatography
Source: Willy Bienvenut
Electrophoretical
separations
Gel
pI pI/Mr Mr
Capillary
pI CZE ≈Mr
Sample preparation
Objetive:
Objetive to allow an efficient separation of the greater number of
proteins in two dimensions.
This procedure is very important and must:
must:
• Solubilise as many proteins as possible including hydrophobic
species
• Prevent protein aggregates and hydrophobic interactions. This
includes denaturing the proteins to break non-covalent interactions, to
break disulfide bonds and to disrupt non-covalent interactions between
proteins and other compounds, such as lipids or nucleic acids
• Remove or digest any RNA or DNA, coenzymes, hormones or
anything else in the cell that could interfere with the proteins separation
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Sample preparation (cont.)
Sample preparation (cont.)
To overcome some of these problems :
 to increase the number of low abundance proteins, two
alternative approaches of enrichment of rare polypeptides may
be used: Subcellular fractionation and protein prefractionation.
 separate membrane proteins, nucleole, cytoplasmic,
cytosolics, etc.
 Specific reagents to increase solubility of hydrophobic
proteins.
 Specific reagents to break disulfid bonds.
 Enzymatic digestion of DNA and ultracentrifugation.
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Gel free approach
• Sub cellular fractionation:
– Differential centrifugation,
– Mechanical techniques…
• Chromatographic approach:
– Ion exchange (cation or anion)
– Gel filtration
– Affinity
• Proteins precipitation:
– Acetone,
– Ammonium sulfate…
Sample pre-fractionation
http://www.chem.uwec.edu/Chem352_S03/Pages/Overheads/C352_lect6_view.pdf
4
Chromatography
• Separation technique that depends on
differential affinity for a mobile and a
stationary phase:
– For protein isolation, the mobile phase is usually
an aqueous solution
– The stationary phase is attracted to a physical
property of the protein:
•
•
•
•
Ion exchange – net charge
Reverse phase – hydrophobicity
Gel filtration - size
Affinity – ligand binding
http://www.chem.uwec.edu/Chem352_S03/Pages/Overheads/C352_lect6_view.pdf
Gel free separation
http://www.chem.uwec.edu/Chem352_S03/Pages/Overheads/C352_lect6_view.pdf
5
Gel-filtration Chromatography
http://www.chem.uwec.edu/Chem352_S03/Pages/Overheads/C352_lect6_view.pdf
Affinity Chromatography
http://www.chem.uwec.edu/Chem352_S03/Pages/Overheads/C352_lect6_view.pdf
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Electrophoresis technique
First dimension:
dimension
Cathode (-)
pH
10
3
electric
+ +
field
+
Basic tampon + sample
R - COOH + OHR - NH2 + OH-
Anode (+)
pH
3
Anode (+)
R - COO- + H2O
R - NH2 + OH10
electric
+ +
field
+
Acid tampon + sample
R - COOH + H3O+
R - COOH + H3O+
+
R - NH2 + H3O
R - NH3 + + H2O
Cathode (-)
First dimension: Isoelectric focusing
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2-D PAGE Second dimension
Isofocalisation
Between first and second
dimensions, there is an equilibrium
phase necessary to:
First dimension
Second dimension
Cathode (-)
• eliminate products of the initial
gel (ampholytes)
• resolubilise the proteins and
• charge the proteins with SDS.
Anode (+)
SDS-PAGE: molecular weight separation
on polyacrylamide gel
See: http://www.rit.edu/~pac8612/electro/Electro_Sim.html
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1-DE separation of proteins (by Mr)
Std
Gel A
(Mr en KDa)
1
2
Gel B
3
4
5
Std
6
(Mr en KDa)
150 KDa
120 KDa
PHS2 (98 KDa)
BSA (65 KDa)
OVAL (45 KDa)
PHS2 (98 KDa)
BSA (65 KDa)
CAH2 (31 KDa)
ITRA (24 KDa)
OVAL (45 KDa)
LYC (14 KDa)
30 KDa
10 KDa
Gel A: 12% Acrylamide,
Gel B: 8% Acrylamide,
Nucleolar protein separation after coomassie blue staining
http://www.expasy.org/cgi-bin/map2/def?NUCLEOLI_HELA_1D_HUMAN
2-DE separation of proteins (by Mr)
pH
pH
Second dimension = molecular weight (Mw)
Human liver proteins
separated by 2-DE , silver stained
Human serum proteins
separated by 2-DE, silver stained
9
IPG with sigmoidal pH
Ecoli 3.5 - 10 pH
3 - 10 linear rule
Final dimensions: 160 x 200 x 1.5 mm, loading capactiy 5 mg of proteins
IPG with 1 or 2 pH unities
Swiss 2DPAGE:
Ecoli 3.5-10, Ecoli 4-5, Ecoli 4.5-5.5, Ecoli 5-6
Final dimensions: 160 x 200 x 1.5 mm, loading capactiy 15 mg of proteins
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Staining
Once separated, the proteins must be detected over the gel to then be
called a proteome map.
Many different colorants:
 Coomassie blue staining
 Amido black staining
 Silver staining
 Fluorescence
 Autoradiography ou fluorography
Silver nitrate
Commassie blue
Source Dr. Jean-Charles Sanchez, LCCC, Geneva
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Analytic 2-D PAGE
Technically, these are the following steps :
 Sample preparation
 First dimension with IPG
 Second dimension with SDS-PAGE
 Fixation of proteins on the gel
 Proteins detection with staining
Preparative 2-D PAGE
Technically, these are the following steps :
 Sample preparation
 First dimension with IPG
 Second dimension with SDS-PAGE
 Transfer to PVDF membrane
 Proteins binding on the membrane
 Proteins detection with staining over PVDF membrane
12
Transfer to PVDF membrane
PVDF = Polyvinylidene difluoride membrane
Thin as a paper sheet (100 µm):
 a more efficient means of storage (proteins are dried).
 proteins are even more concentrated.
 specially when proteins are designated to sequencing,
amino acid composition and mass spectrometry.
Protein identification from gel electrophoresis
Protein in the Gel
Electroblotting
Image analysis
Gel matching
Spot cutting for MS analysis
Electroelution of the proteins
Willy Bienvenut
Protein on the
membrane
Image analysis
Spot cutting for MS analysis &
Edman analysis
Protein immunodetection
AA composition analysis
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