title: Identification of extracellularly phosphorylated membrane proteins.
abstract: Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.
authors.affiliation: Department of Molecular Cardiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Burghoff
authors.affiliation: Department of Molecular Cardiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: Wibke
authors.initials: W
authors.lastname: Willberg
authors.affiliation: Department of Molecular Cardiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schrader
mesh_terms: D001963:Bromelains; D002853:Chromatography, Liquid; D042783:Endothelial Cells / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D020710:RGS Proteins / Q000032:analysis / Q000378:metabolism; D051881:Receptor, Notch1 / Q000032:analysis / Q000378:metabolism; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2 / Q000032:analysis / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D016326:Extracellular Matrix Proteins; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; C000609451:OTOP1 protein, human; D010750:Phosphoproteins; D020710:RGS Proteins; C456322:RGS13 protein, human; D051881:Receptor, Notch1; C112735:USH2A protein, human; D001963:Bromelains; C090899:PTPRD protein, human; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2
keywords: Cell biology; Endothelial cells; Extracellular phosphorylation; K562 cells; Mass spectrometry; Membrane proteins