We investigated the effect of pulsing on the protein band intensity across a range of molecular masses; twelve protein species were separated by 14 % PAGE. The results showed that FIGE increased band intensities (Figure 2a I) compared with the CFE control (Figure 2a II). This notion was further investigated quantitatively using densitometry (Figure 2b). Protein peak heights were increased two-fold for protein molecular mass lower than 66 kDa. The enhancement of intensity for protein with molecular mass larger than 97 kDa was not obvious at this gel percentage and under these pulsing conditions. The increase in band intensities may be a result of reduced band diffusion and trapping of protein molecules in the gel matrix during migration. Diffusion became apparent when a control gel was run and allowed to rest for a further 12 hours within the glass plates at 20°C prior to staining (see Figure 2a III). The differences in band intensities were not caused by artifacts in staining and scanning as the parameters for staining and scanned were identical within the same set of experiments. Diffusion was not due to an increase in temperature as the temperature of gel during pulsing was 5°C higher than that of constant field controls. It was apparent that FIGE reduced band diffusion over a longer run time, since the run time required in this case was approximately 13-fold greater than that of the control. A general reduction in the full-width-half-maximum measure as a result of pulsing with respect to control was observed, suggesting that pulsing improved the overall efficiency of protein separation (Table 1). The improvement in separation efficiency becomes less obvious for relatively high molecular mass (≥ 200 kDa) and relatively low molecular mass (≤ 21.5 kDa) proteins. In summary, protein band intensity could be enhanced using FIGE.

We studied the effects of different ratios of forward pulse times (ta) and backward pulse times (tr) on protein relative mobility under different percentages of PAGs. Relative mobility was defined as the ratio of the migration distance of the target protein to the migration distance of the dye front (R_f). The relative mobility was abbreviated as the percentage of R_f (% R_f). The % R_f did not involve run time as it measured the position of the proteins when the dye front reached the same position regardless of the run time. Biphasic changes of % R_f were observed at 6%, 10%, and 14% of PAGs (Figure 3a to 3c) under the tested pulsing conditions. More reduction in % R_f was observed with protein size of 36 kDa to 66 kDa. The reduction decreased when protein species were more massive than 66 kDa. The apex (i.e., the molecular mass that showed the maximum difference in % R_f) shifted to smaller molecular mass species when the gel percentage increased (Figure 3a, 3b, and 3c). Higher frequencies pulse cycles (ta/tr = 60/16 msec, 150/40 msec, and 300/80 msec) resulted in more obvious differences in relative mobility compared to low frequency pulse cycle (ta/tr = 900/240 msec) (Figure 3a to 3b). This change upon different pulse cycles was observed in the repeat run (Figure 3a and 3b). However, we did not see a simple linear relationship changes in relative mobility to gel concentration, pulse frequency, and protein size. We observed negative values in differences of % R_f (Figure 3c and 3d) relative to CFE at high percentages of PAGs, suggesting that the target proteins revealed a longer migration distance. However, we did not observe a consistent pattern of altered relative mobility at 18 % PAG. The migration of proteins in such a high polyacrylamide concentration was apparently a stochastic process (Figure 3d). In summary, the results showed that the maximum difference in relative mobility was not a simple linear relationship with molecular mass, pulse cycles, and gel percentage.

We tested the applicability of FIGE in proteomics studies such as the 2D PAGE analysis of rat liver lysates with pulsing in the second dimension. The results were subsequently analyzed by image densitometry for spot number and intensity. Image analysis concluded that pulsing increased the overall number of spots by more than 20% as well as a general increase in spot intensity over CFE (Figure 4a and 4b). The total number of spots detected after CFE and FIGE were 477 and 611, respectively. In another set of experiments, gels were stained with Coomassie blue stain and the total number of detectable spots after CFE and FIGE were 190 and 240, respectively (data not shown). These results translated into a 26% increase in the total number of detectable spots.

We next studied the level of enhancement in protein spot intensity using densitometric analysis of selected spots. The enhancement of spot intensity ranged from 12 % to more than 300% on selected protein species (Table 2). We confirmed that this enhancement was not a result of sample loading or focusing error by including an internal control sample spot. Densitometry results of spot A (an internal control of a molecular mass standard protein) showed very similar densitometric values (31998.386 for control and 33593.516 for pulsing; Table 3); these results therefore allowed the dismissal of the observed pulsing effects as a consequence of handling error.

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.

We further explored the application of FIGE in the separation of protein complexes in native gel conditions. We discovered that the purified E. coli GroEL 14-mer complex (840 kDa) could effectively condense and migrate into the gel matrix under pulsing conditions of 900/240 msec (Figure 5a right panel). In contrast, the GroEL 14-mer complex migrated in a stochastic fashion under constant field conditions (see Figure 5a left panel). The difference was not due to an inappropriate buffer composition as the appearance of the GroEL 14-mer complex band was sharper than that in the CFE control. In addition, the protein bands of the MW protein markers were sharper after pulsing than the diffused bands in the control (see Figure 5a). Next, we tested a complex sample using MCF-7 cell line nuclear extract under native PAGE conditions. The results revealed that pulsing effectively condensed protein complexes greater than 2000 kDa in size, with a better resolution and band intensity (Figure 5b). In summary, FIGE might also be used to resolve very high molecular mass (MW > 1000 kDa) protein complexes by native PAGE.

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).

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 1. 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 (R_f) of these proteins under different pulsing methods (different ratios of forward pulse times, ta, to different reverse pulse times, tr) 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 25, the ta/tr 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 26. Escherichia coli chaperonin GroEL was purified as previously described 27 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.

Mouse liver protein lysate was prepared as described previously 28. 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 ta/tr 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).

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 2930. 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.