Tomoyo Nakano

Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

Prefractionation procedures facilitate the identification of lower‐abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one‐tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal‐regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two‐dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK‐activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.

Structural elucidation of cyanobacterial peptides encoded by peptide synthetase gene in Anabaena species

Abstract During our biosynthesis study of cyanobacterial peptides including microcystins, we investigated the metabolic peptides in the hepatotoxic cyanobacteria, Anabaena sp. strains 90 and 202A2, in which the genetic analysis of the peptide synthetase had been carried out. For the exhaustive screening of cyanobacterial peptides, an analytical method using ESI-LC/MS on-line photodiode array detection was successfully developed and applied. Based on the analytical results, two groups of peptides, the cyclic depsipeptides having a 3-amino-6-hydroxy-2-piperidone moiety, anabaenopeptilides, and the cyclic peptides possessing an ureido linkage, anabaenopeptins, were isolated together with microcystins from both strains. Consequently, we confirmed the structures including the stereochemistry of the anabaenopeptilides encoded by the sequencing peptide synthetase genes in Anabaena strain 90.