TiO<sub>2</sub>-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment Tine E. Thingholm Martin R. Larsen Christian R. Ingrell Moustapha Kassem Ole N. Jensen 10.1021/pr800099y.s002 https://acs.figshare.com/articles/journal_contribution/TiO_sub_2_sub_Based_Phosphoproteomic_Analysis_of_the_Plasma_Membrane_and_the_Effects_of_Phosphatase_Inhibitor_Treatment/2924347 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<sub>2</sub>-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<sup>7</sup> 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. 2008-08-01 00:00:00 plasma membrane protein preparation microgram levels phosphorylation sites mass spectrometry membrane proteins cell surface biomarker discovery Phosphopeptide enrichment Phosphatase Inhibitor TreatmentPhosphorylation Plasma Membrane phosphoproteomic analysis sodium carbonate extraction plasma membrane phosphoproteomics 376 phosphoproteins protein phosphatase inhibitors sucrose centrifugation cell cultures protein phosphorylation attenuate plasma membrane transport processes plasma membrane phosphoproteome cell differentiation phosphopeptide populations titanium dioxide chromatography signal transduction pathways plasma membrane proteins phosphopeptide sequencing