title: Integrated approach using multistep enzyme digestion, TiO2 enrichment, and database search for in-depth phosphoproteomic profiling.
abstract: Protein phosphorylation is a major PTM that regulates important cell signaling mechanisms. In-depth phosphoproteomic analysis provides a method of examining this complex interplay, yielding a mechanistic understanding of the cellular processes and pathogenesis of various diseases. However, the analysis of protein phosphorylation is challenging, due to the low concentration of phosphoproteins in highly complex mixtures and the high variability of phosphorylation sites. Thus, typical phosphoproteome studies that are based on MS require large amounts of starting material and extensive fractionation steps to reduce the sample complexity. To this end, we present a simple strategy (integrated multistep enzyme digestion, enrichment, database search-iMEED) to improve coverage of the phosphoproteome from lower sample amounts which is faster than other commonly used approaches. It is inexpensive and adaptable to low sample amounts and saves time and effort with regard to sample preparation and mass spectrometric analysis, allowing samples to be prepared without prefractionation or specific instruments, such as HPLC. All MS data have been deposited in the ProteomeXchange with identifier PXD001033 (http://proteomecentral.proteomexchange.org/dataset/PXD001033).
authors.affiliation: Departments of Biomedical Sciences, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; Biomedical Engineering, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; Institute of Medical & Biological Engineering, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Dohyun
authors.initials: D
authors.lastname: Han
authors.forename: Jonghwa
authors.initials: J
authors.lastname: Jin
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Yu
authors.forename: Kyunggon
authors.initials: K
authors.lastname: Kim
authors.forename: Youngsoo
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D030562:Databases, Protein; D006801:Humans; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D049490:Systems Biology / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014025:Titanium / Q000737:chemistry
chemical_list: D020543:Proteome; C009495:titanium dioxide; D014025:Titanium
keywords: LC-MS/MS; Phosphoproteome; Post-translational modification; Proteome profiling; Systems biology