Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins

Abstract

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

“…Recombinant RBM8A was phosphorylated by GSK-3, as detected by reduced mobility (Fig. 4A, red arrow) in a Phos-tag/polyacrylamide gel, in which a phosphatebinding moiety slows mobility of phosphorylated species relative to non-phosphorylated forms (44).…”

“…PhosTag gels were prepared using PhosTag TM acrylamide reagent (Wako Pure Chemical Industries, Ltd., catalogue no. AAL-107) with ZnCl 2 based on the manufacturer's protocol (44). The composition for optimal separation of NPM1 was 30 M PhosTag TM acrylamide reagent and 10% acrylamide using a acrylamide/bisacrylamide solution (29:1).…”

Protease-resistant modified human β-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model

“…We purified the modB and mod2B with terminal M6P-containing N-glycans by 3-step column chromatography (Supplemental Table 1), including an affinity chromatography step with Phos-tag resin, which is usually used to capture phosphorylated proteins (27). The modB and mod2B purified from the serum-free conditioned medium (CM) were both composed of the precursor forms (β 1 '-P and β 2 '-P) and the mature forms (β 1 '-M and β 2 '-M) as determined by SDS-PAGE under reducing conditions (Supplemental Figure 2, A-C).…”

Large‐scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography

“…The first one is chemical reaction-based methods which include beta-elimination of the phosphate group and subsequent introduction of an affinity tag [11], and then captured by beads and released [12]. The second one is chromatography based methods which include affinity chromatography with immobilized metal ions (IMAC) [13][14][15][16][17][18][19][20][21][22][23], metal oxides such as titanium dioxide (TiO 2 ) and zirconium dioxide (ZrO 2 ) [24][25][26][27][28][29][30][31], and strong cation exchange chromatography (SCX) [32][33][34][35]. Among all of these methods, IMAC with Fe 31 is the most popular technique for the enrichment of phosphorylated peptides.…”