CyDye DIGE Fluor minimal dyesによる細胞表面タンパク質の

CyDye DIGE Fluor minimal dyesによる細胞表面タンパク質の
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Part of GE Healthcare
Application Note 11-0033-92 AB
2-D DIGE
Selective labeling of cell-surface proteins using CyDye
DIGE Fluor minimal dyes
Key words: cell-surface protein labeling Ettan
DIGE CyDye DIGE Fluor minimal dyes cellsurface proteins
Cell-surface proteins are relevant to almost all proteinprotein interactions, for example as inducers of intracellular signaling, environment adaption, and drug
treatment, and are often involved in disease pathogenesis
and pathology (1). Therefore cell-surface proteins and
receptors as potential drug targets are of great interest to
the pharmaceutical industry (1).
Two-dimensional (2-D) electrophoresis is used extensively
for visualization of biomarkers and targets. However, cellsurface proteins, partly due to their low abundance (1–2%
of cellular proteins), can be difficult to detect in a 2-D gel
without fractionation or some other type of enrichment.
They are also often poorly represented in 2-D gels due to
their hydrophobic nature and high molecular weight (2).
In this study, we present a new protocol using CyDye™
DIGE Fluor minimal dyes to visually enrich for and detect
this important group of proteins. This protocol is rapid,
simple to use, and all three CyDye DIGE Fluor minimal dyes
(Cy™2, Cy3 and Cy5) can be used to label cell-surface
proteins. These features allow for multiplexing according to
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE)
using Ettan™ DIGE technology and analysis of protein
expression changes using DeCyder™ 2-D Differential
Analysis Software. In this way, the level of cell-surface
proteins can be studied in different disease states or when
responding to different treatments. Small changes in
abundance can be detected with high accuracy, and
results are supported by defined statistical methods.
Fig 1. Overview of labeling workflow protocols
Materials
GE Healthcare products used
CyDye DIGE Fluor Cy2 minimal dye
RPK0272
CyDye DIGE Fluor Cy3 minimal dye
RPK0273
CyDye DIGE Fluor Cy5 minimal dye
RPK0275
2-D Quant Kit
80-6483-56
IGP Buffer pH 3–11NL
17-6004-40
Immobiline™ DryStrip pH 3–11NL, 24 cm
17-6003-77
Immobiline Reswelling Tray
80-6465-32
DeStreak™ Rehydration solution
17-6003-19
Immobiline DryStrip Cover Fluid
17-1335-01
Ettan IPGphor™ II IEF Unit
80-6505-03
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Ettan IPGphor Manifold
80-6498-38
Cell surface labeling
Ettan DALTtwelve Gel Caster
80-6467-22
Ettan DALTtwelve Large Vertical System
80-6466-27
Typhoon™ 9410 Variable Mode Imager
63-0038-56
DeCyder 2-D Differential Analysis
Software Version 6.0
11-0010-95
ImageQuant™ Analysis Software
Inquire
Carefully detach adherent cells non-enzymatically, count
cells and divide into aliquots of 5–10 × 106 cells. For cells
growing in suspension, omit the detaching step. Centrifuge
the cells at about 800 × g for 5 min and remove the
supernatant containing the medium. Wash the pellets by
resuspension in 1 ml ice cold Hank’s Balanced Salt Solution
(HBSS) pH 8.5 and centrifuge at 800 × g at 4 ºC for 2 min.
Remove the supernatant and resuspend the cell pellet in
200 µl ice cold labeling buffer (HBSS pH 8.5, 1 M urea). Label
the intact cells with 600 pmol CyDye DIGE Fluor minimal dyes
for 20 min on ice in the dark. Quench the reaction by adding
20 µl 10 mM lysine and incubate for 10 min. Wash the
surface-labeled cells twice by resuspension in 500 µl HBSS
pH 7.4 followed by centrifuging at 800 × g at 4 ºC for 2 min.
Urea
17-1319-01
CHAPS
17-1314-01
Dithiothreitol (DTT)
17-1318-01
Bromophenol Blue
17-1329-01
Tris
17-1321-01
Sodium Dodecylsulfate (SDS)
17-1313-01
For comparison with the standard Ettan DIGE protocol see
Figure 1.
PlusOne™ Glycerol 87 %
17-1325-01
Cell lysis and fractionation
PlusOne ReadySol IEF 40% T/ 3% C
17-1310-01
PlusOne TEMED
17-1312-01
PlusOne Ammonium Persulfate
17-1311-01
PlusOne Glycine
17-1323-01
Lyse the surface-labeled cells in 150 µl cold lysis buffer
directly (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris,
5 mM magnesium acetate pH 8.5) and leave on ice for at
least 1 h with occasional vortexing. Centrifuge the lysates at
12 000 × g at 4 °C for 10 min. Transfer the supernatant, which
is the cell surface protein–labeled, non-fractionated sample
(containing proteins from internal and cell surface
membranes, as well as cytosol), to a new tube. Wash the
pellets as above and then fractionate (using a membrane
fractionation kit, Pierce) into membrane and cytosolic
fractions prior to 2-D gel electrophoresis. The membrane
fraction contains proteins from internal and cell-surface
membranes. For whole-cell labeling (Fig 1, protocol 2), follow
the standard Ettan DIGE procedure, and label the lysed cells
before fractionation (3).
Other materials required
General cell culture equipment and suitable cell culture
medium
Cell dissociation media, enzyme free PBS-based
(Invitrogen)
2-D Sample Prep for Membrane proteins (Pierce)
Methods
Cell culture
Grow Chinese Hamster Ovary cells (CHO-K1) using
standard cell culture procedures in F-12 Ham medium
with GlutaMAX™ I containing 10% fetal calf serum,
50 U/ml penicillin, and 50 µg/ml streptomycin sulfate
(Invitrogen). To perform an Ettan DIGE experiment,
exchange the culture medium to a serum-free medium.
Label the cell-surface proteins at different time points
with CyDye DIGE Fluor Cy3 or Cy5 minimal dyes (see cell
surface labeling section). Pool equal fractions from each
time point and label with CyDye DIGE Fluor Cy2 and use
as an internal standard for each 2-D gel.
The majority of the experiments were performed with
CHO-K1 cells, but mouse embryo fibroblasts (3T3 L1) and
mouse ascites lymphoma lymphoblasts (EL4) were also
used. The two latter cell types were grown in DMEM
medium with GlutaMAX II, but otherwise with identical
conditions as for the CHO-K1 cells.
2
Application Note 11-0033-92 AB 2005-04
Protein quantitation
Quantitate the proteins from the different fractions using the
2-D Quant kit.
1-D and 2-D electrophoresis
Rehydrate Immobiline DryStrip gels, pH 3–11NL (24 cm) using
Immobiline DryStrip Reswelling Tray, 24 cm in 450 µl
DeStreak Rehydration solution (0.5% IPG Buffer) overnight.
Apply the CyDye labeled samples onto Immobiline DryStrip
gels by anodic cup loading in the Manifold and perform
isoelectric focusing (IEF) using Ettan IPGphor II IEF System
according to instructions (3). After IEF, equilibrate the strips in
two steps and place on top of large (26 x 20 cm) 12.5%
polyacrylamide gels (SDS-PAGE) and overlay with 0.5%
agarose (in running buffer containing bromophenol blue).
Perform 2-D electrophoresis using Ettan DALTtwelve Large
Vertical System at 5 W/gel for 30 min, and then at 15 W/gel
until the dye front reaches the bottom of the gel.
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Imaging and data analysis
Directly after completing 2-D electrophoresis, scan the gels
using Typhoon 9410 Variable Mode Imager set for Cy2, Cy3,
and Cy5 fluorescence. Compare spot maps from the
membrane fractions, cytosolic fractions, and nonfractionated samples using DeCyder 2-D Differential Analysis
Software (4).
Post-staining
After imaging, silver stain the gels according to standard
procedure (5), or any post-stain such as Deep Purple™ Total
Protein Stain.
Results and discussion
Protein concentration
An overview of the two labeling workflows is shown in Figure
1. Since the cells are still intact when labeled, according to
the cell-surface protein labeling protocol, only the proteins
on the cell surface are exposed to the dye (Fig 1). In the
standard Ettan DIGE protocol, the cells are lysed first and
proteins inside as well as outside the cell are labeled (Fig 1).
The relative amount of dye to protein in the cell-surface
protein labeling protocol is not known, since cell-surface
proteins can not be specifically quantitated. However, it is
known that cell-surface proteins constitute a very low
proportion of the cellular proteins (2). Approximately
5–10 × 106 cells to 600 pmol of dye were used. It may be
possible to use fewer cells since only 12.5–25% of the non-
Non-fractionated
fractionated sample was used for 2-D electrophoresis.
Protein concentrations in the different fractions were
determined using the 2-D Quant Kit. The total protein
amount derived from 10 × 106 CHO-K1 cells was 920 µg in
the non-fractionated sample, 225 µg in the
membrane/hydrophobic fraction and 770 µg in the
cytosolic/hydrophilic fraction. These amounts will most likely
vary depending on cell type and cell size. The proportion of
proteins that are labeled in the cell-surface protocol is
probably higher than the standard Ettan DIGE minimal
labeling which is 2–3% of total protein.
It seems that only one dye is attached per protein molecule
since the spot shape is rounded and there is no vertical
streaking for the low molecular proteins on the gels (Fig 2
and 3). Two and more dye molecules per protein would
cause vertical streaking due to increased molecular weight
of the labeled protein. A range of molecular weight shifts
would be seen on the gel depending on the number of
additional dye molecules resulting in vertical streaking.
This effect would best be seen for the low molecular
weight proteins, since a change in molecular weight results
in a larger shift on the gel compared to high molecular
weight proteins.
Cell-surface protein specific labeling
Two identical samples of cells were surface labeled with
CyDye DIGE Fluor Cy3. One was lysed and used directly for
2-D electrophoresis. The other sample was lysed and
fractionated into membrane and cytosolic fractions. All the
Membrane fraction
Cytosolic fraction
A
3
pl
11
3
pl
11
3
pl
11
B
3
pl
11
3
pl
11
3
pl
11
Application Note 11-0033-92 AB 2005-04
3
Fig 2. Specificity of
cell-surface protein
labeling. The cellsurface proteins of
CHO-K1 cells were
labeled with Cy3 and
fractionated. The
different fractions
were separated by
2-D electrophoresis
and scanned for Cy3
fluorescence (A). The
same gels were then
silver stained (B).
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label appeared in the membrane fraction; the cytosolic
fraction was devoid of any labeled proteins (Fig 2). The
same gel was silver stained and the result showed that
there were proteins in the gel, but they were not labeled
using the cell-surface protein labeling protocol. These
results suggest that this new labeling protocol is cellsurface protein specific. The CyDye DIGE Fluor minimal dye
does not appear to enter the cell or pass through the cell
membrane. The cells are kept on ice prior to the labeling
and this may reduce any active transport across the
membrane. The time for CyDye DIGE Fluor minimal dye
exposure is also relatively short (20 min). Another probable
explanation for the lack of labeling inside the cell is that
even if the dye passes across the membrane, the pH inside
the cell is too low (< pH 7.4) for an efficient labeling
reaction to occur (optimal pH 8.5). The labeling reaction
is quenched followed by washing of the cells, which
further prevents any protein labeling after the cells have
been lysed.
A comparison of these results using DeCyder 2-D software,
revealed over 80 novel proteins spots present only in the
cell-surface protein labeled fraction (Fig 3B). The cell surface
protein labeling protocol improved the detection of many
proteins when compared with the standard method. Cy5
cell-surface labeled samples and Cy3 labeled lysates from
3T3 fibroblasts and EL4 lymphoblasts (growing in suspension)
were also compared using DeCyder 2-D software. Similar
results (50 to 100 new spots) were obtained as for the
CHO-K1 cells (data not shown).
A
Fractionation
There are only minor differences in the spot pattern for the
membrane fractionated sample compared with the nonfractionated sample (Fig 2). The two spot maps were
compared using DeCyder 2-D Differential Analysis
Software and all the spots detected in the membrane
fraction were also present in the non-fractionated sample.
Fractionation, therefore, is not necessary to improve
detection of cell-surface proteins but can be used to verify
lack of labeling of proteins inside the cells.
B
Protocol 2
Protocol 1
Cy3 lysate
Cy5 cell surface
Comparison between protocols
To be able to evaluate the advantages with the new cellsurface protein labeling protocol, a comparison with the
standard Ettan DIGE protocol was performed. Two identical
samples from CHO-K1 cells grown in the same flask were
labeled in parallel with the two different protocols,
respectively (Fig 1). A cell-surface Cy5 labeled sample was
run on the same gel as a Cy3 labeled cell lysate. The green
spots on the gel (Fig 3A) represent the proteins labeled
using the standard Ettan DIGE procedure followed by
membrane fractionation. These spots are presumably
membrane proteins including cell-surface proteins as well
as proteins from membranes inside the cell (ER, Golgi,
mitochondrion, and nucleus). Standard Ettan DIGE labeling
procedure followed by a membrane fractionation step was
chosen for comparison, since it should give the highest
probability for detecting the low-abundant cell-surface
proteins. The red spots on the gel (Fig 3A) are cell-surface
specific proteins labeled according to the new cell-surface
protocol that are not visible with the standard labeling
procedure (green spots). Furthermore, the yellow spots
represent overlapping proteins that occur in both samples
using either procedure (Fig 3A).
Application Note 11-0033-92 AB 2005-04
4
Fig 3. (A) 2-D gel images of a CHO-K1 Cy5 cell-surface labeled sample (red
spots, see protocol 1, Fig 1) and a membrane fractionated Cy3 sample
(green spots, see protocol 2, Fig 1) labeled according to standard Ettan DIGE
protocol run in the same 2-D gel. (B) DeCyder 2-D Differential Analysis
Software views from the 2-D gel showing a cell-surface labeled protein not
visible using the standard Ettan DIGE protocol.
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Multiplexing
To test the cell-surface protein labeling protocol in an Ettan
DIGE experiment using all three dyes (6), a series of
samples from serum depleted CHO-K1 cells were collected
and cell-surface proteins labeled at different time points
(Table 1). Samples were separated by 2-D electrophoresis.
All three CyDye DIGE Fluor minimal dyes labeled cellsurface proteins similarly (data not shown). Changes in
expression during serum starvation for many of cellsurface proteins were detected using DeCyder 2-D
software (Fig 4).
Table 1. An Ettan DIGE experiment was performed using samples from
serum depleted cells labeled according to the cell-surface protein labeling
protocol in figure 1.
Sample
Time of serum
depletion
Labeled
with CyDye
Gel
number
1
-
Cy3, Cy2
1
2
30 min
Cy5, Cy2
1
3
2h
Cy3, Cy2
2
4
4h
Cy5, Cy2
2
5
16 h
Cy5, Cy2
3
Conclusions
The new Ettan DIGE protocol for cell-surface protein
labeling is rapid, simple to use and a specific method
for labeling cell-surface proteins. Many novel cellsurface proteins are only visible using the cell-surface
protein labeling protocol. Over 80 new cell-surface
proteins for CHO-K1 cells were detected using DeCyder
2-D Differential Analysis Software. Multiplexing is
achieved using the three CyDye DIGE Fluor minimal
dyes, and in combination with DeCyder 2-D software,
this new protocol is a powerful tool for studying cellsurface proteins.
Acknowledgements
We thank Professor Dontscho Kerjaschki, Corina
Mayrhofer and Sigurd Krieger at the Institute of Clinical
Pathology, University of Vienna, Austria for their
collaboration.
References
Fig 4. Change in expression of two cell-surface proteins during starvation
of CHO-K1 cells. Spot maps were analyzed using DeCyder 2-D Differential
Analysis software.
5
Application Note 11-0033-92 AB 2005-04
1. Jang, J. H and Hanash, S. M. Profiling of the cell surface proteome.
Proteomics 3, 1947–1954 (2003).
2. Shin, B.K. et al. Global profiling of the cell surface proteome of cancer cells
uncovers an abundance of proteins with chaperone function. J Biol Chem. 9,
7607–16 (2003).
3. Ettan DIGE System User Manual, Amersham Biosciences, 18-1164-40 (2002).
4. Data file: DeCyder 2-D Differential Analysis Software v 6.0, Amersham
Biosciences, 11-0011-98 Edition AA (2004).
5. 2-D Electrophoresis using immobilized pH gradients, principles and methods,
Amersham Biosciences, 80-6429-60 Edition AB (2002).
6. Data file: CyDye DIGE fluors and labeling kits, Amersham Biosciences,
18-1164-84 Edition AB (2003).
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General Electric Company reserves the right, subject to any
regulatory approval if required, to make changes in specifications
and features shown herein, or discontinue the product described at
any time without notice or obligation. Contact your GE
Representative for the most current information. © 2005 General
Electric Company – All rights reserved. GE and GE Monogram are
trademarks of General Electric Company. Amersham Biosciences,
Cy, CyDye, DeCyder, DeStreak, Ettan, ImageQuant, Immobiline,
IPGphor, PlusOne, and Typhoon are trademarks of GE Healhcare Ltd.
GlutaMAX is a trademark of Invitrogen Corporation. CyDye: this
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and other patents pending. Some of these products may only be
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A license to use the CyDye Fluors for commercial purposes is
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GE imagination at work
Application Note 11-0033-92 AB 2005-04
6
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