Transcript PPT - Bioinformatics.ca

2D Gel Analysis
David Wishart
University of Alberta
Edmonton, AB
[email protected]
Lecture 1.3
1
Separation & Display Tools
• 1D Slab Gel Electrophoresis
• 2D Gel Electrophoresis
• Capillary Electrophoresis
• HPLC (SEC, IEC, RP, Affinity, etc.)
• Protein Chips
Lecture 1.3
2
2D Gel Electrophoresis
• Simultaneous
separation and
detection of ~2000
proteins on a 20x25
cm gel
• Up to 10,000 proteins
can be seen using
optimized protocols
Lecture 1.3
3
Why 2D GE?
• Oldest method for large scale protein
separation (since 1975)
• Still most popular method for protein
display and quantification
• Permits simultaneous detection, display,
purification, identification, quantification
• Robust, increasingly reproducible, simple,
cost effective, scalable & parallelizable
• Provides pI, MW, quantity
Lecture 1.3
4
Steps in 2D GE
• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation
Lecture 1.3
5
Steps in 2D GE
Lecture 1.3
6
Sample Preparation
• Sample preparation is key to
successful 2D gel experiments
• Must break all non-covelent proteinprotein, protein-DNA, protein-lipid
interactions, disrupt S-S bonds
• Must prevent proteolysis, accidental
phosphorylation, oxidation, cleavage,
deamidation
Lecture 1.3
7
Sample Preparation
• Must remove substances that might
interfere with separation process
such as salts, polar detergents (SDS),
lipids, polysaccharides, nucleic acids
• Must try to keep proteins soluble
during both phases of electrophoresis
process
Lecture 1.3
8
Cell Disruption Methods
Vigorous Methods
• Sonication
• French press
• Glass bead disruption
Gentle Methods
•
•
•
•
Lecture 1.3
Enzymatic lysis
Detergent lysis
Freeze-thaw
Osmotic lysis
9
Sample Preparation
Proteolysis Protection
Contaminant Removal
• PMSF
• Dialysis
• Pefabloc
• Filtration
• EDTA
• Centrifugation
• EGTA
• Chromatography
• leupeptin
• Solvent Extraction
Lecture 1.3
10
Protein Solubilization
•
•
•
•
•
8 M Urea (neutral chaotrope)
4% CHAPS (zwitterionic detergent)
2-20 mM Tris base (for buffering)
5-20 mM DTT (to reduce disulfides
Carrier ampholytes or IPG buffer (up
to 2% v/v) to enhance protein
solubility and reduce charge-charge
interactions
Lecture 1.3
11
Other Considerations
• Further purification or separation?
– Subcellular fractionation
– Chromatographic separation
– Affinity purification
• Optimizing electrophoresis parameters
– IEF pH gradient, Acrylamide %, loading
• Limits of detection
– ng? (Coomasie stain) pg or fg? (Western)
Lecture 1.3
12
Detergent Fractionation
Cells
Extraction with
Digitonin/EDTA
supernatent
Cytoplasmic
Fraction
pellet
Extraction with
TritonX100/EDTA
supernatent
Organelle
Membranes
pellet
Extraction with
SDS/EDTA
supernatent
Nuclear
Lecture 1.3
pellet
Cytoskeletal (in SDS)
13
Subcellular Fractionation
Human mitochondrial proteins
Lecture 1.3
Human nuclear proteins
14
Differential Solubilization
Protein sample
Extraction with
40mM Tris Base
supernatent
Fraction 1
pellet
Extraction with
8M Urea, 4% CHAPS
supernatent
Fraction 2
pellet
Extraction with
5M Urea, 2M Thiourea
2% CHAPS, 2% SB3
supernatent
Fraction 3
Lecture 1.3
pellet
Extract with SDS
Fraction 4
15
Steps in 2D GE
• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation
Lecture 1.3
16
2D Gel Principles
SDS
PAGE
Lecture 1.3
17
Isoelectric Focusing (IEF)
Lecture 1.3
18
IEF Principles
Increasing pH
A
N
O
D
E
_
_
_
_
_
_
_
_
_
+
+
+
+
+
+
+
+
+
pI = 5.1
Lecture 1.3
pI = 6.4
C
A
T
H
O
D
E
pI = 8.6
19
Isoelectric Focusing
•
•
•
•
•
Separation of basis of pI, not Mw
Requires very high voltages (5000V)
Requires a long period of time (10h)
Presence of a pH gradient is critical
Degree of resolution determined by slope
of pH gradient and electric field strength
• Uses ampholytes to establish pH gradient
Lecture 1.3
20
Ampholytes vs. IPG
• Ampholytes are small,
soluble, organic molecules
with high buffering
capacity near their pI (not
characterized)
• Used to create pH
gradients via user
• Gradients not stable
• Batch-to-batch variation is
problematic
Lecture 1.3
• An immobilized pH
gradient (IPG) is made
by covalently integrating
acrylamido buffer
molecules into
acrylamide matrix at
time of gel casting
• Stable gradients
• Pre-made (at factory)
• Simplified handling
21
IPG Strips
Strip Length
Gel Length
Strip Width
Gel Thickness
pH Gradients
Standard
Overlapping
7.9 cm
7.3 cm
3.3 mm
0.5 mm
11.8 cm
11.0 cm
3.3 mm
0.5 mm
17.8 cm
17.1 cm
3.3 mm
0.5 mm
3-10,4-7 3-10,4-7 3-10,4-7
3-6,5-8 3-6,5-8 3-6,5-8
CH2=CH-C-NH-R
||
O
Acrylamido buffer
R = weakly acidic or basic buffering group
Lecture 1.3
22
Narrow-Range IPG Strips
pH 4
Lecture 1.3
pH 4
pH 5
pH 5
pH 6
pH 9
23
IEF Phase of 2D GE
Rehydrate IPG
strip & apply
protein sample
Lecture 1.3
Place IPG strip
in IEF apparatus
and apply current
24
Steps in 2D GE
• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation
Lecture 1.3
25
SDS PAGE
Lecture 1.3
26
SDS PAGE Tools
Lecture 1.3
27
SDS PAGE Principles
SO4 Na
+
Sodium Dodecyl Sulfate
C
A
T
H
O
D
E
Lecture 1.3
_
_
_
_
_
_
_
_ _ _ _ _ _
_ _
_
_ _
_
_
_
_ _ _
_ _ _
_ _ _ _
_
_
_ _
_
+
+
+
+
+
+
+
A
N
O
D
E
28
SDS-PAGE Principles
Loading Gel
Running Gel
Lecture 1.3
29
Mobility & Acrylamide%
200
200
200
45
200
45
45
200
45
6.5
45
Lecture 1.3
30
Electrophoretic Mobility
45kD
10kD
25kD
m = n/E = q/f
m = electrophoretic mobility
n = velocity of molecule
E = electric field
q = charge of molecule
f = frictional coefficient
Lecture 1.3
31
SDS-PAGE
•
•
•
•
Separation of basis of MW, not pI
Requires modest voltages (200V)
Requires a shorter period of time (2h)
Presence of SDS is critical to
disrupting structure and making
mobility ~ 1/MW
• Degree of resolution determined by
%acrylamide & electric field strength
Lecture 1.3
32
SDS-PAGE for 2D GE
• After IEF, the IPG strip is soaked in an
equilibration buffer (50 mM Tris, pH 8.8,
2% SDS, 6M Urea, 30% glycerol, DTT,
tracking dye)
• IPG strip is then placed on top of pre-cast
SDS-PAGE gel and electric current applied
• This is equivalent to pipetting samples
into SDS-PAGE wells (an infinite #)
Lecture 1.3
33
SDS-PAGE for 2D GE
equilibration
Lecture 1.3
SDS-PAGE
34
2D Gel Reproducibility
Lecture 1.3
35
Trouble Shooting 2D GE
Horizontal streaks
Sample not completely
solubilized prior to application
on IPG, sample poorly soluble in
rehydration solution, ionic
impurities, ionic detergents
Lecture 1.3
Vertical streaks
Insufficient equilibration,
insufficient SDS
36
Advantages and
Disadvantages of 2D GE
• Provides a hard-copy
record of separation
• Allows facile quantitation
• Separation of up to 9000
different proteins
• Highly reproducible
• Gives info on Mw, pI and
post-trans modifications
• Inexpensive
Lecture 1.3
• Limited pI range (4-8)
• Proteins >150 kD not
seen in 2D gels
• Difficult to see
membrane proteins
(>30% of all proteins)
• Only detects high
abundance proteins
(top 30% typically)
• Time consuming
37
2D Gel Protocols & Courses
• Online Protocols
–
–
–
–
http://ca.expasy.org/ch2d/protocols/
http://www.abdn.ac.uk/~mmb023/protocol.htm
http://www.aber.ac.uk/~mpgwww/Proteome/Tut_2D.html
http://www.noble.org/PlantBio/MS/protocols.html
• 1 Day and 1 Week Courses
– http://us.expasy.org/bprg/training/(Geneva)
– http://www.pence.ca (Toronto)
Lecture 1.3
38
Steps in 2D GE
• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation
Lecture 1.3
39
Protein Detection
• Coomassie Stain (100 ng to 10 mg protein)
• Silver Stain (1 ng to 1 mg protein)
• Fluorescent (Sypro Ruby) Stain (1 ng & up)
C2H 5
C2H5
CH2 N
C
O3S
N
CH3
CH2
SO3
Coomassie R-250
N
H 5 C2
Lecture 1.3
C2 H5
40
Gel Stains - Summary
Stain
Sensitivity (ng/spot)
Advantages
Coomassie R-250
50-100
Simple, fast, consistent
Colloidal Coomassie
5-10
Simple, fast
Silver stain
1-4
Very sensitive, awkward
Copper stain
5-15
Reversible, 1 reagent
negative stain
Zinc stain
5-15
Reversible, simple, fast
high contrast neg. stain
SYPRO ruby
Lecture 1.3
1-10
Very sensitive, fluorescent
41
Stain Examples
Coomassie
Lecture 1.3
Silver Stain
Copper Stain
42
Stain Examples
Normal liver
Tumor
Both
SYPRO fluorescent stain
Lecture 1.3
43
Detection via Western Blot
Glioma Silver Stain
Lecture 1.3
Glioma Western Blot
(anti-p53 antibody)
44
Imaging/Scanning Tools
Phosphoimager for
32P and 35S labelled
1D or 2D gels
Lecture 1.3
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
45
Steps in 2D GE
• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation
Lecture 1.3
46
2D-GE + MALDI (PMF)
Trypsin
+ Gel punch
p53
Trx
G6PDH
Lecture 1.3
47
2D-GE + MS-MS
Trypsin
+ Gel punch
p53
Lecture 1.3
48
Typical Results
• 401 spots identified
• 279 gene products
• Confirmed by SAGE,
Northern or Southern
• Confirmed by amino
acid composition
• Confirmed by amino
acid sequencing
• Confirmed by Mw & pI
Lecture 1.3
49
Steps in 2D GE
• Sample preparation
• Isoelectric focusing (first dimension)
• SDS-PAGE (second dimension)
• Visualization of proteins spots
• Identification of protein spots
• Spot pattern evaluation/annotation
Lecture 1.3
50
2D Gel Software
Lecture 1.3
51
Commercial Software
• Melanie 4 (GeneBio - Windows only)
– http://ca.expasy.org/melanie
• ImageMaster 2D Elite (Amersham)
– http://www.imsupport.com/
• Phoretix 2D Advanced
– http://www.phoretix.com/
• PDQuest 6.1 (BioRad - Windows only)
– http://www.proteomeworks.bio-rad.com/html/pdquest.html
Lecture 1.3
52
Common Software Features
•
•
•
•
•
•
•
Image contrast and coloring
Gel annotation (spot selection & marking)
Automated peak picking
Spot area determination (Integration)
Matching/Morphing/Landmarking 2 gels
Stacking/Aligning/Comparing gels
Annotation copying between 2 gels
Lecture 1.3
53
2D Gel Analysis Freeware
CAROL http://gelmatching.inf.fu-berlin.de/Carol.html
Lecture 1.3
54
2D Gel Analysis Freeware
FLICKER http://www-lecb.ncifcrf.gov/flicker
Lecture 1.3
55
Flicker
• Permits comparison of 2 images from
internet sources on web browser
• Comparison via adjustable “flicker” rate of
overlaid gel images
• Images may be enhanced by spatial
warping, 3D projections or relief map,
image sharpening, contrast enhancement,
zooming, complement grayscale transform
Lecture 1.3
56
2D Gel Analysis Freeware
Melanie Viewer http://ca.expasy.org/melanie/Viewer.htm
Lecture 1.3
57
2D Gel Analysis Freeware
http://www.gelscape.ualberta.ca:8080/index.html
Lecture 1.3
58
Federated 2D Gel Databases
•
•
•
•
Remotely queryable via the web
Attainable through SWISS-PROT search
Linked to other 2D databases via web
Image mapped 2D gel spots to support
graphical image query
• Directly reachable from within 2D gel
analysis software
Appel, R.D. et al. Electrophoresis 17: 540-546 (1996)
Lecture 1.3
59
2D Gel Databases
http://ca.expasy.org/ch2d/2d-index.html
Lecture 1.3
60
2D Gel Databases
http://ca.expasy.org/ch2d/2d-index.html
Lecture 1.3
61
Swiss 2D-PAGE
Lecture 1.3
62
Swiss 2D-PAGE
Lecture 1.3
63
Swiss 2D-PAGE
Lecture 1.3
64
Competing Technologies
Capillary Electrophoresis (single & tandem)
Lecture 1.3
65
ICAT vs 2D Gels
Lecture 1.3
ICAT
66
MudPIT
IEX-HPLC
RP-HPLC
Trypsin
+ proteins
p53
Lecture 1.3
67
2D Gels vs Protein Arrays
Lecture 1.3
68
A Triumph For Gels
(Actually Western
Blotting)
Lecture 1.3
69
Yeast Proteome Analysis
Ghaemmaghami S, et al., Nature 425:737-741 (2003).
Lecture 1.3
70
Tap Tagged Western
Lecture 1.3
71
Tap-Tagged Western Sensitivity
Lecture 1.3
72
Yeast Proteome Results
Lecture 1.3
73
The Yeast Proteome
• 80% of the proteome is expressed during
normal growth conditions
• Abundance of proteins ranges from fewer
than 50 to more than 106 molecules per cell
• Many proteins, including essential proteins
and most transcription factors, are present
at levels that are not readily detectable by
other proteomic techniques
Lecture 1.3
74
Conclusions
• 2D gel electrophoresis is still the most
popular and powerful method for protein
display, separation, visualization and
quantitation
• Offers good to excellent sensitivity and is
now very reproducible
• 2D GE is still essential for proteomics
• Running and analyzing 2D gels requires
skill, patience and good software
Lecture 1.3
75
Conclusions
• Web tools are now available that permit
partial analysis and comparison of 2D gels
• Commercial software still is required in
most cases to complete full-scale analysis
• Web-enabled gel databases are now
democratizing & popularizing 2D gel
analysis
• Competing technologies are now emerging
that may offer advantages over 2DE
Lecture 1.3
76