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Proteomes 2013, 1(3), 254-274;

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

Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle/Saale, D-06120, Germany
Riboxx GmbH, Meissner Str. 191, Radebeul, D-01445, Germany
Author to whom correspondence should be addressed.
Received: 15 October 2013 / Revised: 28 November 2013 / Accepted: 28 November 2013 / Published: 5 December 2013
(This article belongs to the Special Issue Plant Proteomics)
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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

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This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Lassowskat, I.; Naumann, K.; Lee, J.; Scheel, D. PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants. Proteomes 2013, 1, 254-274.

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