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Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule.

ABSTRACT: We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3.

SUBMITTER: Nakanishi T

PROVIDER: S-EPMC2392992 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule.

Nakanishi Tsuyoshi T Ando Eiji E Furuta Masaru M Kinoshita Eiji E Kinoshita-Kikuta Emiko E Koike Tohru T Tsunasawa Susumu S Nishimura Osamu O

Journal of biomolecular techniques : JBT 20071201 5

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-4 ...[more]

PMID: 18166671

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Project description:This report describes a synthetic protocols and the crystal structures involving a novel pincer-type H3[NNN] ligand, namely di-μ-bromido-μ-{2-(2,2-di-methylpropanimido-yl)-N-[2-(2,2-di-methyl-propanimido-yl)-4-methyl-phen-yl]-4-methylaniline}-bis-[(diethyl ether)lithium], [Li2Br2(C24H33N3)(C4H10O)2] (1) and a dinuclear metal complex, namely di-μ-bromido-2:3κ4Br:Br-bis-{2-(2,2-di-methylpropanimido-yl)-N-[2-(2,2-di-methyl-propanimido-yl)-4-methyl-phen-yl]-4-methylaniline}-1κ3N,N',N'';4κ3N,N',N''-tetra-μ-iso-propano-lato-1:2κ4O:O;3:4κ4O:O-diiso-propano-lato-1κO,4κO-2,3-dilithium-1,4-dititanium, [Li2Ti2Br2(C24H32N3)2(C3H7O)6] or {[NHNNH]Ti(O i Pr)3(LiBr)2}2 (2). Complex 1, which sits on a twofold rotation axis, is a rare example of a pincer-type ligand which bears ketimine side arms. A unique feature of complex 1 is that the ketimine N atoms have an LiBr(Et2O) fragment bonded to them, with the Li atom adopting a distorted tetra-hedral geometry. This particular fragment creates an LiBr bridge between the two ketimine sidearms, which leads to a cage-type appearance of the ligand. Complex 2 consists of the previously described ligand and a TiIV metal atom in an octa-hedral environment, and is located on an inversion center. Complex 2 crystallizes as a dinuclear species with the metal atoms being bridged by an LiBr entity [the Br atoms are disordered and refined in two positions with their site occupation factors refining to 0.674 (12)/0.372 (12)], and the Li cation being bonded to the isopropoxide O atoms (Li having a tetra-hedral coordination as in 1). The organic ligand of compound 2 exhibits disorder in its periphery groups; isopropyl and tert-butyl groups (occupation factors fixed at 0.6/0.4). The novel [NNN]H3 pincer-type ligand was characterized by multinuclear and multidimensional NMR, HRMS and X-ray crystallography. The dinuclear metal complex 2 was characterized by X-ray crystallography. Although each structure exhibits donor N-H groups, no hydrogen bonding is found in either one, perhaps due to the bulky groups around them. One of the ethyl groups of the ether ligand of 1 is disordered and refined in two parts with site-occupation factors of 0.812 (8) and 0.188 (8). One and a half toluene solvent mol-ecules are also present in the asymmetric unit of 2. The toluene mol-ecules were significantly disordered and could not be modeled properly, thus SQUEEZE [Spek (2015 ▸). Acta Cryst. C71, 9-18] was used to remove their contributions to the overall intensity data.

Dataset Information

Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule.

Publications

Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule.

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