Preparation Of Dendrimer For Detection Of Phosphopeptides In Biological Sample

In eukaryotes, protein phosphorylation is an important dynamic post-translational modification involved in the regulation of a wide variety of cellular events including signal transduction, gene expression, metabolism, and cell growth, division, differentiation and apoptosis. Conversely, abnormal phosphorylation can sometimes contribute to diseases such as cancer. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. Since protein phosphorylation is low stoichiometry, enrichment of phosphorylated peptides from proteolytic mixtures is a challenge prior to mass spectrometric analysis. A variety of metal-binding ligands with metal ions employed in immobilized metal affinity chromatography stationary phases have been devoted to specific capture of phosphopeptide. A well defined and highly branched polyamidoamine dendrimers are selected as enrichment of phosphorylated peptides. Silica gel is functionalized with polyamidoamine dendrimers to chelated titanium ions to improve the specific interaction between the coordinated metal ions and the phosphate groups of phosphopeptides. Optimization of the enrichment conditions, the sample liquid organic solvent concentration of50%acetonitrile, the type of acid of0.1%trifluoroacetic acid, washing times of once and elution of0.4mol/L ammonium hydroxide. The results show that this method has high selectivity phosphorylated peptides, and the detection limit was10-9mol/L. Under optimal conditions, the conditions established enrichment and mass spectrometry method for milk samples and analysis of phosphopeptide enrichment and identification of phosphorylated peptides to milk the sequence coverage of100%. The excellent detection limit for selective purification of phosphorylated peptides from P-casein is as low as femtomole level. The high specificity and efficiency of immobilized titanium ion on polyamidoamine dendrimers adsorbent is performed to enrich phosphopeptides from the mixture β-casein and bovine serum albumin with high molar ratio at1:100. Polyamidoamine solid-functionalized silica is the promising material for efficient enrichment of phosphopeptides by the analysis of phosphopeptides in real samples.