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A two-dimensional immobilized metal affinity electrophoresis method is described here. In this method, ferric ions are immobilized in the second-dimensional polyacrylamide gel to extract the phosphoprotein β-casein from a mixture containing proteins with a broad range of pI and MW. Native 7.5–15% gradient tris-glycine gel with SDS tris-glycine gel running buffer are used so that proteins can be separated according to their molecular mass in the second dimension.
One-dimensional immobilized metal affinity electrophoresis (1-D IMAEP) is a technique developed in our laboratory1,2 to capture phosphorylated proteins. In 1-D IMAEP, metal ions such as ferric ion (Fe+3) and manganese ion are embedded in a distinct area of a native PAGE to entrap phosphoproteins. To extend the usefulness of the 1-D IMAEP method, this technique is now incorporated into the 2-D SDS-PAGE,3–5 which allows for better separation, identification, and quantification of larger numbers of proteins than the 1-D SDS-PAGE. In 2-D SDS-PAGE, proteins are resolved on a polyacrylamide gel using IEF in the first dimension, which separates proteins according to their pIs, followed by electrophoresis in a second dimension in the presence of SDS, which separates proteins according to their molecular masses. In 2-D IMAEP, the Fe+3 is incorporated on top of the second dimensional polyacrylamide gel to capture and immobilize phosphoproteins. These captured phosphoproteins can be readily identified using in-gel trypsin digestion and mass spectrometry. Besides enrichment of phosphoproteins such as the most popular method, metal ion affinity chromatography,6,7 where phosphoproteins or phosphopeptides are immobilized on a metal affinity column, IMAEP also separates the phosphoproteins by their pIs.
The first dimension of the 2-D IMAEP was performed as follows: a protein mixture (10 μg each protein) of amyloglucosidase from Aspergillus niger (pI 3.6, MW 100 kDa), β-casein from bovine milk (pI 4.6, MW 24 kDa), human albumin (pI 5.2, MW 66 kDa), carbonic anhydrase from bovine erythrocytes (pI 6.6, MW 30 kDa), and lysozyme from chicken egg white (pI 9.3, MW 14 kDa) was dissolved in 120 μl 2-D sample buffer (8 M urea, 2 M thiourea, 4% CHAPS, 2 mM EDTA, pH 8.0, 250 mM DTT, 2 mM tributyl phosphine, 0.5% ampholytes, and a trace of bromophenol blue). The protein solution was used to rehydrate a 7-cm immobilized pH gradient (IPG) pH 3–10 strip (GE Bioscience, Piscataway, NJ, USA) overnight. The first dimension IEF was conducted at room temperature on the PROTEAN IEF cell (Bio-Rad Laboratories, Hercules, CA, USA) for a total of ~50,000 volt-hours. Before the second-dimensional SDS-PAGE electrophoresis, IPG strips were equilibrated with an equilibrium solution containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, a trace of bromophenol blue, and DTT (1% w/v) for 20 min, followed by a second equilibration for 20 min in the same equilibrium solution containing iodoacetamide (2.5% w/v) instead of DTT.
The second dimension 7.5–15% gradient IMAEP gel was prepared as follows: native 7.5% (w/v) and 15% (w/v) polyacrylamide gel solutions (3.9 ml) were poured into light and heavy solution reservoir chambers of a gradient gel maker, respectively. The gel solution was prepared by mixing 30.8% stock monomer acrylamide solution (Sigma Chemical Co., St. Louis, MO, USA), 1.5 M Tris-HCl 4× running gel buffer (pH 8.8), H2O, 10% (w/v) ammonium persulfate solution (APS), and tetramethylethylenediamine (TEMED). The second-dimension gel was cast in an empty gel cassette (Invitrogen, Carlsbad, CA, USA), and perforated plastic strips were used to make one larger well of 70 × 8 × 1 mm and one smaller well of 2 × 8 × 1 mm for the protein maker. Following gel formation and removal of the perforated plastic strips, 525 μl native 7.5% polyacrylamide gel solution with APS, TEMED, and 7 μl 1 M Fe+3 solution was added to the larger well and allowed to polymerize. In the control, normal 2-D-PAGE, 7 μl 1 M Fe+3 solution was not added. The gel was run using 500 ml gel running buffer (25 mM Tris, 192 mM glycine, pH 8.3) with PowerEase 500 programmable power supply (Invitrogen). Electrophoresis was carried out at ~3 watts with a current of ~22 mA and a voltage of ~125 volts for 1.5 h. Gels were stained with coomassie blue G-250 or Pro-Q diamond phosphoprotein gel stain (Molecular Probes, Eugene, OR, USA) and documented using a digital camera and a light box or a FX fluorescent imager (Bio-Rad Laboratories). Pro-Q diamond phosphoprotein gel staining is a method for selectively staining phosphoproteins in polyacrylamide gels. Briefly, the gel was fixed in 50% methanol and 10% acetic acid, washed with ultrapure water, and stained with Pro-Q diamond phosphoprotein gel stain. After staining, the gels were destained with 20% acetonitrile, 50 mM sodium acetate, pH 4, and then washed with ultrapure water. Gels were visualized using excitation at λ = 532 nm and 550 nm long-pass filter of the laser-based, gel-scanning Molecular Imager FX.
Fig. 1a shows a normal 2-D-PAGE of the protein mixture of amyloglucosidase, β-casein, human albumin, carbonic anhydrase, and lysozyme. The corresponding Fe+3 2-D IMAEP is shown in Fig. 1C. Data show that Fe+3 ions immobilized on top of the gel are able to catch the phosphoprotein β-casein through metal-phosphate ion-pair interaction, and the β-casein freely migrates down in the normal 2-D-PAGE.
To further confirm the capture of the phosphoproteins, the 2-D IMAEP was stained with Pro-Q diamond phosphoprotein gel stain (Fig. 1B and D). β-Casein was observed in the normal 2-D-PAGE (Fig. 1B). As β-casein forms a Fe+3-phosphate complex in the IMAEP gel, it was not stained by Pro-Q in the IMAEP gel (Fig. 1D). The Fe+3-protein complex was excised from the IMAEP gel and subjected to trypsin in-gel digestion. Tryptic peptides were analyzed by a MALDI-TOF/TOF 4700 Proteomics mass spectrometer (Applied Biosystems, Foster City, CA, USA). Results (data not shown) show that the majority of the tryptic peptides of the Fe+3-protein complex is from β-casein, which demonstrated the selective entrapment of the phosphoproteins by the 2-D IMAEP technique.
In conclusion, the 2-D IMAEP is a powerful technique for capturing phosphoproteins, which can be useful in phosphoproteomics. Such capture methods are vital for understanding biological processes, as phosphorylation is involved in many cellular activities, such as signal transduction, gene expression, cell cycle progression, cytoskeletal regulation, and energy metabolism.8,9 The 2-D IMAEP is simple, easy, and inexpensive to perform. Also, the enriched phosphoproteins in the gel are compatible with the downstream protein identification by mass spectrometry.
We thank the support of the Research Resources Center at the University of Illinois at Chicago.