Complementary Metal-directed Immobilized Metal Ion Affinity Chromatography for Phosphoproteomic Profiling of Human Mesenchymal Stem Cells

摘要

Protein phosphorylation plays an important role in biological process. Despite the success of mass spectrometry in protein identification, comprehensive enrichment of phosphopeptides is crucial prior to mass spectrometric analysis due to low abundances, poor ionization efficiency and heterogeneous modification of phosphoprotein. Taking advantage of the different binding affinity between metal ions (such as Ti~(4+), Zr~(4+) and Fe~(3+)) and phosphopeptides and the presence flexibility of immobilized metal ion with spacer arm linked to silica based beads (nitrilotriacetic acid, NTA) as chelator to reduce the steric hindrance, we designed a metal-directed immobilized metal ion affinity chromatography to increase the coverage in phosphoproteomics. The integrated method will be applied to Raji B cells and human mesenchymal stem cells (HMSC).