A New Method For Selective Enrichment Of Phosphorylated Peptides Based On Two-dimensional Egyptian Blue And Metal Organic Framework Nanosheets

In recent years,phosphorylated proteins played an important role in the regulation of life activities in the phosphoproteomics research.However,the analysis of large-scale phosphorylated proteins still were limited that mainly due to the extremely low concentration of phosphorylated proteins in the organism.To solve the problem,the researchers developed a new kind of sample pretreatment method called "bottom up"and mass spectrometry(MS)analysis strategy which enrichment the phosphopeptides from the tryptic digest of phosphorylated proteins and detected the phosphorylated proteins based on the mass spectrum.However,how to highly selective enrichment of phosphopeptides is the main factor in this method.To date,many materials and methods were used in the enrichment of phosphopeptides,however,few papers were reported in the specific selectivity towards phosphopeptides.In this paper,two-dimensional materials were used in phosphopeptides enrichment,and we developed Egyptian blue nanosheets and metal-organic framework nanosheets in this study.The detailed research contents and the mainly results in this dissertation are as follows:1.An Exfoliated 2-D Egyptian Blue Nanosheet for Highly Selective Enrichment of Multi-Phosphorylated Peptides in Mass Spectrometric AnalysisPhosphopeptide enrichment is important for the phosphoprotein analysis due to the low abundance in complex biological samples.Moreover,selective binding of multi-phosphopeptides is rarely reported.Here we synthesized three two-dimensional(2-D)nanosheets,including CaCuSi4O10 nanosheets,the well-known ancient pigment and its analogues(SrCuSi4O10 nanosheets and BaCuSi4O10 nanosheets),which were employed in the enrichment of phosphopeptides for the first time.Surprisingly,the 2-D CaCuSi4O10 nanosheets was highly selective towards multi-phosphopeptides without enriching the mono-phosphopeptide in a wide range of acidic conditions or buffer compositions.Moreover,ultrathin and well-defined 2-D morphology with abundant Ca(II)of Egyptian blue nanosheets were applied in the hyperphosphorylation Tau protein that associated with Alzheimer's disease.The as-prepared 2-D CaCuSi4O10 nanosheet not only showed specific selectivity but also exhibited high sensitivity(detection limit of 4 × 10-7 M).2.Two-Dimensional Titanium Metal-Organic Framework Nanosheets for Highly Efficient Capture of PhosphopeptidesReversible protein phosphorylation regulates many cellular processes and significantly alters protein activities and functions.Shotgun proteomics based on MS is a powerful tool for detecting phosphoproteins/phosphopeptides.However,the phosphopeptides are usually expressed at extremely low concentration(less than 1 ×10-9 M),thus their detection from complex biological matrixes is always a challenge.Herein,four water-stable MOFs and MOF nanosheets,including Ti-based 3-D MOFs(MIL-125 and NH2-MIL-125),Ti-based 2-D NTU-9 nanosheets and Zr-based 2-D nanosheets were selected as the enrichment materials.Combining the properties of high-valence Ti(IV)and nanosheets intriguing features,the NTU-9 nanosheets demonstrated the best selectivity(1:10000),sensitivity(1 × 10-10 M)and capacity in the enrichment of standard phosphopeptides.Finally,the application of this material is successfully demonstrated with in situ isotope labeling in the analysis of the dysregulated phosphopeptides in serum samples from Type 2 diabetes patients.Finally,NTU-9 nanosheets was used in the enrichment of phosphopeptides from the tryptic digest of rat brain neocortex lysate and rat spinal cord lysate.