title: PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants.
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 fromArabidopsis thalianaleaf 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.
authors.affiliation: Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle/Saale, D-06120, Germany. Ines.Lassowskat@ipb-halle.de.
authors.forename: Ines
authors.initials: I
authors.lastname: Lassowskat
authors.affiliation: Riboxx GmbH, Meissner Str. 191, Radebeul, D-01445, Germany. Kai.Naumann@riboxx.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Naumann
authors.affiliation: Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle/Saale, D-06120, Germany. jlee@ipb-halle.de.
authors.forename: Justin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle/Saale, D-06120, Germany. dscheel@ipb-halle.de.
authors.forename: Dierk
authors.initials: D
authors.lastname: Scheel
keywords: Arabidopsis thaliana; LC-MS; phosphoprotein enrichment; phosphoproteomics