Multi-strips on One Gel
Method to Improve the Reproducibility, Resolution Power and High-throughput of
Two-dimensional Electrophoresis
YUAN Quan, AN Jie, LIU
Ding-Gan, ZHAO Fu-Kun*
( Key Laboratory of
Proteomics， Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences,
Shanghai 200031, China )
Abstract Two-dimensional polyacrylamide gel
electrophoresis is one of the most key separation tools which can reveal
hundreds or even thousands of proteins at a time in proteomic research. In this
paper, we report a new IPG strip application, called multi-strips on one gel
(MSOG) method. By comparing the 2-DE patterns of the same sample, the different
state samples and the same sample in the different second dimensional SDS
running systems (large size and medium size gels), we found this new method can
not only improve the reproducibility and resolution power of 2-DE pattern, but
also achieve high throughput and economical format which is helpful to
automatic proteomic research.
Key words
two-dimensional electrophoresis; multi-strips one gel (MSOG) method;
reproducibility; high-throughput; proteomics
Two-dimensional electrophoresis (2-DE) is
used to study changes in cellular protein expression, for detection of
disease-related proteins, and in a great number of other implications. It is
the only method currently available that is capable of simultaneously
separating thousands of proteins for quantitative comparison at a time[1,2].
Therefore, for the comparison of results within laboratory and between
laboratories, the importance of achieving maximum reproducibility of spot
positions in 2-D patterns cannot be over-emphasized.
Classical 2-D electrophoresis with pH
gradient generated by a carrier ampholyte (CA) was limited in its resolution,
reproducibility and protein-loading capacity[3] because of pH-gradient
instability with prolonged focusing time: the pH gradient moves towards the
cathode (cathode drift). Detailed comparisons of CA-based patterns for the same
cell material in separate laboratories were very difficult, furthermore,
limiting to establish collective databases of 2-D gel information. By contrast,
with introduction of immobilized pH gradients (IPGs), problems of pH-gradient
instability and reproducibility have been largely overcome[4].
However, a series of problems in IPG-Dalt
still remains to be solved for the instability of each operated step of 2-DE.
Although the standardization of the pre-procedure can be confirmed such as
sample preparation (the methods of protein extraction and quantification, the
compositions of lysis buffer), the run condition of IEF and the time controlled
of other steps in one’slaboratory for the same samples, it’shard to precisely
control such procedures as the SDS-PAGE dimension and silver staining. Corbett et
al.[5] reported the SDS-PAGE was not the steady-state methods, so that
variability results not only from differences in gel preparation, but is also
influenced by electrophoretic conditions, for instance, temperature
variability[6].
When the SDS-PAGE is terminated, silver
staining of the gel is still the main choice for protein spots visualization
since the higher sensitive limits of detection can reach the nanogram level
compared with the Coomassie brilliant blue stains. It’smore easily
laboratory-made and much cheaper than Sypro Ruby[7－9] which combines a linear dynamic
range with sensitivity levels. There are lots of published versions of the
silver staining protocols[10,11], in combination with fixation, sensitization,
silver impregnation and development, stopping and preservation, and washing
steps, etc. In general, the time of each step of silver staining can be exactly
controlled except for the development step which strongly depends on the
experience of the operators.
In recent years, some new methods came out
such as fluorescence two-dimensional gel electrophoresis (2-D DIGE)[12, 13].
Through labeling of samples with one of three specially different fluorescent
dyes, Cyanine-2 (Cye2), Cyanine-3 (Cye3) or Cyanine-5 (Cye5), the labeled
samples are then run in one gel and detected individually by scanning the gel
at different wavelengths. After quantitative analysis by the
Phoretix/ImageMaster software, the different expressed proteins can be
obtained. Based on the principle of this method, however, those proteins
without lysine residues can t be labeled and lost. At the same time, the high
cost of the whole system prevents it from spreading out.
Here, we developed a new IPG-Dalt procedure
that two or three IPG strip gels (length ≤13 cm) were run simultaneously on one
SDS-PAGE (25.5 cm×20 cm, Ettan Dalt twelve system from Amersham Biosciences).
This method not only improves the reproducibility and matching efficiency of
2-D electrophoresis, but also achieves higher resolution power comparing to the
former method called one IPG strip gel run on one gel such as using SE 600
system. Furthermore, the final 2-D patterns showed the artificial errors could
be diminished to minimum. By using the MSOG method, we compared the total
proteins extracted from the samples under different conditions and discovered
the differentially expressed proteins of human hepatoma cells in different
states.
1 Materials and Methods
1.1 Apparatus and chemicals
All equipments for IEF and vertical
electrophoresis for SDS-PAGE (IPGphor, SE 600, Ettan Dalt twelve system, IPG
regular strip holder 13 cm, IPG cup loading strip holder)，13 cm IPG strips pH 4－7 or pH 3－10 NL, IPG Buffer pH 4－7 or pH 3－10 NL, ammonium persulfate, SDS,
urea, Tris-base, glycerol, glycine, acrylamide, N, N′-methylenebisacrylamide,
CHAPS, TEMED, agarose and bromophenol blue were from Amersham Biosciences
(Uppsala, Sweden). DTT, iodoacetamide, AgNO3 and thiourea were purchased from
Sigma (St. Louis, MO, USA). Other chemicals are domestic products (analytical
grade).
1.2 Sample preparation
Cultured cells of human hepatoma SMMC7721
and mouse 3T3, provided by Prof. LIU Ding-Gan and Prof. ZHAO Mu-Jun,
respectively, were collected and washed 3 times with PBS. The cell pellets were
thawed and during the procedure rapidly mixed with 7 mol/L urea, 2 mol/L
thiourea, 4% CHAPS, 40 mmol/L DTT, 40 mmol/L Tris-base and 2% Pharmalyte pH 4－7 or pH 3－10 NL. After 1 hour of gentle
stirring at ambient temperature, samples were centrifugated at 40 800 g,
at 4 ℃ for 1 h to remove the precipitated nucleic acids. The supernatants were
stored in small aliquots in 1.5 mL Eppendorf tube at －78 ℃ and the concentration of
proteins was determined by the modified Bradford method[14].
1.3 2-D gel electrophoresis
1.3.1 Isoelectric focusing (IEF) of
proteins in IPG strips and IPG strip equilibration
The cell lysate (80 μg protein) was solubilized in 250 μL of a rehydration
solution containing 8 mol/L urea, 2% CHAPS, 20 mmol/L DTT, a trace of
bromophenol blue and 0.5% Pharmalyte pH 4－7 or pH 3－10 NL. Rehydration and IEF were carried out automatically according
to the programmed settings: 30 V 6 h; 60 V 6 h; 500 V 1 h; 1000 V 1 h; 8000 V 3
h.
After IEF, the IPG strips were immediately
equilibrated for 2×15 min with gentle shaking in 10 ml of a solution containing
Tris-HCl buffer (50 mmol/L, pH 8.8), 6 mol/L urea, 30% glycerol, 2% SDS, and a
trace of bromophenol blue. 1% DTT was added at the first, and 4% iodoacetamide at
the second equilibration step. After equilibration, the IPG strips were aligned
on filter paper along on edge for 1 min to remove excess liquid before they
were applied to the SDS gels[15, 16].
1.3.2 IPG strips transfer and SDS-PAGE Owing to the actual length of one IPG strip (13 cm pH 4－7 or pH 3－10 NL, from Amersham Biosciences)
which removed the support film was about 14 cm, two 13 cm IPG strips (actually
length: 14 cm×2=28 cm) were longer than the whole distance (25.5 cm) of Ettan Dalt
twelve gel. Two ends (acidic and basic ends) of each IPG strip which contacted
with the electrodes of the IPG holder should be cut out about 1 cm,
respectively. And the final length of each strip was adjusted around to 12 cm,
which makes it possible that two 13 cm IPG strips can be run on the one gel
(gel size: 25.5 cm×20 cm×1 mm, Ettan Dalt twelve system) (Fig.1). Then, the two
IPG strips were abreast inserted between the glass plates with a spatula and
brought in close contact with the upper edge of one SDS gel. After the sealed
agarose was cooled down, the second dimensional SDS-PAGE could be carried out
(step 1: 4 W/gel, 45 min; step 2: 20 W/gel, 5 h 30 min; temperature: 20 ℃)
1.4 Silver staining and image analysis
When the SDS-PAGE was finished, the gels
were fixed in ethanol/acetic acid/water (4∶1∶5) overnight. Silver staining was
carried out according to the modified protocol[10]. Computerized 2-D gel
analysis (spot detection, spot editing, pattern matching) was performed with
Image Master 2D Elite 3.1 software package.
2 Results and Discussion
2.1The evaluation of the 2-DE patterns
from the same sample
Fig.1 Schematic representation of multi-strips one gel (MSOG) method
After equilibration of the IPG strips in
the loading buffer, two ends (acidic and basic ends) of each IPG strip were cut
out about 1 cm. Then, the two cut IPG strips were abreast inserted between the
glass plates with a spatula and brought in close contact with the upper edge of
one SDS gel. After SDS-PAGE, the final 2-DE patterns were achieved by silver
staining.
Fig.1 showed the whole course of two IPG
strips running on one SDS gel. Although the patterns of three IPG strips
running on one gel were not shown, they can be easily transferred onto one SDS
gel. Even the cutting step of IPG strip could be omitted (7 cm×3=21 cm <25.5
cm). Reproducibility of the final 2-DE patterns runs through each step of 2-DE
protocol including sample preparation, sample application, the condition of IEF
and SDS-PAGE, gel staining, choice of the analysis software, even the quality
of the various chemicals used. Out of question, it's the most important and
difficult work in the approach of 2-DE/MS of proteomic research. With the introduction
of Immobilized pH gradient (IPG) gels, the reproducibility of IEF has been
greatly improved. However, as for most of researchers taking it as a first
choice in the staining methods to analyze 2-DE patterns, the developing step of
silver staining is hard to control especially in its developing step. Even to
the same researcher, two different 2-DE patterns of the same protein sample
might be obtained only when the developing time of silver staining was changed.
Obviously, it'simpossible to immobilize this time. Owing to the inherently
complex nature of silver staining procedures, spot intensities may easily vary
as much as 20% from batch to batch[17]. We have ever put two SDS gels in one
staining vessel to carry out the silver staining protocol. But the result
showed the sensitivities and intensities of spots were decreased comparing to
one gel in a staining box. One of the probable reasons was the deficient dying
solution in between the two gels. As to comparative proteomic research, the
main task is to find the reproducible differential expressed proteins and then
identify them. When the diversities of background of samples exist or the
intensities of spots vary, the accuracy of quantity for differentially
expressed proteins will decrease. And the possibility of taking these
artificial spots as really differential spots will increase, even for totally
the same sample but at the different running states. Unfortunately, it will
happen, sometimes, that differential protein spots on the 2-D gel are even more
than same ones. To examine whether the identical 2-DE patterns including the
same gel background and the same positions of the protein spots in the gel
could be obtained, the identical samples (80 μg proteins from SMMC7721) were
evaluated[see Fig.2(A)]. As a result of the ImageMaster 2D Elite 3.1 analysis,
the exact match ratio of protein spots is over 95%. The numerical value depends
on the chosen analysis software and the worker. However, we can get the rough
information on reproducibility of the different gels. The result indicated the
backgrounds of gels and the intensities of most matched spots were concurrent.
Fig.2 2-DE patterns of cultured cells from human hepatoma cancer (A) and mouse
3T3 (B) by MSOG method
Two 13 cm IPG strips (the same samples) running on one
SDS-polyacrylamide gel simultaneously. The same running condition of the two 13
cm IPG strips in the first dimension: IPG strip pH 4－7 (A) and pH 3－10NL (B); the
effective separation distance of each IPG strip: 120 mm, sample application by
in-gel rehydration (80 μg protein); IEF was performed on the IPGphor; second
dimension: vertical SDS-PAGE on Ettan Dalt twelve system (12.8% T constant);
silver staining.
The step of background subtraction also
could be omitted when using software to analyze 2-DE patterns. It meant the
dynamic range of silver staining was shortened. In other words, the tendency of
dynamic change of silver staining in one gel was identical. Once the artificial
protein spots are diminished to the least, the real differentially expressed
proteins will be much more easily discovered. It will greatly helpful to reduce
the number of garbage data and save time. Also, to examine the universality of
this method, the mouse 3T3 cells using IPG strip of pH 3－10 NL were evaluated. The resemble
outcome was achieved[see Fig.2(B)]. It illuminated that this method fitted in
with not only the sample running in narrow pH gradient, but also in wide pH
gradient.
2.2 The comparison of 2-DE patterns of
human hepatoma cells under different conditions
Based on the high reproducibility, we
further compared 2-DE patterns of cultured cells of human hepatoma under different
conditions in order to identify the differentially expressed proteins. As we
know, quantitative protein variation revealed by 2-DE can be used to dissect to
some extent the network of regulatory elements acting on individual proteins.
However, SDS-PAGE is not a steady-state method, so that variability results not
only from differences in gel preparation, but is also influenced by
electrophoretic conditions[5]. A major factor limiting the reproducibility of
SDS-PAGE is controlled over termination of the electrophoresis.
The migration of the tracking dye is very
sensitive to pH and temperature, which results in that reproducibility of spot
positions along the molecular weight axis is still a problem. As for silver
staining followed SDS-PAGE, the narrow dynamic range also limited the accurate
quantity of differential proteins of the two state samples. Fig.3(A) and 3(B)
show the complete 2-DE patterns’ comparison of culture cells between two
different state human liver cancers by MSOG method. Owing to the completely
running and staining condition in parallel, not only the migrations of the two
gels’ tracking dye, but also the grayscales of the two gels by silver staining
are totally identical. Therefore, artificially differential proteins will be
diminished to the least. From the comparison between Fig.3(C) and Fig.3(D), we
can easily observe that spot 1 and spot 2 are two differentially expressed
proteins. However, spot 3 could be lost since there is only a little change in
expression between two different states if the normal method is used.
Therefore, it will greatly facilitate in finding much more functional proteins
in comparative proteomics.
2.3 The comparison of resolution power
of 2-DE patterns of the same sample between Ettan Dalt twelve and SE 600 system
Resolution power is another key parameter
for 2-DE pattern. Fig.4(A) and 4(B) displayed the comparison of the same sample
which was run on two different systems. One was run on Ettan Dalt twelve system
(Amersham Biosciences) by using MSOG method, the other on the SE 600 system
(Amersham Biosciences). The results of evaluation with ImageMaster 2D Elite 3.1
revealed that the number of spots detectable on the 2-DE pattern shown on
Fig.4(A) exceeded the number of spots present on the 2-DE pattern of Fig.4(B)
by more than 30%. From the comparison between Fig.4(C) and 4(D), same area
section of Fig.4(A) and 4(B) respectively, it is obvious that many protein
spots in Fig.4(C) which have molecular weight from 70 kD to 100 kD and pIs from
4.5 to 5.0 are lost in Fig.4(D). Moreover, a large number of high Mr proteins
in the range is hard to be effectively separated. It can be easily understood
since the 20 cm separated length of Ettan Dalt twelve system is much longer
than 15 cm valid separated length of SE 600. Although the two sides of IPG
strips were cut out about 1 cm respectively, there were no difference between
Fig.4(A) and 4(B). Although this advantage mainly results from the length of
second gel, obviously, it is not an advisable action of transferring just one
IPG strip of no more than 13 cm to 24 cm SDS gel only for higher resolution.
Therefore, in our opinion, SE 600 should completely be replaced by the Ettan
Dalt twelve system once the new method is carried out to run 7 cm, 11 cm or 13
cm IPG strips of 2-DE patterns.
2.4 Other advantages and applications
In contrast to former results with the SE
600 which can only run two SDS gels simultaneously, Ettan Dalt twelve system
can handle up to 12 large, second-dimension gels (26 cm×20 cm) in a simple,
efficient, and reproducible manner. MSOG procedure improved this advantage,
which has the ability to run up to 24 gels for 11 cm or 13 cm IPG strips and up
to 36 gels for 7 cm IPG strips simultaneously. It should be emphasized that not
all researchers support the conclusion that the longer IPG strips are used, the
better outcome will be achieved. Ducret et al.[17] found that mini 2-D
gel (7 cm IPG strip) could obtain sufficient resolution and reproducibility to
study complex biological samples while considerably increasing throughput and
lowering costs compared to larger number of samples. In other words, the 2-DE
pattern of 24 cm IPG strip may not always get much higher resolution power and
reproducibility than that of 7 cm IPG strip especially in some special complex
which only contains hundreds of protein components. The choice depends on
experimental purpose and sample source. Besides in one SDS gel, up to three
different 2-DE patterns can be intuitively compared, MSOG method farthest save
running time and reduces the consumption of chemical reagents.
Fig.3 Comparison of 2-DE patterns of cultured cells from human hepatoma under
two differential conditions [(A) and (B)] by multi-strips one gel (MSOG) method
First dimension: IPG 4－7; separation distance of each IPG strip: 120 mm, sample application by
in-gel rehydration (80 μg protein); IEF was performed on the IPGphor; second
dimension: vertical SDS-PAGE on Ettan Dalt twelve system (12.8% T constant);
silver staining. (C) and (D) are enlargements of gel areas from (A) and (B).
Three differentially expressed protein spots (spot 1, spot 2 and spot 3) are
indicated with arrows.
For instance, the old second dimensional
system consumed at least 4 liters electrophoretic buffer to run two gels, but
only 10 L were enough to achieve 36 patterns of 7 cm IPG strips simultaneously.
Obviously, it highly fits the demand of high-throughput and automation in the
proteomic research[18].
2.5 Concluding remarks
Taken together, the reproducible,
high-resolution and high-throughput 2-DE to separate complex protein mixtures
is crucial for successful proteomic research.
Fig.4 Comparison of resolving power of 2-DE patterns from the same sample
between Ettan Dalt twelve and SE 600 system
The same running conditions of first dimension: IPG strips pH 4－7; sample
application by in-gel rehydration (80 μg protein of culture cells of human
Repatoma); IEF was performed on the IPGphor. Second dimension: (A) vertical
SDS-PAGE in Ettan Dalt twelve system (12.8% T constant) by MSOG method;
effectively separated distance: 200 mm. (B) vertical SDS-PAGE in SE 600 system
by old method; effectively separated distance: 150 mm. (C) and (D) are
enlargements of gel areas from (A) and (B). The protein spots indicated by
arrowheads in (C) cannot be resolved and detected in (D).
In this paper,
we firstly put forward a new 2-DE running protocol――MSOG method that not only improved
the reproducibility, resolution power and high-throughput of 2-DE, but could be
conveniently mastered and applied by most of laboratories. Therefore, we
suggest that MSOG method is one of the choices to obtain excellent 2-DE pattern
for 7 cm, 11 cm and 13 cm commercial IPG dry strips including narrow pH and
wide pH gradient, especially when comparing the differentially expressed
proteins of different state cells, organelle and biomolecular complex. Since it
could offer a much more rapid and economical, and intuitive format, at the same
time, it is extremely useful for range finding and sample preparation
optimization.
Acknowledgements We
wish to thank Prof. ZHAO Mu-Jun for affording mouse 3T3 Cell, Dr. DENG Hai-Teng
for critical reading of the manuscript, Mrs. MAO Jun-Ling for fruitful
discussion and Amersham Biosciences, Inc. for technical assistance.
References
1 Klose
J．Protein
mapping by combined isoelectric focusing and electrophoresis of mouse tissues.
A novel approach to testing for induced point mutations in mammals.
Humangenetik, 1975, 26(3)：231－243
2 O’Farrell
PH． High
resolution two-dimensional electrophoresis of proteins. J Biol Chem, 1975,
250(10)： 4007－4021
3 Klose
J, Kobalz U. Two-dimensional electrophoresis of proteins: An updated protocol
and implications for a functional analysis of the genome. Electrophoresis,
1995, 16(6)： 1034－1059
4 Gorg
A. Two dimensional electrophoresis. Nature, 1991, 349(6309): 545－546
5 Corbett
JM, Dunn MJ, Posch A, G�rg A． Positional reproducibility of protein spots in two-dimensional
polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric
focusing in the first dimension: An interlaboratory comparison.
Electrophoresis, 1994, 15(8-9)： 1205－1211
6 Gorg
A, Postel W, Friedrich C, Kuick R, Strahler JR, Hanash SM. Temperature
dependent spot positional variability in two-dimensional polypeptide patterns.
Electrophoresis, 1991, 12(9)： 653－658
7 Lopez
MF, Berggren K, Chernokalskaya E, Lazarev A, Robinson M, Patton WF． A comparison of silver stain and
SYPRO Ruby protein gel stain with respect to protein detection in
two-dimensional gels and identification by peptide mass profiling． Electrophoresis, 2000, 21(17)： 3673－3683
8 Rabilloud
T, Strub JM, Luche S, Dorsselaer AV, Lunardi J． A comparison between Sypro Ruby and ruthenium II tris
(bathophenanthroline disulfonate) as fluorescent stains for protein detection
in gels. Proteomics, 2001, 1(5)： 699－704
9 Rabilloud
T． A comparison
between low background silver diammine and silver nitrate protein stains． Electrophoresis, 1992, 13(7)： 429－439
10 Yan JX, Wait R, Berkelman T, Harry RA,
Westbrook JA, Wheeler CH, Dunn MJ． A modified silver staining protocol for visualization of proteins
compatible with matrix-assisted laser desorption/ionization and electrospray
ionization-mass spectrometry． Electrophoresis, 2000, 21(17)： 3666－3672
11 Mortz
E, Krogh TN, Vorum H, G�rg A． Improved silver staining protocols for high sensitivity protein
identification using matrix-assisted laser desorption/ionization-time of flight
analysis. Proteomics, 2001, 1(11)： 1359－1363
12 Tonge
R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F et al.
Validation and development of fluorescence two-dimensional differential gel
electrophoresis proteomics technology． Proteomics, 2001, 1(3)： 377－396
13 Zhou
G, Li H, DeCamp D, Chen S, Shu H, Gong Y, Flaig M et al．2D differential in-gel
electrophoresis for the identification of esophageal scans cell cancer-specific
protein markers． Mol Cell
Proteomics, 2002, 1(2)： 117－124
14 Ramagli
LS．Quantifying
protein in 2-D PAGE solubilization buffers． Methods Mol Biol, 1999, 112, 99－103
15 Gorg
A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The
current state of two-dimensional electrophoresis with immobilized pH gradients.
Electrophoresis, 2000, 21(6)： 1037－1053
16 Gorg
A, Obermaier C, Boguth G, Csordas A, Diaz JJ, Madjar JJ． Very alkaline immobilized pH
gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins． Electrophoresis, 1997, 18(3-4)： 328－337
17 Ducret
A, Desponts C, Desmarais S, Gresser MJ, Ramachandran C. A general method for
the rapid characterization of tyrosine-phosphorylated proteins by mini two
dimensional gel eletrophoresis. Electrophoresis, 2000, 21(11)： 2196－2208
18 Quadroni
M, James P． Proteomics
and automation．
Electrophoresis, 1999, 20(4-5)： 664－677
___________________________________________
Received: February 12, 2003 Accepted:
April 5, 2003
This work was supported by the grants from
the National Natural Science Foundation of China (No. 39990600), Foundation of
the Chinese Academy of Sciences (No. KSCX 2-2-06) and Shanghai Sciences of
Developing Foundation (No. ODJC14018)
*Corresponding author: Tel, 86-21-54921155;
Fax, 86-21-64331090; e-mail, [email protected]