Lysozyme Nanomembrane Functionalized Magnetic Nanomaterials For Enrichment Of Phosphorylated Peptides

Protein phosphorylation is ubiquitous in organisms because it can regulate the proliferation,differentiation and apoptosis of cells in organisms,signal transduction and material transfer,and cell recognition and fusion,and other physiological functions and biological processes.Phosphopeptides/proteins as biomarkers for identifying many diseases,are particularly important in the study of phosphopeptidomics.Mass spectrometry(MS)plays a vital role in identifying phosphorylation sites and quantifying their dynamic changes due to its advantages such as fast analysis speed,wide dynamic range,and high sensitivity.However,due to the inevitable non-phosphopeptide/protein interference,as well as the low ionization efficiency and low abundance of the phosphopeptide/protein,it seriously hinders the comprehensive identification of phosphopeptides in mass spectrometry analysis.Therefore,the preparation of functionalized materials for the highly selective capture of phosphopeptides before MS analysis is an essential link for in-depth study of the phosphoproteome.In recent years,the surface functionalization of nanomaterials has played an increasingly important role in various fields.The currently reported methods for surface functionalization of nanomaterials mostly rely on chemical bonding,usually involving poisonous reagents and multistep treatments,which takes a long time and requires harsh conditions.Lysozyme is a cheap antibiotic enzyme on the market and can undergo an unique configuration changes in neutral TCEP buffer and quickly self-assemble into phase transformations lysozyme(PTL)nanofilms on various particle surface.The formed lysozyme nanofilms provides strong interface bonding can resist mechanical and chemical peeling under harsh conditions,and the surface multifunctional groups support various sequences of surface chemical derivatization.In view of this,we first uses lysozyme to functionalize the surface of nanoparticles,then performs targeted modification on the surface of the formed PTL nanofilms,and finally applies it to the highly selective enrichment of phosphopeptides in complex biological samples.This paper includes the following three chapters:Part 1:IntroductionThis chapter mainly summarizes the phosphopeptide enrichment methods and enrichment materials.First,the definition,type and research significance of phosphoproteins are explained,and then the common methods of enriching phosphopeptides,common matrix materials for separation and enrichment,and the mechanism of one-step aqueous self-assembly of lysozyme nanofilms are introduced.Finally,the purpose and main content are briefly introduced.Part 2:Immobilized Ti4+ functionalized magnetic nanoparticles based on lysozyme nanofilms for effective enrichment of phosphopeptidesIn this chapter,the PTL nanofilms self-assembled on the surface of SiO2@Fe3O4 and C@Fe3O4 magnetic nanoparticles under physiological conditions to prepared PTL-coated magnetic nanoparticles.The abundant functional groups on the surface of PTL nanofilms,such as-NH2,-COOH,-OH and-SH,can be used as effective linkers for chelating Ti4+,which shows strong interaction with the phosphate groups in the phosphopeptides,so as to achieve effective enrichment of phosphopeptides from complex biological samples.Then the ?-casein digests was used as the model to investigate the enrichment performance of the synthetic material in detail.The resulting the material can achieve rapid enrichment within 3 min,high sensitivity(0.01 fmol/?L),good selectivity(?-casein:BSA,1:400),and reproducibility and stability,successfully applied to enrich the phosphopeptide in human serum.Part 3:AuNPs functionalized magnetic nanoparticles based on lysozyme nanofilms for efficient enrichment of phosphopeptidesSince the surface of the PTL nanofilms contains-SH,AuNPs can grow in situ on the surface,then the cysteamine is connected to the AuNPs on the surface of the nanoparticles through the Au-S bond.Finally,the amino group and the phosphate ion on the cysteamine are used strong interaction to enrich the phosphopeptides,the ?-casein digests was used as model,and the enrichment performance of the synthetic material was investigated in detail.The synthesis method is simple,fast,efficient and does not require complex organic reagent processing.It can achieve the enrichment of phosphopeptides within a few minutes,and the sensitivity and selectivity are better than reported literature.The experimental results show that the material can also be used to efficiently enrich the phosphopeptides in standard proteins and actual samples.