title: Isolation of phosphopeptides by pI-difference-based electrophoresis.
abstract: Effective proteomics studies of protein phosphorylation require an efficient enrichment method for phosphopeptides, which remains a challenge. Here, we describe the discovery of pI differences between methylated phosphopeptides (typically <7.4) and methylated nonphosphorylated peptides (typically >9.0). This pI difference allows isolation of methylated phosphopeptides from the methylated nonphosphopeptides by in-solution isoelectric focusing. We proved the principle of such a novel approach by isolating a phosphorylated peptide from nonphosphorylated tryptic digest of myoglobin. While the principle for pI-based, in-solution electrophoresis is proven, it requires further development for practical application. The method described here provides a stepping stone toward more reliable, convenient method for efficient isolation of phosphopeptides.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
authors.forename: Yingda
authors.initials: Y
authors.lastname: Xu
authors.forename: Robert
authors.initials: R
authors.lastname: Sprung
authors.forename: Sung Won
authors.initials: SW
authors.lastname: Kwon
authors.forename: Sung Chan
authors.initials: SC
authors.lastname: Kim
authors.forename: Yingming
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000818:Animals; D004586:Electrophoresis / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D008745:Methylation; D009211:Myoglobin / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification*; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
chemical_list: D009211:Myoglobin; D010748:Phosphopeptides; D011506:Proteins; D014357:Trypsin