Phosphorylation is one of the most important protein post-translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non-specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β-casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non-fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research.
Wenrui Wu,Ruizhi Tang,Zhouxian Li,Yehua Shen,Shujuan Ma,Junjie Ou
It is prerequisite to efficient extraction of phosphopeptides in bottom-up strategy for protein phosphorylation research. A kind of Ti 4+ -immobilized polydispersed phosphate-rich microsphere was fabricated via inverse suspension polymerization by employing vinylphosphonic acid (VPA) and N,N -methylenebisacrylamide (MBA) as functional monomer and crosslinker, respectively. The resulting microsphere demonstrated excellent sensitivity (as low as 10 fmol), selectivity (the mass ratio of β-casein to BSA digests is 1/500), and adsorption capacity (up to 200 mg g −1 ). What's more, 113 unique phosphopeptides assigned to 25 unique phosphoproteins were indiscriminately identified from 5 μL of pasteurized milk digest, exhibiting great performance in capturing phosphopeptides. In this approach, only two steps were required to synthesize Ti 4+ -IMAC (Ti 4+ -Immobilized metal affinity chromatography). Compared with other methods required multistep modifying process, this strategy is simple and time-saving, offering a prospect of pilot production and commercialization. It is expected that the application of IMAC in milk and other food samples will still make it possible to unravel the huge complexity of the Foodome in the near future.
Protein glycosylation and phosphorylation are two important post-translational modifications and play significant roles in various biological processes. However, both glycoproteins and phosphoproteins are in low abundance in complex samples, which hinders their characterization. In this work, we developed a novel method for the simultaneous adsorption of glycoproteins and phosphoproteins based on solid phase extraction. By virtue of the unique structure of hydrophilic titanium (IV) ion immobilized cotton fiber functionalized with phytic acid (denoted as Ti4+-PA-cotton), glycoproteins, and phosphoproteins could be efficiently enriched in one-step incubation and eluted in sequence. The newly prepared Ti4+-PA-cotton was well characterized, and the adsorption isotherms and kinetics were studied. The maximum adsorption capacities of Ti4+-PA-cotton for β-casein and HRP were 833.3 mg/g and 384.6 mg/g with the adsorption rate of 259.1 and 63.1 mg/g·min. Standard protein mixture of BSA, HRP, and β-casein was used to test the enrichment ability as a proof of concept. The SDS-PAGE results demonstrated that Ti4+-PA-cotton had an excellent enrichment performance and possessed an outstanding application potential in the analysis of glycoproteins and phosphoproteins.
危险品化学品经营许可证（带存储）