Isolation of phosphopeptides by pI-difference-based electrophoresis.
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.
Xu
Yingda
Y
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
Sprung
Robert
R
Kwon
Sung Won
SW
Kim
Sung Chan
SC
Zhao
Yingming
Y
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
D009211:Myoglobin; D010748:Phosphopeptides; D011506:Proteins; D014357:Trypsin