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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(7): 611-618
CN 31-1300/Q |
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 )
Key words
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 ones laboratory for the same samples, its hard 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. Its more 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 cant 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, its impossible 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.
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___________________________________________
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]