PROTOCOL Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE Eiji Kinoshita, Emiko Kinoshita-Kikuta & Tohru Koike Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima, Japan. Correspondence should be addressed to E.K. ([email protected]). Published online 24 September 2009; doi:10.1038/nprot.2009.154 We provide a standard phosphate-affinity SDS-PAGE (Mn2+–Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins (B150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted natureprotocols / migration bands on the improved Mn2+–Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of m o electrophoresis requires about 4 h in this protocol. c . e r u t a n INTRODUCTION . w Reversible phosphorylation is a key signaling mechanism for studies after electrophoresis, using radioactive compounds of w w 32 32 / modulating the functional properties of proteins involved in gene [g- P]-labeled ATP and [ P]-labeled orthophosphate. A meta- / : p t expression, cell adhesion, cell cycle, cell proliferation or cell bolic radiolabeling protocol has been successfully used in many t h 1 differentiation . There are more than 500 protein kinase genes laboratories to identify phosphoproteins; however, this approach is p 2,3 u within the human genome , a number that clearly reflects the limited to specimens that are amenable to labeling, and it raises o r importance of protein phosphorylation. Abnormal protein phos- certain problems with regard to safety and waste disposal. The G phorylation is closely involved with many human diseases, includ- second approach involves immunoblotting with antibodies against g n i ing cancer, diabetes mellitus, neurodegeneration and immune/ phosphorylated amino acids. Unfortunately, the lack of specificity h 4 s i inflammatory disorders . Experimental procedures for the deter- of some antibodies can be a problem in some cases. Anti-phospho- l b mination of the phosphorylation status of certain proteins are tyrosine monoclonal antibodies are widely used because they react u P therefore very important in relation to studies on a diverse range of selectively with various proteins containing phosphorylated tyro- e r physiological and pathological processes. sine residues. In contrast, monoclonal antibodies against phospho- u t a Separation of a phosphorylated protein and its nonphosphory- serine or phosphothreonine residues are unpopular because their N lated counterpart by using gel-based electrophoresis can facilitate affinity and specificity are less than optimal. To achieve a precise 9 0 0 an understanding of the phosphorylation status. The SDS-PAGE characterization of signaling events, it is desirable to raise a high- 2 procedure5, in which proteins are separated by electrophoresis on a quality antibody against a phosphorylated residue; however, raising © polyacrylamide gel, is often used to identify phosphorylated this type of antibody is costly, time-consuming and often unsuc- proteins because phosphoproteins show different extents of elec- cessful. A more global quantification of phosphoproteins has been trophoretic migration compared with their nonphosphorylated achieved newly using a phospho-specific Pro-Q Diamond gel/blot counterparts. Uniform binding of SDS to the phosphoprotein stain6,7 (Invitrogen) or a phospho-specific Phos-tag gel/blot fluoro- can be disrupted by the presence on the phosphoprotein of stain8–12 (Perkin-Elmer). In addition, an immobilized metal a negatively charged phosphate group that can decrease the charge affinity gel-based electrophoresis has been developed recently as a density on the phosphoprotein compared with that on its nonphos- useful tool for separation of phosphoproteins from the nonphos- phorylated counterpart. If there is a sufficient difference in charge phorylated counterparts13,14. Presumably, it would be a challenge density between the phosphorylated and nonphosphorylated to implement these techniques for detection of phosphoprotein forms, the phosphorylated protein will show a retarded migration isotypes. and will appear at a position corresponding to a higher molecular weight on the gel compared with its nonphosphorylated counter- Mn2+–Phos-tag SDS-PAGE for mobility shift detection of part. This observation of a shift in mobility has sometimes been phosphoprotein isotypes used as an index of protein phosphorylation in certain biological We previously found that a dinuclear metal complex (i.e., 1,3- events; however, the shift in mobility on phosphorylation depends bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) com- on protein-specific structural characteristics, and the number of plex) acts as a phosphate-binding tag molecule in an aqueous phosphoproteins that can be analyzed by conventional SDS-PAGE solution15. We called this zinc complex ‘Phos-tag.’ The Phos-tag is limited. molecule has a vacancy on its two zinc ions that will accept a There are two principal approaches to the detection of protein phosphomonoester dianion as a bridging ligand. In an aqueous phosphorylation by combinations of the conventional SDS-PAGE solution, the dinuclear zinc(II) complex strongly binds to the À8 and other techniques. One approach involves autoradiography phenyl phosphate dianion (Kd ¼ 2.5 Â 10 M) at a neutral pH. NATURE PROTOCOLS | VOL.4 NO.10 | 2009 | 1513 PROTOCOL Figure 1 | Phosphate-affinity Mn2+-Phos-tag SDS-PAGE for the mobility-shift a Polyacrylamide H R detection of phosphoproteins. (a) Structure of the polyacrylamide-bound N R H 2+ O N Mn –Phos-tag and scheme of the reversible capturing of a phospho- O O O O O 2À 2+ monoester dianion (R-OPO3 )byMn –Phos-tag and (b) schematic P P 2+ – – – representation for the principle of Mn –Phos-tag SDS-PAGE. The polya- N N O O – O N O N 2+ 2+ crylamide-bound Mn –Phos-tag shows preferential trapping of the 2+ Mn 2– 2+ 2+ Mn O– R-OPO Mn Mn N N 3 – phosphorylated proteins, which is because of separation of the phospho- N N N O N proteins from their nonphosphorylated counterparts. In this paper, we focus N N 2+ 2– 2+ on the utility of the phosphate-affinity SDS-PAGE for the separation of large Mn –Phos-tag R-OPO3 –Mn –Phos-tag phosphoprotein isotypes with molecular masses of more than 200 kDa. b P P Nonphosphorylated protein P 2À À À P The anion selectivity indexes against SO ,CHCOO ,Cl and P 4 3 P Phosphorylated protein P À 3 4 5 6 R-OSO3 at 25 1Care5.2Â 10 ,1.6Â 10 ,8.0Â 10 and 42 Â 10 , P respectively. A manganese(II) homolog of Phos-tag (Mn2+–Phos- P Phosphate P tag) can capture a phosphomonoester dianion, such as phospho- binding site P N 2+ B 2+ Mn serine or phosphotyrosine, at alkaline pH values ( 9) (Fig. 1a). Mn – N O N This finding has led to the development of phosphate-affinity gel N N Phos-tag electrophoresis for detecting shifts in the mobility of phospho- Phosphate-affinity SDS-PAGE gel natureprotocols / proteins in comparison with their nonphosphorylated counter- m 16–20 2+ o parts . We used an acrylamide-pendant Mn –Phos-tag as a c . e novel additive in a separating gel for normal SDS-PAGE. In a separation of large phosphoproteins with molecular masses of r u t separating gel containing co-polymerized Phos-tag, the degrees of more than 200 kDa. Although a highly porous polyacrylamide a n . migration of phosphoproteins are less than those of their nonpho- gel is generally used for the separation of high-molecular-mass w w sphorylated counterparts because the tag molecules trap phospho- proteins, B5% (wt/vol) of polyacrylamide is the minimum con- w / / : proteins reversibly during electrophoresis. On the basis of this centration that permits handling of the gel after electrophoresis. p t t principle, we recently established a novel type of gel electrophoresis, In this protocol introduced, this problem was circumvented h 2+ Mn –Phos-tag SDS-PAGE, for the separation of phosphoproteins by homogeneous addition of 0.5% (wt/vol) agarose to a tender p u from their corresponding nonphosphorylated analogs (Fig. 1b). SDS-PAGE gel containing 3% (wt/vol) polyacrylamide and 20 mM o r The Mn2+–Phos-tag SDS-PAGE protocol offers the following Mn2+–Phos-tag36. The agarose gels or agarose–polyacrylamide G g significant advantages: (i) no radioactive or chemical labels are composite gels are usually used for the separation of high- n i 37–39 h required for kinase and phosphatase assays; (ii) the time course molecular-mass proteins . The SeaKem Gold Agarose gel s i l quantitative ratio of phosphorylated to nonphosphorylated pro- (Lonza, Rockland, ME, USA), especially provided for large DNA b u teins can be determined; (iii) several phosphoprotein isotypes, separation, has been reported to work best among various types of P e depending on the phosphorylation status, can be detected as agarose or polyacryamide gels for detecting giant myofibrillar r u multiple migration bands; (iv) the phosphate-binding specificity proteins, such as titin (3,000–4,000 kDa) and nebulin isoforms t a is independent of the kind of phosphorylated amino acid; (v) His- (600–900 kDa)38,39. The improved procedure using a SeaKem Gold N 2+ 9 and Asp-phosphorylated proteins involved in a two-component Agarose–Polyacrylamide composite gel containing Mn –Phos-tag 0 0 signal-transduction system can be detected simultaneously in their permitted the separation of phosphoprotein isotypes having mole- 2 © phosphotransfer reactions; (vi) separation of phosphoprotein iso- cular masses of 200–350 kDa within 2 h.