Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe 3 O 4 @polydopamine-Nb 5+ (denoted as Fe 3 O 4 @PD-Nb 5+ ) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe 3 O 4 @PD-Ti 4+ microspheres, the Fe 3 O 4 @PD-Nb 5+ microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.
Wang Bing,Zhang Xiaoya,Wang Baichun,Feng Quanshou,Luo Yiting,Wang Weimin,Ding Chuan-Fan,Yan Yinghua
A Ti 4+ functionalized β-cyclodextrin covalent organic framework nanoparticle (named as β-CD-COF@Ti 4+ ) was synthesized using a one-pot method successfully realizing the enrichment of phosphorylated peptides and exosomes based on the immobilized metal ion affinity chromatography strategy. The functionalized β-CD-COF@Ti 4+ exhibited superior performance on the enrichment of phosphopeptides, including high selectivity (1:1000), low detection limit (0.5 fmol), and loading capacity for phosphopeptides (100 mg·g −1 ). After treatment with β-CD-COF@Ti 4+ , 9 phosphopeptides from defatted milk, 29 phosphopeptides related to 23 phosphoproteins from normal group serum, and 24 phosphopeptides related to 22 phosphoproteins from the serum of uremia patients were captured. Through the analysis of Gene Ontology, the captured phosphoprotein is closely related to kidney disease, including lipoprotein metabolism, very-low-density lipoprotein particle, high-density lipoprotein particle, and lipid binding activity process. Furthermore, western blot verification showed that this nanoparticle could successfully capture exosomes from human serum. This study demonstrates great prospects for the enrichment of phosphopeptides and exosomes from actual bio-samples. Graphical Abstract
Inspired by the enrichment preferences of metal ions for phosphopeptides, a layered assembly method was adopted to introduce hydrophilic polyhydroxy ligands tannic acid (TA) to prepare magnetic bimetal ion-functionalized nanomaterials denoted by Fe3O4@TA@Ti4+/Zr4+ to provide the comprehensive enrichment of phosphopeptides. Benefiting from the excellent ion immobilization ability and unbiasedness for phosphopeptide enrichment, Fe3O4@TA@Ti4+/Zr4+ shows excellent properties for the enrichment and identification of global phosphopeptides. The results show that the material possesses high efficiency, outstanding selectivity (the maximum molar ratio β-casein to bovine serum albumin is 1:1000), a low detection limit of 0.4 fmol/μL, and the ability to be recycled at least 10 times. In addition, excellent performance was also demonstrated in the analysis of skimmed milk and human saliva: 13 and 31 phosphopeptides were successfully captured. The results demonstrate that the material has high potential for practical application in phosphoproteomics.
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