title: TiO(2)-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment.
abstract: Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO(2)-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing. We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10(7) cells), achieving more than 70% yield of membrane proteins. Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein phosphatase inhibitors produces distinct phosphopeptide populations and an increase of 10-40% of the number of detected and sequenced phosphoserine, phosphothreonine and phosphotyrosine containing peptides. In summary, our analytical strategy enables functional phosphoproteomic analysis of stem cell differentiation and cell surface biomarker discovery using very low amounts of starting material.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Tine E
authors.initials: TE
authors.lastname: Thingholm
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Christian R
authors.initials: CR
authors.lastname: Ingrell
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002845:Chromatography / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D059630:Mesenchymal Stem Cells / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D053719:Tandem Mass Spectrometry; D014025:Titanium
chemical_list: D008565:Membrane Proteins; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium; D010744:Phosphoric Monoester Hydrolases