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A rapid screening assay to search for phosphorylated proteins in tissue extracts.

ABSTRACT: Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO(2) enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.

SUBMITTER: Garaguso I

PROVIDER: S-EPMC3499474 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A rapid screening assay to search for phosphorylated proteins in tissue extracts.

Garaguso Ignazio I Borlak Juergen J

PloS one 20121115 11

Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strateg ...[more]

PMID: 23166814

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Project description:Background:Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results:In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. Conclusions:This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

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A rapid screening assay to search for phosphorylated proteins in tissue extracts.

Publications

A rapid screening assay to search for phosphorylated proteins in tissue extracts.

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