PAPE Prefractionation-Assisted Phosphoprotein Enrichment: A Novel Approach for Phosphoproteomic Analysis of Green Tissues from PlantsReportar como inadecuado


PAPE Prefractionation-Assisted Phosphoprotein Enrichment: A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants


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1

Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle-Saale, D-06120, Germany

2

Riboxx GmbH, Meissner Str. 191, Radebeul, D-01445, Germany





*

Author to whom correspondence should be addressed.



Abstract Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and-or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment PAPE, to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography MOAC step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel potential phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. View Full-Text

Keywords: phosphoproteomics; LC-MS; Arabidopsis thaliana; phosphoprotein enrichment phosphoproteomics; LC-MS; Arabidopsis thaliana; phosphoprotein enrichment





Autor: Ines Lassowskat 1, Kai Naumann 2, Justin Lee 1,* and Dierk Scheel 1

Fuente: http://mdpi.com/



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