Subcellular Fractionation
in the context of proteomics
Lukas A. Huber
Biocenter, Innsbruck Medical
University
[email protected]
Subcellular Fractionation &
Proteomics
• Allows access to intracellular organelles and multi-protein
complexes
• Enrichment of low abundant proteins and signaling
complexes
• Reduced sample complexity
• Flexible and adjustable approach
• Most efficiently combined with functional analysis
• Combineable with 2-DE and gel-independent techniques
Subcellular Fractionation remains
a major bottle neck….
• Similar physical properties
• Differences tissue vs. cultured cells
Subcellular Fractionation
• Organelles, Membrane Transport
• Fractionation of Organelles
– Homogenization
– Organelle Separation
•
•
•
•
•
Density Gradients
Density Shifts
Free- Flow Electrophoresis
Immunoisolation
Fluorescence Activated Organelle Sorting
Membrane Traffic
The Endocytic Pathway
EARLY ENDOSOME
ECV / MVB
LATE ENDOSOME
LYSOSOME
Of course….
Specific markers are required to
follow the fractionation procedure
Markers for the Endocytic Pathway
HRP
Rab4, Rab11
Tfn-R,
EEA1
EARLY ENDOSOME
Rab5, Tfn-R
ECV / MVB
LATE ENDOSOME
Rab7
β-hexosaminidase
LYSOSOME
Internalization into Endosomes
COMPARTMENT
TIMES AT 37°C MICROTUBULES
Early endosomes
5 min
with/without
Endosomal carrier
vesicles [ECVs]
nocodazole]
5 min + 40 min
without MT
[+ 10 µM
Late endosomes
5 min + 40 min
with MT
Homogenization (I)
• Gentle conditions of homogenization should be
used to limit possible damage to endosomal
elements, particularly when using fluid phase
markers.
• Clearly, the markers should remain entrapped in
vesicles (latent) after homogenization.
• Harsh conditions should however always be
avoided in order to limit the breakage of
lysosomes and consequent proteolysis due to
released hydrolases.
All Steps on Ice!
Homogenization (II)
4°C
4°C
Homogenisation
Confluent cell culture
Scrape and collect
by centrifugation (500g)
• Cells are released from the dish by scraping
with the sharp edge of a rubber policeman.
• Homogenization is easier at a relatively high
density of cells, typically 20-30% [vol/vol].
• It is wise to monitor each step of the
homogenization process under phase contrast
microscopy.
Scraping with a Rubber
Policeman
Homogenization (II)
4°C
4°C
Homogenisation
Confluent cell culture
Scrape and collect
by centrifugation (500g)
• The cells are then homogenized by passage through a
needle or the tip of a pipette and then a post-nuclear
supernatant (PNS) is prepared. Under gentle conditions
of homogenization, 50-60% of a fluid phase marker is
recovered in the PNS. The rest, which consists partially
of unbroken cells, is lost to the nuclear pellet (NP).
Homgenization with a Needle
Homogenization (III)
4°C
4°C
Homogenisation
Confluent cell culture
Scrape and collect
by centrifugation (500g)
• When working with cells in suspension, eg after
trypsin treatment, homogenization may require
harsher conditions. The protocol then remains
essentially the same, except that a tight-fitting
glass-glass Potter or a Dounce homogenizer is
used. Up to 15-20 passages of the pestle may be
required to achieve sufficient cell breakage.
Nitrogen Decompression
(Nitrogen Cavitation)
• Large quantities of nitrogen are first
dissolved in the cell under high pressure
within a suitable pressure vessel. Then,
when the gas pressure is suddenly released,
the nitrogen comes out of the solution as
expanding bubbles that stretch the
membranes of each cell until they rupture
and release the contents of the cell.
Nitrogen Decompression
(Nitrogen Cavitation)-1
Nitrogen Decompression
(Nitrogen Cavitation)-2
Nitrogen Decompression
(Nitrogen Cavitation)-3
Nitrogen Decompression
(Nitrogen Cavitation)-4
Nitrogen Decompression
(Nitrogen Cavitation)-5
…has several advantages
• Gentle method without chemical and physical
stress.
• There is no heat damage due to friction.
• There is no oxidation.
• Any suspending medium can be used.
• Each cell is exposed only once.
• The product is uniform.
• Easy to apply.
Subcellular Fractionation
• Organelles, Membrane Transport
• Fractionation of Organelles
– Homogenization
– Organelle Separation
•
•
•
•
•
Density Gradients
Density Shifts
Free- Flow Electrophoresis
Immunoisolation
Fluorescence Activated Organelle Sorting
Density Gradients (I)
• Organelles are separated according to their
physical properties
• Problem
– Some compartments share similar physical properties
Homogenization
Centrifugation
Sucrose Gradient
Cells, Tissue
Subcellular organelles
Density Gradients (II)
H o m o g e n iz a t io n
1
1 0
2 5
E a r ly
%
%
b u ffe r
m l
S in
S in
D
H
2
2
O o r
O
e n d o s o m e s
1 .5
1 6 %
3 5 %
S in
S in
1 .0
m l
D
H
2
2
O o r
O
m l
L o a d : P N S in
4 0 .6
%
S
The PNS is brought to 40.6 % sucrose [S] and loaded at the bottom
of an SW 60 tube. The load is the overlaid sequentially with 16
%sucrose in heavy water [or 35 % sucrose], 10 % sucrose in heavy
water [or 25% sucrose] and finally with homogenization buffer. The
gradient is run for 60 min at 35K rpm. Early endosomes and late
endosomes [+ carrier vesicles] are collected as indicated.
…next step: Gradient-1
Pellet (3,000g)= nuclei
Supernatant=PNS
4°C
165,000g
10 %
Sucrose gradient
40 %
Collect intact membranes
and vesicles
…next step: Gradient -2
Pellet (3,000g)= nuclei
Supernatant=PNS
4°C
165,000g
10 %
Sucrose gradient
40 %
Collect intact membranes
and vesicles
…next step: Gradient-3
Pellet (3,000g)= nuclei
Supernatant=PNS
4°C
165,000g
10 %
Sucrose gradient
40 %
Collect intact membranes
and vesicles
Purification of Endosomes
Subcellular
fractionation
Subcellular fractionation allows access to
low abundant and organelle specific
proteins
200
PNS (Cy2, blue),
Early endosomes (Cy3, green)
Late endosomes (Cy5, red)
Mr
4
pI
9
305 (2 to 120 fold) protein spots
enriched in late endosomal
fraction
292 (2 to 25 fold) spots enriched
in early endosomal fraction
286 proteins increased (2 to 10
fold) in late vs early endosomes
8
Stasyk and Huber, Proteomics, 2005
Subcellular Fractionation
Pasquali et al.,1999 J. Chromatography B
Huber et al., 2003, Circulation Res.
4°C
4°C
4°C
Homogenisation
Confluent cell culture
Scrape and collect
by centrifugation (500g)
1. Marker analysis
2. Na2Co3 Extraction at
Pellet (3,000g)= nuclei
Supernatant=PNS
(Western Bl.,
Enzymes)
4°C
165,000g
10%
high pH (peripheral vs.
integral membrane
proteins)
Sucrose gradient
40%
3. Organelle Proteome
Analysis (2D-GE,
Chromatography, Mass
Spec.)
Collect intact membranes
and vesicles
Na2Co3 extracted Membrane proteins
100,000 g pellet
integral membrane proteins
100,000 g supernatant
peripheral membrane proteins
Subcellular Fractionation
Pasquali et al.,1999 J. Chromatography B
Huber et al., 2003, Circulation Res.
4°C
4°C
4°C
Homogenisation
Confluent cell culture
Scrape and collect
by centrifugation (500g)
1. Marker analysis
2. Na2Co3 Extraction at
Pellet (3,000g)= nuclei
Supernatant=PNS
(Western Bl.,
Enzymes)
4°C
165,000g
10%
high pH (peripheral vs.
integral membrane
proteins)
Sucrose gradient
40%
3. Organelle Proteome
Analysis (2D-GE,
Chromatography, Mass
Spec.)
Collect intact membranes
and vesicles
Quality Control
• In all fractionation experiments, a balance
sheet should be established for the
distribution of protein and markers (eg
bHRP) in all fractions.This provides the
only appropriate means to judge the
homogenization /fractionation steps and to
compare different preparations.
Balance Sheet
(separation of early and late endosomal
fractions on the flotation gradient)
Vol.
(ml)
HRP
OD
Protein Sp. Act
(mg)
Yield
%
RSA
0.4
0.4
4.0
0.4
100
67
14.7
1.2
1.0
1.0
10.0
1.2
0.3
0.2
0.4
6.9
100
58
1.3
25.0
1.0
0.9
1.6
27
A) Early endosomes (5 min at 37°C)
Homog.
PNS
Early fract.
Late fract.
0.7
0.6
0.4
0.3
4.5
3.0
0.6
0.06
11.3
7.37
0.15
0.12
B) Late endosomes (5 + 30 min at 37°C)
Homog.
PNS
Early fract.
Late fract.
0.7
0.6
0.5
0.6
2.7
1.6
0.04
0.6
10.8
7.2
0.09
0.09
Density Gradients (III)
(continous gradients)
A
1 .5 0
E
L
b
a
1 .2 5
E
E
lP M
pP M
1 .0 0
0 .7 5
(
n
g
/µ
g
)
H
R
P
/p
r
o
te
in
0 .5 0
0 .2 5
0 .0 0
0
2
4
6
8
1 0
1 2
1 4
1 6
1 8
2 0
2 2
2 4
f r a c t io n s
0 .0 3
b e ta -h e x .
C y t.C -R e d u c ta s e
G a lT
0 .0 2
s
p
e
c
.
a
c
ti
v
it
y
B
0 .0 1
0 .0 0
0
2
4
6
8
1 0
1 2
1 4
1 6
1 8
2 0
2 2
f r a c t io n s
Sucrose
10%
40%
Subcellular Fractionation & Organelle Proteome Analysis
+ EGF
0; 5; 40 min
Murine
mammary
epithelial
EpH4 cells
4°C
Homogenisation
Scrape and collect
by centrifugation (500g)
4°C;
Integral
100,000g
membrane
proteins
Na2CO3
Extraction at high pH
4°C
Pellet (3,000g) = nuclei
Supernatant = PNS
4°C; 165,000g
Cytosol
Total membranes
Peripheral
proteins
Marker
analysis
(Western
Blotting)
4°C
Sucrose gradients
Continuous
Discontinuous
10%
8%
I
LE
LE
25%
EE
EE
35%
40%
42%
Collect intact
membranes LE=late endosomes
and vesicles EE=early endosomes 8%
35%
42%
Organelle Proteome Analysis (2D-GE,
Chromatography, Mass Spectrometry)
II
Crude
Endosomes
..... ..... ......
.....
..... ......
Huber et al.,.....
Circulation
Res., 2003
.....
Huber, Nature
Rev.,
..... .....
...... Moll Cell Bio, 2003
.....
Stasyk and Huber,
Proteomics, 2004
..... ..... ......
Subcellular Fractionation
• Organelles, Membrane Transport
• Fractionation of Organelles
– Homogenization
– Organelle Separation
•
•
•
•
•
Density Gradients
Density Shifts
Free- Flow Electrophoresis
Immunoisolation
Fluorescence Activated Organelle Sorting
Density Shifts
• Endosomes loaded with colloidal gold
bound to a ligand (eg Transferrin) are
separated by centrifugation [Hopkins]
• Endosomes loaded with HRP bound to a
ligand are separated by centrifugation after
DAB reaction (cross-link of lumenal
proteins) [Courtoy].
Subcellular Fractionation
• Organelles, Membrane Transport
• Fractionation of Organelles
– Homogenization
– Organelle Separation
•
•
•
•
•
Density Gradients
Density Shifts
Free- Flow Electrophoresis
Immunoisolation
Fluorescence Activated Organelle Sorting
Free-Flow Electrophoresis (I)
• Free flow electrophoresis is a powerful
preparative separation tool for protein
enrichment, especially suited for complex
protein mixtures.
• Isolation of subcellular compartments or
pre-fractionation of complex protein
mixtures using narrow pH gradients can be
performed.
Free-Flow Electrophoresis (II)
• Lysosomes and endosomes can be separated from
other organelles in an electrical field [Mellman,
Fuchs etc.].
• Sample submission:
– Protein samples should be provided in buffer or salt
solutions not exceeding 100 mM. Samples should be
free of insoluble material and organic solvents.
Free-Flow Electrophoresis (III)
Free-Flow Electrophoresis (III)
Subcellular Fractionation
• Organelles, Membrane Transport
• Fractionation of Organelles
– Homogenization
– Organelle Separation
•
•
•
•
•
Density Gradients
Density Shifts
Free- Flow Electrophoresis
Immunoisolation
Fluorescence Activated Organelle Sorting
Immunoisolation
• Organelles are separated with antibodies
according to their antigenic properties,
rather than their physical properties
[Gruenberg].
• Is most efficiently combined with density
gradient centrifugation as means for
prefractionation
Immunoisolation
Principal
linker antibody
specific antibody
fraction
solid support
antigen
Experimental Strategies
INDIRECT
DIRECT
ANALYSIS
Experimental Conditions
yield [act]
specific
non-specific bk
2
4
time [hr]
6
Antigen (I)
• The epitope must be exclusively present on
the surface of the desired compartment.
– Immunoisolation can occur (albeit less
efficiently) with a single epitope per vesicle! It
is, therefore, very difficult to carry out
"differential" immuno-isolation, ie to separate
membranes containing different densities of the
antigen (molecules/µm2 membrane surface
area).
Antigen (II)
• The epitope must be exposed on the surface
of the desired compartment (and accessible
to the immobilized antibody).
• Immunoisolation is better achieved with a
relatively abundant epitope.
• However, we find that immunisolation is
efficient with ≈ 50-100 molecules/µm2
membrane surface area.
Antibodies (I)
• Linker Antibody
– Increases the flexibility of the specific
antibody.
– The coupling of a generic anti-Fc antibody (eg
against the Fc domain of mouse IgG) to the
particles/beads increases the proportion of
correctly oriented specific antibodies, hence
organelle binding.
Antibodies (II)
• Specific Antibody
– Antibody raised against an epitope exposed on
the surface of the desired compartment.
– "Good" antibody (Kd ≤ 10-8).
– Selection of an antibody: immunoisolation only
is the real test. It is often dificult to predict,
particularly with monoclonals, whether a given
antibody will be efficient in immunoisolation.
– Polyclonal: affinity purification is required in
most cases.
Solid Supports: Criteria (I)
• Composition
– hydrophobic surfaces are more sticky
– chemical attachement of antibody (eg gentle
coupling of proteins to -OH groups with ptoluene sulfonyl chloride)
– aggregation properties in the absence of
cellular extracts (some latex aggregate easily)
Solid Supports: Criteria (II)
• Flexibility
– correct positioning of the antibody
• Sedimendation, Aggregation
– low speed (eg 3000 X g), so that organelles do
not co-sediment- very small particles (< 0.5
µm) aggregate easily
– heterodisperse particles show higher
aggregation properties than monodisperse
particles
Solid Support: Types
TYPES
ADVANTAGES
DISADVANTAGES
Fixed S.aureus cells
expressing ProteinA
- high capacity
(high S/V ratio)
- monodisperse
- commercially avail.
- high speed sedimentation
- non specific adsorption
- SDS-gels difficult
Magnetic beads
- low background
- low capacity
- NO sedimentation
- monodisperse
- commercially avail.
Cellulose fibers
- high capacity
- high flexibility
- low speed sediment.
- high background
(entrapment)
- not commercial. avail.
Eupergit particles
- high capacity
- ± monodisperse
- commercially avail.
- high background
- only some Ags
Immunoisolation of endosomes
Subcellular Fractionation
• Organelles, Membrane Transport
• Fractionation of Organelles
– Homogenization
– Organelle Separation
•
•
•
•
•
Density Gradients
Density Shifts
Free- Flow Electrophoresis
Immunoisolation
Fluorescence Activated Organelle Sorting
Fluorescence Activated
Organelle Sorting (FAOS) (I)
• Flow cytometry was adapted to sort and
analyze intracellular organelles after
labeling with fluorescent dyes.
• Conventional subcellular fractionation
techniques was combined with high speed
organelle sorting in a FACS.
Fluorsecent activated sorting:
technical principle
Fluorescence Activated
Organelle Sorting (FAOS) (II)
• Labeling intracellular organelles, e.g.
mitochondria, Golgi, ER, plasma
membrane, phagosomes, endosomes, with
fluorescent membrane dyes or fluorescently
labeled ligands, allows purification due to
biological properties rather than physical
densities
Criteria
• For organelle sorting, sensitivity is obviously a
major concern, since small structures, e.g.
intracellular organelles, usually have only a small
number of dye molecules associated with them.
• Besides the physical properties of the dye
(absorption coefficient, quantum efficiency)
increased background signals can be a critical
limitation.
For which organelles?
• A good example for organelle sorting are,
once again, endosomes, since they can be
accessed from outside the cell and loaded
transiently with fluorescent membrane dyes
or fluorescently labeled ligands under
different conditions.
But...
• Today a diverse array of cell-penetrating
fluorescent stains that selectively associate
with intracellular organelles or the
cytoskeleton, in living cells, is available.
• In addition green fluorescent protein (GFP)
of jellyfish Aequorea victoria can be fused
to known organelle markers and used for
FAOS
TMA-DPH
Trimethyl
ammonium DPH
H
C
C
H
H
C
H
C
C
H
C
H
N(CH3) 3
cationic DPH analog with a charged substituent
as surface anchor
TMA-DPH
Features
• can be removed from plasma membrane by
washing
• can be used to study endocytosis
• non fluorescent in water and binds in
proportion to the available membrane
surface
• excitation 355nm, emission 450nm
TMA-DPH
Internalization
Starting Fraction (PNS)
FAOS enriched Endosomes
The cell map
Organelle proteomics - a new resolution to cellular processes
…more than 90%
of the phagosome
proteins would
have been
undetected by
analysis of the total
cell lysate…
Brunet S. et al. Organelle proteomics:
looking at less to see more.
Trends Cell Biol. 2003:629-38.
Acknowledgements
BioCenter
Div. Cell Biology
Sandra Morandell
Taras Stasyk
Hong-Lei Huang
and
all other members of the
Huber group
Günther K. Bonn
Isabel Feuerstein
all members of the group
Karl Mechtler
Elisabeth Roitinger
Thomas Lindhorst
Zlatko Trajanoski, TU Graz
Florian Überall, Med Uni Ibk
Jakob Troppmair
Stephan Geley
Manuela Baccarini
Jacques Pouysségur
Andy Catling