Complementary multiple hydrogen-bond-based magnetic composite microspheres for high coverage and efficient phosphopeptide enrichment in bio-samples.

Abstract

Due to the number of phosphorylation sites, mono- and multiple-phosphopeptides exhibit significantly different biological effects. Therefore, comprehensive profiles of mono- and multiple-phosphopeptides are vital for the analysis of these biological and pathological processes. However, the most commonly used affinity materials based on metal oxide affinity chromatography (MOAC) show stronger selectivity toward mono-phosphopeptides, thus losing most information on multiple-phosphopeptides. Herein, we report polymer functionalized magnetic nanocomposite microspheres as an ideal platform to efficiently enrich both mono- and multiple-phosphopeptides from complex biological samples. Driven by complementary multiple hydrogen bonding interactions, the composite microspheres exhibited remarkable performance for phosphopeptide enrichment from model proteins and real bio-samples. Excellent selectivity (the molar ratio of nonphosphopeptides/phosphopeptides was 5000 : 1), high enrichment sensitivity (2 fmol) and coverage, as well as high capture rates of multiple-phosphopeptides revealed their great potential in comprehensive phosphoproteomics studies. More importantly, we successfully captured the cancer related phosphopeptides (from the phosphoprotein Stathmin-1) and identified their relevant phosphorylation sites from oral carcinoma patients' saliva and tissue lysate, demonstrating the potential of this material for phosphorylated disease marker detection and discovery.