A New Method For Selective Enrichment Of Monophosphorylated Peptides Based On Metal-organic Framework Materials

Protein phosphorylation could be divided into monophosphorylation and multi-phosphorylation,and the functions of monophosphorylation and multi-phosphorylation in cells were different.The technology of biological mass spectrometry was a powerful tool to research phosphoproteomics,and the bottom-up strategy was used to analysis phosphoproteomics at the peptide level.However,due to the low abundance and low ionization efficiency of phosphopeptides,a research strategy of effectively capture phosphopeptides was imperative to develope before mass spectrometry detection.The phosphopeptides could be divided into monophosphopeptides and multi-phosphopeptides.Moreover,considering the strong affinity of multi-phosphopeptides with enrichment materials,many highly hydrophilic enrichment materials have been reporeted and used to enrich multi-phosphopeptides.However,the selective enrichment of monophosphopeptides was almost no reported,so finding selective and effective enrichment materials toward monophosphopeptides was extremely urgent.Based on this method,the enrichment materials of metal-organic frameworks?MOFs?were used to selectively and effectively capture monophosphopeptides.The detailed research contents were as follows:1.Sn-based Metal-Organic Framework for Highly Selective Capture MonophosphopeptidesThe Sn-based metal-organic framework?Sn-MOF?was synthesized through a bottom-up approach,the Sn?IV?as the metal center and the isophthalic acid as the organic ligand.The Sn-MOF was first used to enrich phosphopeptides and exhibit selective enrichment toward monophosphopeptides.Moreover,the Sn-MOF demonstrated good sensitivity and selectivity,and the Sn-MOF was also feasible in complex biological samples.2.Highly Selective Capture of Monophosphopeptides by Two-Dimensional Metal-Organic Framework NanosheetsThe 2-D Hf-BTB nanosheets were synthesized through a bottom-up approach,the Hf?IV?as the metal cluster and the 1,3,5-tris?4-carboxyphenyl?benzene?H₃BTB?as the organic ligand.Through experiments,we found the 2-D Hf-BTB nanosheets exhibited unique enrichment selectivity toward monophosphopeptides in the digest of model proteins.At the same time,the 2-D Hf-BTB nanosheets demonstrated excellent sensitivity and selectivity.Moreover,the 2-D Hf-BTB nanosheets were also feasible in complex biological samples and mouse brain cortical samples.Subsequently,the2-D Hf-BTB nanosheets could also be used to detect the relative content of phosphopeptides in patients'serum with anal canal inflammationu and healthy adult serum using the in situ isotope dimethyl labeling method.Finally,in order to further reasearch the enrichment mechanism of 2-D Hf-BTB nanosheets toward monophosphopeptides,the other six MOFs were synthesized as enrichment materials to further study.After enriching the digest of model protein and comparing the structures of these MOFs,we found the hydrophobicity of organic ligands and the distance between adjacent metallic clusters were very important for the selective enrichment of phosphopeptides.When the strong hydrophobicity of organic ligand and the large distance between adjacent metallic clusters,the unique balance between metallic affinity of clusters and hydrophobicity from ligands could retain the intermediate monophosphopeptides and exclude both non-phosphopeptides and multi-phosphopeptides,exhibited unique selectivity toward monophosphopeptides.