Increase in local protein concentration by field-inversion gel electrophoresis
© Tsai et al; licensee BioMed Central Ltd. 2007
Received: 03 February 2007
Accepted: 26 September 2007
Published: 26 September 2007
Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).
Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.
The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.
S odium d odecyl s ulfate-p olya crylamide g el e lectrophoresis (SDS-PAGE) is an indispensable technique in protein separation. This technique has only changed marginally over the past three decades . Despite its popularity, SDS-PAGE, as well as native PAGE for protein separation, suffers the basic limitation of band broadening by diffusion and trapping of biomolecules in gel matrices. Nevertheless, protein separation by SDS-PAGE interfaced with mass spectrometry (MS) has currently emerged as the method of choice in the forefront of proteomics. Thus, new tools for upstream gel electrophoresis that can improve protein separation efficiency and subsequent detection will possibly lead to new discoveries in downstream processes.
Pulsed-field gel electrophoresis (PFGE) is an elegantly simple and universally accepted technique for the separation of large DNA molecules . Several variant forms of PFGE with different experimental electrophoretic configurations now exist [3–5]. One variant is the orthogonal-field-alternation gel electrophoresis (OFAGE) technique . Another variant is the field-inversion gel electrophoresis (FIGE) in which net forward molecular migration is achieved by either employing a longer net forward field time or a higher forward field strength compared with the reverse direction . Application of FIGE in the separation of large DNA molecules was first reported two decades ago . Among the various pulsed-field gel electrophoresis techniques, FIGE is likely the easiest to perform with minimal special equipment that generates a highly uniform electric field across the gel . Both methods have been optimized to maximize efficiency (band width in the dimension of separation) and selectivity (distance between the center of two bands) for large DNA molecules. Systematic investigations of FIGE on the separation of DNA molecules in agarose gels were previously reported [6–10]. However, published studies of the use of FIGE for protein separation are still lacking.
Attempts were made to enhance the separation of proteins by means of PFGE . Subsequent applications of PFGE were used to resolve either specific model protein species [12, 13] or high molecular mass muscle myosin heavy chain isoforms . However, these approaches were limited to the application of alternating cycles of on-and-off electric fields across a slab gel. This approach inevitably allowed diffusion to occur during the off times. Unwanted band broadening as a result of diffusion compromises general separation efficiency.
There are currently three models to explain molecular migration during PAGE: I) The Extended Ogston (EO) model assumes an overall sphere-like conformation for native or small proteins in which mobility is a function of available gel pores in a regular lattice fashion . II) The reptation model assumes that molecules go through a rather disordered matrix, such as polyacrylamide , accounting for the snake-like movement of beads-on-a-string shaped polymers, such as protein-SDS complexes. These two models only apply to polypeptides within a certain molecular mass (MW) range at a given cross-linked polyacrylamide concentration. Any deviation from this linearity implies a change or transition in molecular shape (i.e., the radius) and net charge. III) The door-corridor (DC) model explains the behavior of polypeptides above a critical MW where the relative mobility of a protein becomes independent of the cross-linked polyacrylamide concentration . Effective trapping of migrating molecules by the matrix predominates in this model and electrokinetic energy is required to overcome the trapping effect .
The intensities of protein bands increase upon pulsing
Peak variance (σ2) of proteins separated as in Figure 2a I by FIGE (Pulsing) and Figure 2a II by CFE (Control).
σ2 (Peak variance*, mm2)
1.85 ± 0.07
0.41 ± 0.05
0.85 ± 0.12
0.87 ± 0.07
1.32 ± 0.15
4.51 ± 0.24
4.51 ± 0.38
8.51 ± 1.00
4.51 ± 0.15
1.52 ± 0.05
1.62 ± 0.05**
1.62 ± 0.15
2.63 ± 0.13
5.86 ± 0.28
5.86 ± 0.45
6.94 ± 0.42
4.51 ± 0.20
1.52 ± 0.11
Altered protein relative mobility depend on pulse time settings and PAG percentages
Pulsing during the second dimension separation enhances overall data output of 2D PAGE
Selective comparison of densitometric results from 2D PAGE analyses under FIGE versus CFE conditions
Percent Increase in Intensity (%)
Malate dehydrogenase, cytoplasmic
Glutathione S-transferase kappa1
Fatty acid-binding protein, liver
Selective comparison of MS results of protein spots identified from 2D PAGE under FIGE versus CFE conditions
Malate dehydrogenase, cytoplasmic
Glutathione S-transferase kappa1
Fatty acid-binding protein, liver
The spacing between spots 3 (37.2 kDa) and 4 (36.5 kDa) in the CFE control (Figure 4a) was much narrower than the spacing of matching spots in the pulsed condition (Figure 4b). We took into consideration that the migration distance of the dye front was the same for both cases. This observation suggested that FIGE might be used to separate proximal MW protein species or isoforms within a particular MW range under the influence of a given PAG concentration and pulse regime.
To confirm that the increase in spot intensity was not an artifact of pulsing, we further analyzed spots from identical positions between the pulsed and control gels with peptide sequencing using tandem mass spectrometry (MS). The MS results on nine selected protein spots revealed that pulsing increased the overall protein identification scores over control (Table 3). Weak intensity spots in CFE became apparent after pulsing (spots 5 to 9 in Figure 4a). The MS data allowed identification of these apparent spots with high ion scores (see Table 3 spots 5 to 9). These results confirmed the observation of increased band intensities as a consequence of increase in local protein concentrations of the same migrating protein species. In summary, pulsing in the second dimension enhanced local protein concentration during migration and, thus enhancing downstream processes such as mass spectrometry analysis.
Native PAGE upon pulsing improves data output
We report here the first attempt to investigate certain common parameters on the separation of protein species by FIGE. These parameters include protein size, pulsing time ratios, pulsing frequencies, and cross-linked polyacrylamide percentage.
Despite longer running time and a higher running temperature, the protein species under FIGE conditions showed better separation efficiency than CFE. Higher intensity of protein bands or spots after pulsing could be explained by reduction of trapping of protein molecules by gel matrix along the direction of migration. When initial finite populations of polypeptide molecules comigrate in the direction of the electric field, a fraction of polypeptide molecules are continuously trapped along the gel matrix. FIGE relaxes and releases the trapped polypeptide molecules upon field reversal and thus renews the migration path. This field reversal increases the probability of the same polypeptide species to comigrate as a coalesced population. The detrapping hypothesis was in concordance with the observation in Figure 2a) and 2b) and Figure 5a) that protein intensity was consistently higher in pulsed condition despite the same amount of proteins were loaded. However, more massive proteins experience more constriction in migrating through the gel matrix and hence they are less likely to be detrapped upon pulsed condition. This hypothesis may be used to explain the similar protein intensity of more massive protein peaks A, B, and C in Figure 2b. Higher intensity could not be addressed by diffusion disentanglement process because it is dynamically time consuming [18, 19], leading to diffusion to all directions from the origin. Therefore, one should expect lower local protein concentration by diffusion upon longer time or higher temperature. The consistent observation of enhanced protein intensity irrespective of running temperature suggests that the increased local concentration of protein was a genuine concentrating effect by FIGE.
Improved separation efficiency was also observed in the nonlinear focusing of DNA macromolecules according to the temporal ratchet hypothesis in which a short reverse pulse or spike reduced band broadening . This implies that a similar operating mechanism may operate in the separation of protein molecules in PAGs. Furthermore, it was shown that asymmetric periodic electric fields led to the self-focusing of DNA within a gel . This principle achieved a zero-integrated field electrophoresis phenomenon . Similarly, every reversion of the electric field in FIGE generates a drift velocity. In the first case, where t a is longer than t r, the time-averaged drift velocity will be in the forward direction. At an intermediate point, where the forward and reverse drift velocities are equivalent and a zero drift velocity is attained, the molecules are focused in a condition termed virtual trap . Intuitively, a greater focusing effect can be observed if the value of t a/t r is closer to 1. Our preliminary trial found that the best range of ta/tr lied between 1.07 and 3.75. The deviation from the best theoretical ta/tr ratio of 1 may be addressed by the fact that this process is in a non-equilibrium state. A dynamic interaction of proteins with fluxes of electrolytes in the buffer may add additional mobility driving force to the target protein molecule. A future study should involve the detailed investigation of the kinetics of electrolyte fluxes during pulsing.
Pulsing condition in general retards the relative mobility of extremely low mass proteins and accelerate the relative mobility of medium mass proteins (Figure 2b) under the right PAG concentration. In contrast, the relative mobility of extremely massive proteins remains unchanged. The shift in relative mobility cannot be simply explained by differences in thermodynamic energy generated during electrophoresis. The experimental temperature during the band intensity analysis was higher under pulsing conditions (Figure 2) while the temperature for generating Figure 3 data was controlled at 10°C. A consistent mobility shift was observed when pulsing was applied even under different temperatures, suggesting that temperature may not play a major role in the phenomenon.
We attempted to apply the three models to explain the altered relative mobility of polypeptides in FIGE. The EO model can be applied to the proteins with mass smaller than i) 66 kDa in 6% PAG concentration or ii) 36 kDa in 10% PAG concentration (Figure 3a and Figure 3b). The assumption of the EO model is that protein-SDS complexes are sphere-like structure. They migrate through relatively large pores in the gel matrix. Pulsing causes the protein molecules to reorientate in a periodic fashion. The process of reorientation leads to a reduction in relative mobility. As protein mass increases so does the reorientation time, resulting in a reduced relative mobility. Reptation model can be used to explain the migration of proteins with mass smaller than 100 kDa through the gel matrix at 14% PAG concentration (Figure 3c). In this model, the conformation of SDS-protein molecules may appear as a semi-chain like structure. Proteins adapt the most thermodynamically favorable conformations during migration through the gel matrix. The favorable conformation leads to an increase in relative mobility with increased detrapping. Apparently, the detrapping effect was plateaued for proteins with mass larger than 100 kDa because highly massive proteins (MWs higher than 100 kDa in 10 % PAG concentration) experience greater resistance from the relatively small pores in the gel matrix (Figure 3b). Their mobilities are constricted by gel matrix and therefore pulsing does not cause any significant changes in the relative mobility. A minimal decrease in the relative mobility may be the consequence of mostly localized reorientation under the given pulse regimes. At 18% PAG concentration, detrapping of polypeptides by FIGE is not realized. The average pore size of the gel is too small for the polypeptides to overcome local constriction even in the presence of pulsing. Polypeptides migrate through the gel according to the DC model in this high percentage gel condition. This observation is consistent with the behavior of DNA migration in previous study . The relative mobility shift observed upon pulsing is more likely to be contributed by the interplay of the three protein migration models mentioned earlier. However, the inadequacy of all the models lies in the assumption of the properties of gel matrix. All models consider the gel to be either a rigid lattice of tubes, possibly allowing small variation in the size, or a collection of point obstacles in two dimensions. Study of molecular structure of polyacrylamide gel revealed that the pores exhibited certain degree of elasticity . The change in properties of gel matrix under different pulsing conditions was not known. The elastic properties of gel matrix may contribute to a faster relative mobility of the protein molecules in one pulsing setting but a slower relative mobility in another pulsing setting.
FIGE suffers certain disadvantages when compared to other PFGE platform. Although simple in instrumental set up, FIGE suffers the limitation of lower resolving power than contour-clamped homogenous electric field (CHEF) electrophoresis. The resolution of very complex genomics structure can be achieved using two-dimensional CHEF electrophoresis . This lower resolution of FIGE may be explained by the shorter migration route and suboptimal reorientation of migrating molecules. We have not studied in detail the separation of two protein species of similar molecular masses. In theory, the enhanced separation of two protein species with similar molecular masses can also be achieved with appropriate gel concentrations and pulsing conditions. It remains to be tested the lower limit of minimal molecular mass differences of two protein species that can be resolved using FIGE under optimized conditions. Another unaddressed general issue of PFGE linked to the increase in the width of band as seen transverse to the direction of migration. We observed a more prominent widening of spots at the left and right ends of 2D PAGE (Figure 4a and 4b). It was attributed by the difference in chain migration direction and the average direction of motion along the molecules . Association of this difference with the pulsing condition needs further investigation. We can foresee that further improvements on limiting the transverse molecular motion can further improve the local concentration of protein species.
The present investigation tried to set the motion of establishing the potential application of FIGE in proteomics. If needed, further comprehensive analyses of upstream protein separation may be performed. We limited our investigation only to varying the t a/t r ratios under conditions of a constant electric field in different percentages of PAGs. It is, by no means, an exhaustive list of parameters tested. The present investigation suggests that FIGE is a bona fide protein separation technique that has the potential to complement existing CFE techniques in the area of SDS-PAGE, 2D PAGE, and native PAGE. FIGE aids in the reduction of thermo-diffusion within gels. Furthermore, preliminary results also demonstrated that FIGE could enhance the recovery of proteins during electroelution (data not shown). In future perspective, whether it will enjoy widespread acceptance as its counterpart in DNA analysis will depend on continued improvements and optimizations of different parameters. This further development requires parallel exploitation of experimental and computational modeling approaches to understanding FIGE in proteomics and eventually lead to the advent of new applications.
FIGE enhances protein separation by improving local protein concentrations during SDS-PAGE or under native gel conditions. The increased local protein concentration thus improves the observable intensity of protein species in PAGs and also improves the success rate of downstream peptide sequencing using MS. Taken together, FIGE can be used to complement constant field gel electrophoresis for better protein separation and detection.
A schematic representation of the pulsing circuitry in conjunction with an external electrophoresis unit is provided in Figure 1. A picture of the prototype can be found in supplementary data (see additional file 1). Forward and reverse switching of the electric field supply to the gel was achieved by interfacing the voltage supply (Power Pac 1000 power supply, Bio-Rad, Hercules, CA) with an 240 V alternate current (AC) relay (MY2, OMRON, Japan) that could handle a current of 5 Amp. The rate of the forward and reverse switching was controlled by a MD4E-AP programmable 110–240 V AC switching device (Fuji Electric, Japan) that was able to deliver pulses as short as one msec. Demonstration of the different settings can be found in additional file 2 and additional file 4. The performance of this simple instrumentation in regard to square wave form, amplitude, length, and stability of the pulses was checked and ascertained by a Tektronix oscilloscope model TDS 1000 (see additional file3 and see additional file 5).
One-dimensional (1D) PAGE
We adopted a symmetric voltage method by varying both the forward and reverse pulse lengths in different percentages of cross-linked polyacrylamide. FIGE is herein referred to as pulsing and constant field electrophoresis (CFE) is herein referred to as control. Protein standards (2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL) from 2.5 kDa to 200 kDa (Mark12, Invitrogen Inc., Carlsbad, CA) were used as model proteins. A mini-PROTEAN 3 cell system (Bio-Rad Laboratories Inc., Hercules, CA) was used for the self-cast gel. The Criterion cell system (Bio-Rad Laboratories Inc., Hercules, CA) was employed for pre-cast gels used in the second dimension during 2D PAGE. Working solutions for gel casting were prepared from a 30% (w/v) acrylamide stock solution with 37.5:1 and 29:1 ratios of acrylamide to N, N'-methylene-bis-acrylamide for SDS-PAGE and native-PAGE, respectively. Percentages of slab gels used for protein separation were selected based on the sizes of the different proteins under investigation with reference to the migration chart for the protein standards obtained from the vendor (Bio-Rad Laboratories Inc., Hercules, CA). Final compositions of working solutions for the separating gel were based on the Laemmli buffer system . A 1 × SDS running buffer or tank buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) was used with the Multitemp temperature control (GE Healthcare, Piscataway, NJ) at 10°C except as otherwise mentioned. All samples were mixed with 4 × sample loading buffer [0.24 M Tris- HCL, pH 6.8, 2% SDS, 3 mM bromophenol blue, 50.4% glycerol, 0.4 M dithiothreitol (DTT)] at a final concentration of 1 ×. All samples were heated to a temperature of 95°C for 1 minute prior to loading. All gels were run at 200 V until the dye-front reached the end of the gel. The temperature of the running buffer bathing the gel was taken at the beginning and at the end of each run. Subsequently, plots of the relative mobilities from the dye front (Rf) of these proteins under different pulsing methods (different ratios of forward pulse times, t a, to different reverse pulse times, t r) and different percentages of cross-linked polyacrylamide were examined. Based on a series of trial and error experiments (data not shown) coupled with theoretical calculations , the t a/t r ratios tested were between 1.07 and 3.75. Gels were stained with colloidal Coomassie blue (Simply Blue, Invitrogen Inc., Carlsbad, CA) according to the vendor's instructions and subsequently scanned using the Labscan system (GE Healthcare, Piscataway, NJ) at a resolution of 600 dpi.
In the case of native PAGE, SDS was omitted from the working solution as described before . Escherichia coli chaperonin GroEL was purified as previously described  and samples were stored at -86°C until use. Native 2–5% polyacrylamide minigels were cast using the Bio-Rad mini-Protean 3 multi-casting chamber (Bio-Rad Laboratories Inc., Hercules, CA) and gradient former (Model 485, Bio-Rad Laboratories Inc., Hercules, CA). Treatment of samples prior to loading was similar to those for SDS-PAGE except that SDS, DTT, and heating were omitted.
Two-dimensional (2D) PAGE
Mouse liver protein lysate was prepared as described previously . Protein concentrations were measured using the MicroBCA method (Pierce, Rockford, IL) with bovine serum albumin as a standard. Typically 100 μg of liver sample were resuspended in a final volume of 200 μL for the 11 cm ReadyStrip IPG strip, (immobilized non-linear pH 3–10 gradient from Bio-Rad) with rehydration solution (8 M urea, 2 M thiourea, 1% w/v CHAPS, 20 mM DTT, and 0.5% v/v Pharmalyte 3–10). The strips were then covered with mineral oil. Rehydration and isoelectric focusing (IEF) were performed in an IPGphor apparatus (GE Healthcare, Piscataway, NJ) at 20°C. The strips were actively rehydrated for 12 hr at a maximum of 30 V and subsequently focused using the following four steps: 500 V for 500 Vhr, 3000 V for 6000 Vhr, 5000 V for 10 kVhr, and maintained at 8000 V for 60–100 kVhr. The IPG strips were equilibrated twice for 20 min after IEF with gentle shaking in 10 mL equilibration buffer (50 mM Tris-HCl, pH 6.8, 6 M urea, 30% glycerol, 2% (w/v) SDS, and trace bromophenol blue). Two percent (w/v) DTT (Sigma Inc., St Louis, MO) was added to the first equilibration step followed by the addition of 2.5% (w/v) iodoacetamide (Sigma Inc., St Louis, MO) into the second equilibration step. The IPG strips were placed on top of the SDS pre-cast gels and sealed with 0.5% (w/v) agarose in SDS electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS) before the second-dimensional separation. Precision protein standards (Bio-Rad Laboratories Inc., Hercules, CA) were used as molecular mass markers. Criterion Tris-HCl precast gels contained 10–20% cross-linked polyacrylamide with 1.0 mm thickness and a 2D well for an 11-cm IPG strip (Bio-Rad Laboratories Inc., Hercules, CA). The voltage was kept constant at 200 V and the temperature was kept at 10°C. The run was stopped when the dye front reached the bottom of the gel. The t a/t r was set at 400/100 msec for FIGE in the second dimension. Gels were stained with Simply Blue (Invitrogen Inc., Carlsbad, CA) and were subsequently scanned using the Labscan system (GE Healthcare, Piscataway, NJ) at 600 dpi. SYPRO-Ruby (Bio-Rad Laboratories Inc., Hercules, CA)-stained gels were imaged with the molecular imager FX system from Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA).
Image and data analyses
Scanned 2D gel images were analyzed using either PDQuest 2D gel analysis software (Bio-Rad Laboratories Inc., Hercules, CA) or Z3 2D analysis software (Compugen, Israel). Scanned 1D gel images were analyzed using Quantity One 1-D analysis software (Bio-Rad Laboratories Inc., Hercules, CA).
Separation efficiency (i.e., band width or peak width, w, in the direction of separation) was obtained from graphic output employing the Quantity One software. The full-width-half-maximum (FWHM) was measured on the Quantity One graphic output. Assuming a Gaussian distribution, peak variance, σ2, related to FWHM as; σ2 = (FWHM)2/5.54
Protein bands or spots of interest on gels were excised and cut into 1- to 2-mm2 pieces. All gel pieces were washed in ultra-pure water and were subsequently subjected to in-gel trypsin digestion [29, 30]. The resulting peptides from each sample were extracted from the gel pieces twice with 50 μL of 50% (v/v) acetonitrile/0.1 % (v/v) trifluoroacetic acid and the supernatants were pooled. The extracted peptides were dried in a Speed-Vac prior to dissolution in 5% formic acid for LC-MS/MS analysis.
Typically 20–40 μL of each digest was applied to an Agilent 1100 series Nano-HPLC coupled to an Agilent LC/MSD XCT Plus ion trap mass spectrometer. Briefly, peptides were trapped on a Zorbax 300SB-C18 enrichment column (Agilent, Germany) before eluted onto a column packed with 5-μm Magic C18 particles (Michrom BioResources, Auburn, CA). The C18 column output led to an integrated, 15-μm orifice, fritted nanospray source (Agilent Technologies, Inc. Santa Clara, CA). The Nano-LC was operated in micro mode at a flow rate of 300 nL/min. The gradient was maintained at 5% buffer B (0.1% formic acid in acetonitrile) for 2 minutes before increasing to 40% buffer B at 40 minutes. The gradient was further increased to 60% B at 45 minutes, 95% B at 50 minutes, and maintained for 3 minute before decreasing to 5% B for another 2 minutes for equilibration. The MS was operated at a spray voltage of 1.5 kV, with dry gas (200°C) flowing at 2.0 L/min. MS/MS was performed in a data-dependent manner in that three of the most intense peaks from each MS spectrum were selected for subsequent MS/MS analysis. Active exclusion was activated after 1 spectrum and released after 60 seconds.
The mass spectra were later processed using Agilent Data Analysis software. Compounds were first created with selection criteria of a signal/noise threshold of 5 and a relative intensity and area threshold of 10%. The spectra were subsequently deconvoluted and exported as MASCOT generic files for searching the SwissProt database via an in-house MASCOT server. The taxonomical criterion was set to metazoan (animals) with a maximum of 2 missed cleavages using trypsin. Carbaminomethylation was set as the only fixed modification and tolerance was set to 2.0 Da and 0.8 for peptides and MS/MS values, respectively.
- The abbreviations used are:
FIGE, f ield-i nversion g el e lectrophoresis
pulsed-field gel electrophoresis
- 2D PAGE:
two-dimensional polyacrylamide gel electrophoresis
- t a:
forward pulse time
- t r:
reverse pulse time
- σ2 :
The authors would like to thank Rangdong Yuan for during the initial phase of the study, Carolyn Pena for comments on the manuscript, and the Agency for Science Technology and Research (ASTAR) of Singapore for financial support.
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