subcellular fractionation in the context of proteomics · pdf filenitrogen decompression...

Subcellular Fractionationin the context of proteomics

Lukas A. HuberBiocenter, Innsbruck Medical

University Lukas.A.Huber@uibk.ac.at

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 remainsa 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

ECV / MVB

LYSOSOME

LATE ENDOSOME

EARLY ENDOSOME

Of course….

Specific markers are required to follow the fractionation procedure

Markers for the Endocytic Pathway

ECV / MVB

LYSOSOME

LATE ENDOSOME

EARLY ENDOSOME

Rab4, Rab11Tfn-R, EEA1

Rab5, Tfn-R

Rab7β-hexosaminidase

HRP

Internalization into EndosomesCOMPARTMENT TIMES AT 37°C MICROTUBULES

Early endosomes 5 min with/without

Endosomal carrier 5 min + 40 min without MTvesicles [ECVs] [+ 10 µM nocodazole]

Late endosomes 5 min + 40 min with MT

Homogenization (I)

• Gentle conditions of homogenization should beused to limit possible damage to endosomalelements, particularly when using fluid phasemarkers.

• Clearly, the markers should remain entrapped in vesicles (latent) after homogenization.

• Harsh conditions should however always beavoided in order to limit the breakage of lysosomes and consequent proteolysis due to released hydrolases.

All Steps on Ice!

Homogenization (II)

• Cells are released from the dish by scrapingwith 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 thehomogenization process under phase contrastmicroscopy.

Homogenisation4°C 4°C

Scrape and collectby centrifugation (500g)

Confluent cell culture

Scraping with a Rubber Policeman

Homogenization (II)

• The cells are then homogenized by passage through a needle or the tip of a pipette and then a post-nuclearsupernatant (PNS) is prepared. Under gentle conditionsof homogenization, 50-60% of a fluid phase marker isrecovered in the PNS. The rest, which consists partiallyof unbroken cells, is lost to the nuclear pellet (NP).

Homogenisation4°C 4°C

Scrape and collectby centrifugation (500g)

Confluent cell culture

Homgenization with a Needle

Homogenization (III)

• When working with cells in suspension, eg aftertrypsin treatment, homogenization may requireharsher conditions. The protocol then remainsessentially the same, except that a tight-fittingglass-glass Potter or a Dounce homogenizer isused. Up to 15-20 passages of the pestle may berequired to achieve sufficient cell breakage.

Homogenisation4°C 4°C

Scrape and collectby centrifugation (500g)

Confluent cell culture

Nitrogen Decompression(Nitrogen Cavitation)

• Large quantities of nitrogen are firstdissolved in the cell under high pressurewithin a suitable pressure vessel. Then, when the gas pressure is suddenly released, the nitrogen comes out of the solution as expanding bubbles that stretch themembranes of each cell until they ruptureand 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 physicalstress.

• 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 theirphysical properties

• Problem– Some compartments share similar physical properties

Cells, Tissue

Homogenization Centrifugation

Sucrose GradientSubcellular organelles

Density Gradients (II)

1 6 % S i n D 2 O o r3 5 % S i n H 2 O

1 . 5 m l

1 0 % S i n D 2 O o r2 5 % S i n H 2 O

1 m l

E a r l y e n d o s o m e s

L o a d : P N S i n 4 0 . 6 % S1 . 0 m l

H o m o g e n i z a t i o n b u f f e r

The PNS is brought to 40.6 % sucrose [S] and loaded at the bottomof an SW 60 tube. The load is the overlaid sequentially with 16 %sucrose in heavy water [or 35 % sucrose], 10 % sucrose in heavywater [or 25% sucrose] and finally with homogenization buffer. Thegradient is run for 60 min at 35K rpm. Early endosomes and lateendosomes [+ carrier vesicles] are collected as indicated.

…next step: Gradient-1

Sucrose gradient10 %

40 %

Pellet (3,000g)= nucleiSupernatant=PNS

4°C165,000g

Collect intact membranesand vesicles

…next step: Gradient -2

Sucrose gradient10 %

40 %

Pellet (3,000g)= nucleiSupernatant=PNS

4°C165,000g

Collect intact membranesand vesicles

…next step: Gradient-3

Sucrose gradient10 %

40 %

Pellet (3,000g)= nucleiSupernatant=PNS

4°C165,000g

Collect intact membranesand vesicles

Purification of Endosomes

Subcellularfractionation

Subcellular fractionation allows access to low abundant and organelle specific

proteins200

Mr

8

305 (2 to 120 fold) protein spots enriched in late endosomalfraction

292 (2 to 25 fold) spots enrichedin early endosomal fraction

286 proteins increased (2 to 10 fold) in late vs early endosomes

PNS (Cy2, blue),

Early endosomes (Cy3, green)

Late endosomes (Cy5, red)

Stasyk and Huber, Proteomics, 2005

4 pI 9

Subcellular Fractionation

Sucrose gradient

Homogenisation

4°C 4°C

Scrape and collectby centrifugation (500g)

4°C

Pellet (3,000g)= nucleiSupernatant=PNS

4°C165,000g

Collect intact membranesand vesicles

1. Marker analysis(Western Bl., Enzymes)

2. Na2Co3 Extraction at high pH (peripheral vs. integral membrane proteins)

Confluent cell culture

3. Organelle Proteome Analysis (2D-GE, Chromatography, Mass Spec.)

Pasquali et al.,1999 J. Chromatography BHuber et al., 2003, Circulation Res.

10%

40%

Na2Co3 extracted Membrane proteins

100,000 g pellet integral membrane proteins

100,000 g supernatant peripheral membrane proteins

Subcellular Fractionation

Sucrose gradient

Homogenisation

4°C 4°C

Scrape and collectby centrifugation (500g)

4°C

Pellet (3,000g)= nucleiSupernatant=PNS

4°C165,000g

Collect intact membranesand vesicles

1. Marker analysis(Western Bl., Enzymes)

2. Na2Co3 Extraction at high pH (peripheral vs. integral membrane proteins)

Confluent cell culture

3. Organelle Proteome Analysis (2D-GE, Chromatography, Mass Spec.)

Pasquali et al.,1999 J. Chromatography BHuber et al., 2003, Circulation Res.

10%

40%

Quality Control

• In all fractionation experiments, a balancesheet should be established for thedistribution of protein and markers (egbHRP) in all fractions.This provides theonly appropriate means to judge thehomogenization /fractionation steps and to compare different preparations.

Balance Sheet(separation of early and late endosomal

fractions on the flotation gradient)

A) Early endosomes (5 min at 37°C)

Homog. 0.7 4.5 11.3 0.4 100 1.0PNS 0.6 3.0 7.37 0.4 67 1.0Early fract. 0.4 0.6 0.15 4.0 14.7 10.0Late fract. 0.3 0.06 0.12 0.4 1.2 1.2

B) Late endosomes (5 + 30 min at 37°C)

Homog. 0.7 2.7 10.8 0.3 100 1.0PNS 0.6 1.6 7.2 0.2 58 0.9Early fract. 0.5 0.04 0.09 0.4 1.3 1.6Late fract. 0.6 0.6 0.09 6.9 25.0 27

Vol. HRP ProteinSp. Act Yield RSA (ml) OD (mg) %

Density Gradients (III)(continous gradients)

10% 40%Sucrose

2 22 01 81 61 41 21 08642000 . 0 0

0 . 0 1

0 . 0 2

0 . 0 3

f r a c t i o n s

s p e c . a c t i v i t y

b e t a - h e x .C y t . C - R e d u c t a s eG a lT

2 42 22 01 81 61 41 21 08642000 . 0 0

0 . 2 5

0 . 5 0

0 . 7 5

1 . 0 0

1 . 2 5

1 . 5 0E EL Eb lP Ma p P M

f r a c t i o n s

H R P / p r o t e i n ( n g / µ g )

A

B

..... ..... ......

.....

..... ..... ......

.....

..... ..... ......

.....

..... ..... ......

Huber et al., Circulation Res., 2003Huber, Nature Rev., Moll Cell Bio, 2003 Stasyk and Huber, Proteomics, 2004

Sucrose gradients

Homogenisation4°C

Scrape and collectby centrifugation (500g)

4°C Pellet (3,000g) = nucleiSupernatant = PNS

4°C; 165,000g

10%

40%

Subcellular Fractionation & Organelle Proteome Analysis

LEEE

Collect intact membranesand vesicles

Continuous DiscontinuousPeripheral proteins

Integral membrane

proteins

Marker analysis(Western Blotting)

CytosolTotal membranes

Na2CO3

Extraction at high pH

4°C;

100,000g

Organelle Proteome Analysis (2D-GE, Chromatography, Mass Spectrometry)

LE=late endosomesEE=early endosomes

Murinemammary epithelial

EpH4 cells

+ EGF0; 5; 40 min 4°C

8%

35%LE

42%

25%EE

I

8%

35%CrudeEndosomes

42%

II

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) areseparated by centrifugation [Hopkins]

• Endosomes loaded with HRP bound to a ligand are separated by centrifugation afterDAB reaction (cross-link of lumenalproteins) [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 powerfulpreparative separation tool for proteinenrichment, especially suited for complexprotein mixtures.

• Isolation of subcellular compartments orpre-fractionation of complex proteinmixtures using narrow pH gradients can beperformed.

Free-Flow Electrophoresis (II)

• Lysosomes and endosomes can be separated fromother 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 befree 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 antibodiesaccording to their antigenic properties, rather than their physical properties[Gruenberg].

• Is most efficiently combined with densitygradient centrifugation as means forprefractionation

ImmunoisolationPrincipal

solid support

linker antibody specific antibody

antigen

fraction

Experimental StrategiesDIRECT INDIRECT

ANALYSIS

Experimental Conditions

2 4 6time [hr]

yiel

d [a

ct]

specific

non-specific bk

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! Itis, therefore, very difficult to carry out "differential" immuno-isolation, ie to separate membranes containing different densities of theantigen (molecules/µm2 membrane surfacearea).

Antigen (II)• The epitope must be exposed on the surface

of the desired compartment (and accessibleto the immobilized antibody).

• Immunoisolation is better achieved with a relatively abundant epitope.

• However, we find that immunisolation isefficient 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 theparticles/beads increases the proportion of correctly oriented specific antibodies, henceorganelle 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 givenantibody 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 p-toluene 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 higheraggregation properties than monodisperseparticles

Solid Support: Types

TYPES ADVANTAGES DISADVANTAGES

Fixed S.aureus cells - high capacity - high speed sedimentationexpressing ProteinA (high S/V ratio) - non specific adsorption

- monodisperse - SDS-gels difficult- commercially avail.

Magnetic beads - low background - low capacity

- NO sedimentation- monodisperse- commercially avail.

Cellulose fibers - high capacity - high background- high flexibility (entrapment)- low speed sediment. - not commercial. avail.

Eupergit particles - high capacity - high background- ± monodisperse - only some Ags- commercially avail.

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 ActivatedOrganelle 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 ActivatedOrganelle 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-DPHTrimethyl

ammonium DPH

N(CH3)3

C C

H

H C

H

C

H

H C C

H

cationic DPH analog with a charged substituentas surface anchor

TMA-DPHFeatures

• can be removed from plasma membrane bywashing

• can be used to study endocytosis• non fluorescent in water and binds in

proportion to the available membranesurface

• excitation 355nm, emission 450nm

TMA-DPHInternalization

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.

Sandra MorandellTaras StasykHong-Lei Huangandall other members of the Huber group

Günther K. BonnIsabel Feuerstein all members of the group Zlatko Trajanoski, TU Graz

Florian Überall, Med Uni Ibk

Jakob TroppmairStephan GeleyManuela BaccariniJacques PouysségurAndy Catling

Acknowledgements

Karl MechtlerElisabeth Roitinger

BioCenterDiv. Cell Biology

Thomas Lindhorst