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An immobilized metal affinity electrophoresis (IMAEP) method is described here. In this method, metal ions are immobilized in a native polyacrylamide gel to capture phosphoproteins. The capture of phosphoproteins by IMAEP is demonstrated with immobilized metals like iron, aluminum, manganese, or titanium. In the case studies, phosphoproteins α-casein, β-casein, and phosvitin are successfully extracted from a protein mixture by IMAEP.
Immobilized metal affinity column (IMAC) is one of the most popular methods used to extract phosphoproteins or phosphopeptides.1–5 Here, we incorporate the principle of IMAC into affinity electrophoresis by embedding metal ion into the polyacrylamide gel. In this immobilized metal affinity electrophoresis (IMAEP) technique, the metal ion is immobilized in the gel matrix during polymerization. The entrapment of phosphoproteins is tested with many metals including iron (III) chloride, iron (II) chloride, nickel (II) chloride, manganese (II) chloride, magnesium (II) chloride, silver chloride, tin (II) chloride, scandium (III) oxide, aluminum (III) chloride, vanadium (III) chloride, gallium (III) nitrate, cupric sulfate, chromium (III) chloride, zirconium (IV) hydrogen phosphate, sodium chloride, potassium chloride, titanium (III) acetate, calcium (II) chloride, and titanium (IV) oxide nanopowder. The capture of phosphoproteins using IMAEP is demonstrated by analyzing a protein mixture of nonphosphoproteins such as human albumin, carbonic anhydrase, dephosphorylated α-casein, and human hemoglobin and phosphoproteins such as α-casein, β-casein, and phosvitin. All proteins are from Sigma (Saint Louis, MO) and used without further purification. Phosvitin, α-casein, and β-casein were chosen because they are readily available and widely used as phosphoprotein standards. Phosvitin is one of the most highly phosphorylated protein present naturally. Out of 216 amino acid residues present in the sequence of phosvitin, 109 are phosphorylated.6 Also, α-casein and β-casein have eight and five phosphorylated sites, respectively.7–8
The IMAEP gel was prepared as follows: A native 7.5% (w/v) polyacrylamide gel solution (8 mL) was poured into an empty gel cassette (Invitrogen, Carlsbad, CA) and perforated plastic strips were used to make the wells 3 × 8 × 1 mm. The gel solution was prepared by mixing 7.5 mL 30.8% stock monomer acrylamide solution (Sigma, St. Louis, MO), 7.5 mL 1.5 M Tris-HCl 4X resolving gel buffer (pH 8.8), 14.8 mL H2O, 150 μL 10% (w/v) ammonium persulfate solution (APS), and 10 μL of tetramethylethylenediamine (TEMED). Following gel formation and removal of the plastic strips, 75 μL of the native 7.5% polyacrylamide gel solution with APS, TEMED, and 1.5 μL of 1 M metal ion solution (in case of solubility problems, metal ions are either saturated solutions or dispersed nanopowder) was added to the wells and allowed to polymerize. The gel was run using 500 mL of gel running buffer (25 mM Tris, 192 mM glycine, pH 8.3) with PowerEase 500 programmable power supply. Electrophoresis was carried out at ~3 watts with a current of ~22 mA and a voltage of ~125 volts for 1.5 hours. Gels were stained either with Coomassie blue G-250 or Zn+2-reverse staining9 and documented using a digital camera and a light box.
Figure 1a shows IMAEP of three metal ions without loading the protein sample before and after electrophoresis. The colors of the metal ions including yellow (Fe+3), burgundy (Mn+2), and blue (Cu+2) stay fixed in the gel through the electrophoresis confirming that the metal ions have been immobilized. IMAEP of human albumin, carbonic anhydrase, dephosphorylated α-casein, human hemoglobin, α-casein, β-casein, and phosvitin is shown in Figure 1b–d. Data show that among the many metal ions tested here, only Al+3, Ti+3, Fe+3, Fe+2, and Mn+2 are able to catch the phosphoproteins through metal–phosphate ion-pair interaction. Since Cu+2 fails to capture any phosphoprotein, Cu+2 is used as a negative control.
To further confirm the capture of the phosphoproteins, the metal–protein complex was excised from the IMAEP gel and subjected to trypsin in-gel digestion. For the digestion,10 the protein gel was washed with 50% acetonitrile in 50 mM ammonium bicarbonate pH 8.0 and was shrunk with neat acetonitrile. The gel was then dried with a speedvac. Reduction of the disulfide bond and alkylation of the free sulfhydryl group were done by dithiothreitol and iodoacetamide in 50 mM ammonium bicarbonate, pH 8.0, respectively. After alkylation, gel was washed with 50 mM ammonium bicarbonate, pH 8.0, and then shrunk with acetronitrile and dried with a speedvac. Trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate, pH 8.0, solution at a concentration of 2 μg/100 μL was then added to the dried gel. Digestion of proteins in the excised bands was performed at 37°C for 24 hours, after which the digestion solution was extracted. ZipTips, packed with C18 resin, were used to prepare the sample for mass spectrometric analysis using cyano-4-hydroxycinnamic acid as the matrix. Samples were spotted onto a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) target and analyzed by a positive-ion Voyager-DE PRO Mass Spectrometer (Applied Biosystems, Forest City, CA) equipped with a nitrogen laser. Figure 2 shows the MALDI-TOF mass spectrum of the in-gel trypsin digest of the Al+3-protein complex from Figure 1b, lane 2. Peptide mass maps were measured at an instrument resolution of 10,000 in a reflector mode with delayed extraction over the m/z range 700–4000. The spectrum was externally and then internally calibrated. Computer software from protein prospector (http://prospector.ucsf.edu) was used to interpret the mass spectrometric results. Results show that majority of the tryptic peptides of the Al+3-protein complex are from α-casein and β-casein, which demonstrates the selective entrapment of the phosphoproteins of the IMAEP technique.
In conclusion, the IMAEP is a powerful technique for capturing phosphoproteins, which will be useful in proteomics. Among many metal ions tested here, only Al+3, Ti+3, Fe+3, Fe+2, and Mn+2 are able to catch the phosphoproteins. Also, SDS-PAGE can be used in this IMAEP method (data not shown). Other applications and gel running parameters of IMAEP are being investigated in this laboratory.
We thank the Research Resources Center at the University of Illinois at Chicago for its support.