title: Phosphorylation of extracellular matrix tenascin-X detected by differential mass tagging followed by nanoLC-MALDI-TOF/TOF-MS/MS using ProteinPilot software.
abstract: Reversible protein phosphorylation represents a major mechanism of signal transduction in a variety of cellular functions. An understanding of proteome-wide phosphorylation dynamics is important to obtain an overview of the whole signal transduction network. However, a systematic analysis for differentially expressed phosphoproteins under serum-stimulated response is lacking. Here, an easy and fast approach for the identification of differentially expressed phosphoproteins was used. After enrichment of phosphoproteins from serum-stimulated cell lysates by immobilized metal affinity chromatography, a quantitative proteomic approach with isobaric tag for absolute and relative quantitation labeling in combination with nanoLC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis was used. Consequently, 506 differentially expressed phosphoproteins were identified. Among them, 22 proteins that had a reproducible phosphorylation site at Ser or Thr were identified. Out of these 22 phosphoproteins, 7 are mainly involved in splicing. Among the 22 proteins, it was found that extracellular matrix tenascin-X is phosphorylated, although there is little quantitative change by the serum stimulation. MS/MS analysis revealed a novel phosphorylation site of tenascin-X, Thr1841, located in the loop region between the 10th and 11th fibronectin type III repeats. The phosphorylation of tenascin-X would be considered in clarifying its function in the future.
authors.affiliation: Department of Biosignaling and Radioisotope Experiment, Center for Integrated Research in Science, Shimane University, Izumo, Japan. matumoto@med.shimane-u.ac.jp
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Matsumoto
mesh_terms: D002851:Chromatography, High Pressure Liquid; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D036103:Nanotechnology / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D044967:Serum / Q000378:metabolism; D015398:Signal Transduction; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019063:Tenascin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D019063:Tenascin; C081865:tenascin X