Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins*S

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn2+ or Mn2+) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn2+- and Mn2+-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn2+-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn2+-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE).

Phosphorylation is a fundamental covalent post-translational modification that regulates the function, localization, and binding specificity of target proteins (1,2). Organisms utilize this reversible reaction of proteins to control many cellular activities, including signal transduction, apoptosis, gene expression, cell cycle progression, cytoskeletal regulation, and energy metabolism. Abnormal protein phosphorylations are deeply related to carcinogenesis and neuropathogenesis. Methods for determining the phosphorylation status of proteins are thus very important with respect to the evaluation of diverse biological and pathological processes.
Recently we have reported that a dinuclear metal complex (i.e. 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) acts as a novel phosphate-binding tag (Phos-tag) 1 (commercially available at www.phos-tag.com) in an aqueous solution at a neutral pH (e.g. K d ϭ 25 nM for phenyl phosphate dianion) (4). The Phos-tag (see Fig. 1) has a vacancy on two metal ions that is suitable for the access of a phosphomonoester dianion as a bridging ligand. The resulting 1:1 phosphate-binding complex, ROPO 3 2Ϫ -(Zn 2ϩ -Phostag) 3ϩ , has a total charge of ϩ1. The anion selectivity indexes of the phenyl phosphate dianion against SO 4 2Ϫ , CH 3 COO Ϫ , Cl Ϫ , and the bisphenyl phosphate monoanion at 25°C are 5.2 ϫ 10 3 , 1.6 ϫ 10 4 , 8.0 ϫ 10 5 , and Ͼ2 ϫ 10 6 , respectively. These findings have contributed to the development of procedures for MALDI-TOF-MS for the analysis of phosphorylated compounds (e.g. phosphopeptides and phospholipids) (5)(6)(7), IMAC for the separation of phosphopeptides and phosphorylated proteins (8), and surface plasmon resonance (SPR) analysis for reversible peptide phosphorylation (9). In this study, we demonstrated two novel applications of the Phostag molecules. One is the chemiluminescence detection of whole phosphorylated proteins on electroblotting membranes using biotinylated Zn 2ϩ -Phos-tag and HRP-SA. Another is simple SDS-PAGE for the separation of a phosphorylated protein and the corresponding nonphosphorylated one where polyacrylamide-bound Mn 2ϩ -Phos-tag was used as a phosphate-binding moiety.
Apparatus-IR spectrum was recorded on a Horiba FT-710 infrared spectrometer with a KCl pellet (Real Crystal IR Card) at 20 Ϯ 2°C. 1 H (500-MHz) and 13 C (125-MHz) NMR spectra at 25.0 Ϯ 0.1°C were recorded on a JEOL LA500 spectrometer. Tetramethylsilane (in CDCl 3 ) (Merck) was used as an internal reference for 1 H and 13 C NMR measurements. MALDI-TOF-MS spectra (positive reflector mode) were obtained on a Voyager RP-3 BioSpectrometry work station (PerSeptive Biosystems) equipped with a nitrogen laser (337 nm, 3-ns pulse). Time-to-mass conversion was achieved by external calibrations using peaks for ␣-cyano-4-hydroxycinnamic acid (m/z 190.05 for M ϩ H ϩ ) and a peptide, Ac-Ile-Tyr-Gly-Glu-Phe-NH 2 (m/z 691.31 for M ϩ Na ϩ ). The pH measurement was conducted with a Horiba F-12 pH meter (Kyoto, Japan) and a combination pH electrode (Horiba-6378), which was calibrated using pH standard buffers (pH 4.01 and 6.86) at 25°C. Fluorescence gel images were acquired on an FLA 5000 laser scanner (Fujifilm, Tokyo, Japan). Pro-Q Diamond dye (10) was detected by 532-nm excitation with a 575-nm bandpass emission filter. SYPRO Ruby dye (11) was detected by 473-nm excitation with a 575-nm bandpass emission filter. A LAS 3000 image analyzer (Fujifilm) was used for the observation of chemiluminescence.

COOH, and 40% (v/v) MeOH) for 1 h and then washed in an aqueous solution containing 25% (v/v) MeOH and 10%
(v/v) CH 3 COOH until the background was clear. For SYPRO Ruby staining (11), fixed gels or Pro-Q Diamond-stained gels were incubated with SYPRO Ruby protein gel stain for 2 h and then washed in Two-dimensional Polyacrylamide Gel Electrophoresis-A431 cell lysate solved in a radioimmune precipitation assay buffer was desalted using Microcon YM3 filter units and resolved in a sample buffer for IEF (9.5 M urea, 2% (w/v) Nonidet P-40, 2% (w/v) Ampholine, and 5% (v/v) 2-mercaptoethanol) to become 2.5 g of protein/l. An IEF disc gel (2-mm diameter and 12 cm long) consisted of 9.2 M urea, 4% 1.5% (w/v) Nonidet P-40, 2% (w/v) Ampholine, and 0.015% (w/v) ammonium persulfate. IEF was performed using Atto SJ-1060DCII. The electrode buffer of positive pole (11 mM H 3 PO 4 , 0.75 liter) was poured into the lower chamber, and the disc gels were set. Sample solutions (50 g of protein/20 l) were applied, 20 l of a sample protection buffer (4.5 M urea and 1% (w/v) Ampholine) was put on the sample layer, and electrode buffer of negative pole (25 mM NaOH, 450 ml) was poured into the upper chamber. IEF was carried out at 400 V for 16 h at room temperature without cooling. After IEF electrophoresis, a disc gel was soaked twice for 15 min with a 0.25 M Tris-HCl (pH 6.8) buffer containing 2.5% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 10% (w/v) glycerol. And then SDS-PAGE (12.5% (w/v) polyacrylamide) was performed at 50 mA/gel at room temperature using a 1-mm-thick, 13.5-cm-wide, and 13.5-cm-long gel (model AE-6200, Atto).
Electroblotting-The separated proteins in a polyacrylamide gel were electroblotted to PVDF membranes for 2 h using a semidry blotting system (Nippon Eido NB-1600, Tokyo, Japan) at 2 mA/cm 2 with three kinds of blotting solutions (solutions A, B, and C). After normal SDS-PAGE, the gel was soaked in solution B (25 mM Tris and 5% (v/v) MeOH) for 10 min. After Mn 2ϩ -Phos-tag SDS-PAGE, the gel was soaked in solution B containing 1.0 mM EDTA for 10 min and then soaked in solution B for 10 min. Three No. 3MM papers were soaked in solution A (25 mM Tris, 40 mM 6-aminohexanoic acid, and 5% (v/v) MeOH) and piled on the negative pole board. The gel, the PVDF membrane, and a piece of No. 3MM paper were soaked in solution B and piled up in order. Then two pieces of No. 3MM paper soaked in solution C (0.30 M Tris and 5% (v/v) MeOH) were piled up. Finally they were covered with the positive pole board, and electricity was supplied.
Probing with Zn 2ϩ -Phos-tag-bound HRP-SA-A protein-blotted PVDF membrane was soaked in a TBS-T solution at least for 1 h. The membrane was incubated with the Zn 2ϩ -Phos-tag-bound HRP-SA solution (1 ml/5 cm 2 ) in a plastic bag for 30 min and washed twice with a TBS-T solution (10 ml/5 cm 2 ) for 5 min each time at room temperature. The chemiluminescence was observed using an appropriate volume of ECL Plus solution.
Probing with Antibody-A blotting membrane was blocked by 1% (w/v) bovine serum albumin in TBS-T solution for 1 h. For detection of phosphorylated proteins on tyrosine residue, the membrane was probed with HRP-conjugated anti-Tyr(P) monoclonal antibody (clone PY20) (0.5 g/ml in TBS-T, 1 ml/5 cm 2 ) in a plastic bag for 1 h and washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, and then the chemiluminescence was observed. For detection of phosphorylated proteins on serine residue, the membrane was probed with rabbit anti-Ser(P) polyclonal antibody (1 g/ml in TBS-T, 1 ml/5 cm 2 ) in a plastic bag for 1 h, washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, probed with HRP-conjugated anti-rabbit IgG antibody (0.1 g/ml in TBS-T, 1 ml/5 cm 2 ) in a plastic bag for 1 h, and washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, and then the chemiluminescence was observed. For detection of phosphorylated MBP on threonine residue, the membrane was probed with anti-pMBP monoclonal antibody (clone P12) (0.5 g/ml in TBS-T solution, 1 ml/5 cm 2 ) in a plastic bag for 1 h, washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, probed with HRPconjugated anti-mouse IgG antibody (0.1 g/ml in TBS-T, 1 ml/5 cm 2 ) in a plastic bag for 1 h, and washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, and then the chemiluminescence was observed. For phosphorylated MAP kinase 1/2 (Erk1/2) detection, the membrane was probed with anti-phospho-MAP kinase 1/2 antibody (clone 12D4) (0.1 g/ml in TBS-T solution, 1 ml/5 cm 2 ) in a plastic bag for 1 h, washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, probed with HRP-conjugated anti-mouse IgG antibody (0.1 g/ml in TBS-T, 1 ml/5 cm 2 ) in a plastic bag for 1 h, and washed twice with TBS-T solution (10 ml/5 cm 2 ) each for 10 min, and then the chemiluminescence was observed.
Reprobing of the Blotting Membranes-For elimination of biotinpendant Zn 2ϩ -Phos-tag and HRP-SA or antibodies from the blotting membrane after ECL analysis, the membranes were incubated with a stripping buffer (25 ml/5 cm 2 ) consisting of 62.5 mM Tris-HCl (pH 6. Phosphatase Assays-Dephosphorylated samples of phosphoproteins were prepared using a 50 mM Tris-HCl buffer (pH 9.0, 0.20 ml) containing 1.0 mM MgCl 2 , 50 g of protein, and 3.3 units of alkaline phosphatase (incubation at 37°C for 12 h and then mixing with 0.10 ml of 3ϫ SDS-PAGE loading buffer). Time-dependent dephosphorylation of bovine milk ␤-casein was performed in a 50 mM Tris-HCl buffer (pH 9.0, 0.14 ml) containing 1.0 mM MgCl 2 , 0.7 mg of protein, and 165 microunits of alkaline phosphatase at 37°C. Phosphorylated Abltide-GST was prepared as mentioned above, and the solution of the kinase reaction was used in the tyrosine phosphatase assay. Time-dependent dephosphorylation of phosphorylated Abltide-GST was performed in 50 mM Tris-HCl buffer (pH 7.0, 0.20 ml) containing 20 l of phosphorylated Abltide-GST (2 g) solution, and 0.3 unit of TC-PTP at 30°C. The dephosphorylation reactions were stopped by addition of a half-volume of 3ϫ SDS-PAGE loading buffer. Zero time samples were prepared using the same components without the enzymes.

Specific Visualization of Phosphorylated Proteins on a Blot-
ting Membrane-To visualize phosphorylated proteins, we determined the potency of biotin-pendant Zn 2ϩ -Phos-tag (9) (see Fig. 1) as a phosphate-binding biotin derivative for Western blotting analysis. As the first example, phosphorylated proteins (i.e. ␣-casein, ␤-casein, ovalbumin, and pepsin) spot-ted on a PVDF membrane were specifically detected at nanogram levels using an ECL system and a 4:1 complex of biotin-pendant Zn 2ϩ -Phos-tag and HRP-SA without a blocking treatment of the membrane. A typical ECL image by dot-blotting analysis is shown in Fig. 2a. No ECL signal was detected on the spots of the corresponding dephosphorylated proteins and the nonphosphorylated proteins (i.e. bovine serum albumin, human serum albumin, carbonic anhydrase, and ␤-galactosidase). In the absence of the zinc(II) ions (i.e. detected on the spots of phosphorylated proteins (data not shown). Thus, the phosphate-selective ECL signals were produced by the complex of biotin-pendant Zn 2ϩ -Phos-tag and HRP-SA via the interaction between the zinc(II) ions and the phosphomonoester dianion.
Next we applied the complex of biotin-pendant Zn 2ϩ -Phostag and HRP-SA to an electroblotting analysis after SDS-PAGE. This application did not require a blocking treatment of a PVDF membrane. The phosphorylation of Abltide-GST incubated with a tyrosine kinase, Abl, and the dephosphorylation of phosphorylated Abltide-GST incubated with a tyrosine phosphatase, TC-PTP, were visualized on PVDF membranes. The Abltide-GST is a recombinant fusion protein of an Abl substrate peptide (Abltide, Glu-Ala-Ile-Tyr-Ala-Ala-Pro-Phe-Ala-Lys-Lys-Lys) tagged by a GST. The Tyr 4 residue (italicized) of the Abltide section is selectively phosphorylated by Abl. The time courses of the phosphorylation and dephosphorylation are shown in Fig. 2, b and c, respectively. Both ECL detections were successively confirmed by a conventional procedure, immunoprobing with the anti-Tyr(P) monoclonal antibody (clone PY20) of the same blots. We also demonstrated similar electroblotting analyses of the phosphorylation of MAP kinase (phosphorylated on the Thr 183 and Tyr 185 residues) incubated with MEK1 (a Tyr and Ser/Thr dual kinase) and the phosphorylation of histone H1.2 incubated with protein kinase A (a Ser/Thr kinase) (Supplemental Fig. 1). These results indicate that biotin-pendant Zn 2ϩ -Phos-tag captures various phosphorylated proteins bound on a PVDF membrane.
Visualization of the Protein Phosphorylation Status of A431 Cells-We extended the phosphate-specific detection to the analysis of the phosphorylation status of A431 human epidermoid carcinoma cells before and after EGF stimulation. The EGF-dependent protein phosphorylations in the A431 cell have been well established (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23); therefore, we selected the cell as the first biological sample. For one-dimensional SDS-PAGE (Fig. 3, a-g), the molecular weight standards (lane M), the lysate of the cells before (lane 1) or after EGF stimulation (lane 2), and the AP-treated lysate of the cells after EGF stimulation (lane 3) were sequentially applied. Fluorescent gel staining with SYPRO Ruby (Fig. 3a) showed that the total proteins of the lysate applied in each lane were almost equal. Pro-Q Diamond gel staining (Fig. 3b) showed the increase of phosphorylated proteins by EGF stimulation and the decrease of dephosphorylated proteins by AP treatment. Electroblotting analysis using the 4:1 complex of biotin-pendant Zn 2ϩ -Phos-tag and HRP-SA (Fig. 3c) demonstrated that the ECL signals can represent the phosphorylation status of A431 cells more clearly than the fluorescent gel staining (Fig. 3b). The ECL signals increased by EGF stimulation (lane 2) were greatly diminished by AP treatment of the lysate (lane 3), indicating that the visualized proteins as ECL signals are phosphorylated proteins. The electroblotting analysis also detected a phosphorylated protein, ovalbumin (45 kDa), in the molecular weight standards (Fig. 3b, lane M). In electro-blotting analysis using only HRP-SA, some proteins (e.g. a biotinylated endogenous protein) in the lysate were detected as ECL signals (Fig. 3d). Electroblotting analysis using the 4:1 complex of the biotin-pendant Phos-tag ligand and HRP-SA in the presence of 1 mM EDTA (Fig. 3e) showed, however, that the ECL signals (Fig. 3, c and d) disappeared completely. These facts suggested that the zinc(II) complexation and the binding of the biotin-pendant to HRP-SA are essential for phosphate-specific detection. The proteins of the cell lysate were also analyzed by immunoblotting using the anti-Tyr(P) antibody (Fig. 3f) and the anti-Ser(P) polyclonal antibody (Fig.  3g). With regard to monitoring the change in the phosphorylation status, the data of the blotting analysis using biotinpendant Zn 2ϩ -Phos-tag were compatible with these results obtained with conventional methods using antibodies. In all electroblotting analyses, a nonspecific ECL signal of a marker protein, lysozyme (14 kDa in lane M), was observed using the ECL system (Fig. 3, c-g).
Next we determined the protein phosphorylation status of the EGF-stimulated cells by two-dimensional (2-D) IEF/SDS-PAGE followed by an electroblotting analysis as shown in Fig.  3h. The ECL signal intensity and number of spots on the membrane remarkably increased in comparison with those before EGF stimulation (see Supplemental Fig. 2). After the ECL detection with biotin-pendant Zn 2ϩ -Phos-tag, the same membrane was sequentially reprobed with the anti-Tyr(P) antibody and the anti-Ser(P) antibody. Superimposed images of the area surrounded with a dotted square in Fig. 3h were obtained by using biotin-pendant Zn 2ϩ -Phos-tag (green) and the anti-Tyr(P) antibody (magenta) (Fig. 3i) and biotin-pendant Zn 2ϩ -Phos-tag (green) and the anti-Ser(P) antibody (magenta) (Fig. 3j). Some proteins detected by both biotin-pendant Zn 2ϩ -Phos-tag and the antibodies appear as white spots in the superimposed images.
Separation of Phosphorylated Proteins in a Polyacrylamide Gel-We synthesized an acrylamide-pendant Phos-tag ligand (see Fig. 1 and "Experimental Procedures") as a novel additive (i.e. a copolymer) of the separating gel for the visualization of phosphorylated proteins by SDS-PAGE. The principle of this detection is the mobility shift of phosphorylated proteins due to reversible phosphate trapping by Phos-tag molecules immobilized in the gel. First we applied a polyacrylamide-bound Zn 2ϩ -Phos-tag to SDS-PAGE according to the widely accepted Laemmli method (12). However, the expected phosphate-selective mobility shift was not observed under such general SDS-PAGE conditions (data not shown). Presumably the alkaline condition (more than pH 9 during the electrophoresis) is beyond the optimum pH (ϳ7) for the phosphate trapping of Zn 2ϩ -Phos-tag (4). Therefore, we investigated the other metal complex acting as a phosphate-trapping molecule at an alkaline pH. The phosphate binding of the Phos-tag metal complex at pH 9 was confirmed by MALDI-TOF-MS using phenyl phosphate, phosphoserine, and phosphotyrosine as typical phosphates (ROPO 3 2Ϫ ) in the presence of 2 eq. of metal(II) ions (i.e. Mn 2ϩ , Cu 2ϩ , Co 2ϩ , Fe 2ϩ , and Ni 2ϩ ). The acrylamide-pendant Phos-tag ligand showed remarkable MS signals of the 1:1 phosphate-binding manganese(II) complexes (i.e. ROPO 3 2Ϫ -[acrylamide-pendant Mn 2ϩ -Phos-tag] 3ϩ , see Supplemental Fig. 3). Similar MALDI-TOF-MS signals for corresponding 1:1 phosphate complexes with Zn 2ϩ -Phos-tag have been reported at a neutral pH (5).
On the basis of the MALDI-TOF-MS results, we conducted SDS-PAGE using polyacrylamide-bound Mn 2ϩ -Phos-tag. Fig.  4 shows the effect of Mn 2ϩ -Phos-tag on the mobility of phosphorylated proteins (␣-casein, ␤-casein, and ovalbumin) in SDS-PAGE by subsequent CBB staining. In the absence of polyacrylamide-bound Mn 2ϩ -Phos-tag (i.e. normal SDS-PAGE), the mobilities of ␣-casein and dephosphorylated ␣-casein (Fig. 4a, lane at 0 M), of ␤-casein and dephosphorylated ␤-casein (Fig. 4b, lane at 0 M), and of ovalbumin and dephosphorylated ovalbumin (Fig. 4c, lane at 0 M) are almost the same. In the presence of polyacrylamide-bound Mn 2ϩ -Phos-tag (50, 100, and 150 M), a difference in mobility between the phosphorylated proteins and the corresponding dephosphorylated proteins (i.e. a mobility shift of the phosphorylated protein against the dephosphorylated protein) is observed. The polyacrylamide-bound Phos-tag ligand without Mn 2ϩ ions did not show any mobility shifts of phosphorylated proteins (data not shown). The R f values of all samples become smaller dose-dependently in comparison with those in the absence of Mn 2ϩ -Phos-tag; this is possibly due to electrostatic interaction between cationic Mn 2ϩ -Phos-tag and an- ionic SDS-bound proteins. Interestingly ␤-casein appears as eight bands using 100 M Mn 2ϩ -Phos-tag (see Fig. 4b), indicating the existence of at least eight isotypes with a different number of phosphomonoester dianions.
Simultaneous Determination on Phosphorylated and Dephosphorylated Proteins-We quantitatively monitored the enzymatic incorporation or removal of phosphate into proteins using Mn 2ϩ -Phos-tag SDS-PAGE and CBB staining (see Fig. 5). Normal SDS-PAGE analyses showed that the total protein (the amount of a substrate for kinase or phosphatase) applied at each incubation time was almost equal. In the kinase assays of Abl (Fig. 5a, Abltide-GST as a substrate) and MAP kinase (Fig. 5b, MBP as a substrate) followed by 100 M Mn 2ϩ -Phos-tag SDS-PAGE and CBB staining, the slower (higher) migration bands increased time-dependently, whereas the faster (lower) migration bands decreased. Successive immunoblotting analyses using the anti-Tyr(P) antibody (Fig. 5a) and the anti-pMBP antibody (which recognizes the phosphorylated Thr 97 residue) (Fig. 5b) showed that the slower migration bands are phosphorylated proteins. Treatment of the Mn 2ϩ -Phos-tag SDS-PAGE gel with EDTA before the immunoblotting analyses is necessary for efficient transfer of the phosphorylated proteins to a PVDF membrane without trapping by the Phos-tag molecule (see "Experimental Procedures"). In the phosphatase assays of AP (Fig. 5c, ␤-casein as a substrate) and TC-PTP (Fig. 5d, phosphorylated Abltide-GST as a substrate) followed by Mn 2ϩ -Phos-tag SDS-PAGE and CBB staining, the slower (higher) migration bands decreased time-dependently, whereas the faster (lower) migration bands increased. These results indicate that acrylamide-pendant Mn 2ϩ -Phos-tag preferentially captures phosphomonoester dianions bound to Ser, Thr, and Tyr residues as well as biotin-pendant Zn 2ϩ -Phos-tag. Thus, Mn 2ϩ -Phos-tag SDS-PAGE has enabled the simultaneous determination of phosphorylated and corresponding dephosphorylated proteins in a polyacrylamide gel. DISCUSSION In this report, we have described two methods using Phostag molecules for the visualization of protein phosphorylation and dephosphorylation. The methods are independent on the amino acid residues; thus, protein phosphorylation can be comprehensively detected. One method is the application of biotin-pendant Zn 2ϩ -Phos-tag and HRP-SA to Western blotting analysis: protein samples are first separated by electrophoresis and then electroblotted to a PVDF membrane and detected as ECL signals. We succeeded in the sensitive and specific detection of phosphorylated proteins on serine, threonine, and tyrosine residues using the ECL system. The blotstaining method has a general advantage over gel-staining methods with regard to the long term storage of proteinblotted membranes. The other method is the application of polyacrylamide-bound Mn 2ϩ -Phos-tag to SDS-PAGE for the separation of phosphorylated proteins in the gel. By means of the subsequent general method of CBB staining, phosphorylated proteins can be visualized as a slower migration band compared with a corresponding dephosphorylated protein. This quantitative method revealed the existence of the isotypes of a multiphosphorylated protein (e.g. ␤-casein) as different migration bands. Additionally it is appropriate for the screening of an activator or an inhibitor of protein kinase and phosphatase.
The biotin-pendant Zn 2ϩ -Phos-tag was reported as a novel probe of multiphosphopeptides to detect the on-chip phosphorylations by the SPR imaging technique (9). Previously another SPR detection system with the anti-Tyr(P) antibody was proposed (24). Antibodies whose epitopes are Ser(P), Thr(P), or Tyr(P) are commercially available and can be applied to phosphorylation detection on the SPR. However, multiple antibodies are required for the peptide array on which many kinase substrates are immobilized. Therefore, the Phostag molecule, which is not dependent on the amino acid residue, was very useful in a system with an array format. In a similar fashion, it is worthwhile to consider using the biotinpendant Zn 2ϩ -Phos-tag in Western blotting analysis for evaluating whole phosphorylated proteins from the cell lysate on a PVDF membrane. In addition, the application can increase the chances of finding a new phosphorylated protein. Classical detection of phosphorylated proteins usually requires the use of autoradiography after the incorporation of isotopic [ 32 P]orthophosphate into cultured cells or subcellular fractions by protein kinases (14 -21). However, this approach is limited to specimens amenable to radiolabeling and poses certain safety and disposal problems. Furthermore phosphorylated proteins on a PVDF membrane can be detected by immunoblotting analysis using antibodies against phosphorylated amino acid residues (25)(26)(27). Unfortunately the specificity of antibodies depends on the quality of the antibody, and the antibodies are often sensitive to the amino acid sequence context. Although a few high quality clones of anti- bodies to phosphorylated tyrosine are commercially available, the binding specificities of some anti-Ser(P) and anti-Thr(P) antibodies are dependent on the microenvironmental structures of phosphorylated residues in the proteins. Compared with these traditional approaches, our established method offers significant advantages. (i) The radioactivity is avoided. (ii) The blocking treatment of a PVDF membrane is not necessary. (iii) The binding specificity of the Phos-tag molecule is independent of the amino acid sequence context. Furthermore the Phos-tag method can be fully followed by downstream procedures, such as antibody reprobing, mass spectrometry, or Edman sequencing.
The Mn 2ϩ -Phos-tag was utilized as a novel phosphate affinity SDS-PAGE. The polyacrylamide-bound Mn 2ϩ -Phos-tag showed preferential trapping of the phosphorylated proteins without disordering (waving or tailing of protein bands) of the migration image. The 1:1 phosphate-bound Mn 2ϩ -Phos-tag complexes were confirmed by MALDI-TOF-MS at an alkaline pH for the general SDS-PAGE. A relevant x-ray crystal structure of the dimanganese complex with the Phos-tag ligand (alkoxide form) was reported as an acetate-bridging species (28). Our established method requires a general minislab PAGE system and an additive, acrylamide-pendant Mn 2ϩ -Phos-tag without any special apparatuses, radioisotopes, or fluorescent probes. The Mn 2ϩ -Phos-tag SDS-PAGE can identify the time course ratio of phosphorylated and dephosphorylated proteins in an SDS-PAGE gel.
The phosphorylation status of a particular protein is deter-mined by the equilibrium of the opposing activities of protein kinase and phosphatase. Perturbations in the equilibrium fundamentally affect the numbers of cellular events and are also involved in many diseases. Therefore, the development of a more specific and efficient method to detect protein phosphorylation has attracted great interest toward phosphoproteome studies in the biological and medical fields. We believe that phosphoproteomics would progress greatly by combining our Phos-tag technology and existing methods using high quality antibodies (27) and convenient mass spectrometers (3,29,30).
* This work was supported by Grants-in-aid for Scientific Research (B) 15390013 and for Young Scientists (B) 17790034 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a research grant from Hiroshima University Fujii Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.