Preparation Of Magnetic Organic Porous Framework And Their Applications In Phosphorotein And Glycoprotein Analysis

Protein post-translational modifications（PTMs）are an important cellular regulatory mechanism that affect the folding,conformation,activity,and other properties of proteins,thereby influencing their functions.PTMs are a dynamic biochemical process,and linking modifications to physiological and cellular events is still highly challenging.Bottom-up proteomics of post-translationally modified proteins typically involves enzymatic cleavage of the modified protein into smaller peptide fragments using proteases,followed by identification and characterization of modified motifs and sites using mass spectrometry（MS）technology.However,the dynamic range of concentrations,complexity of actual samples,and low ionization efficiency of post-translationally modified peptides make their comprehensive analysis complex,posing significant challenges to direct MS analysis.Therefore,effective pre-enrichment of low-abundance post-translationally modified peptides in complex systems is key to achieving large-scale proteomics analysis of post-translationally modified proteins before MS detection.Magnetic solid phase extraction（MSPE）is a sample pre-treatment technique based on magnetic materials.This technique utilizes the affinity of magnetic materials for target compounds to selectively extract and separate them,achieving the purpose of separation and enrichment.MSPE technology has the advantages of easy operation,high efficiency,speed,greenness,and high sensitivity,and has been widely used.In MSPE,some modification of magnetic adsorbents with functional molecules or materials is essential to achieve rapid separation and efficient enrichment.In PTMs analysis,functional magnetic nanoparticles（MNPs）are widely used because of their unique physical properties（such as size,shape,magnetism）and the ability to enhance affinity with proteins through surface chemical modification.Functional MNPs provides researchers with an efficient,rapid,and highly selective sample pre-treatment method,playing an important role in the fields of biomedical and analytical chemistry.Porous materials have long been an excellent platform for scientists and engineers to explore new multifunctional materials.Among them,crystalline porous framework materials such as metal organic frameworks（MOFs）,covalent organic frameworks（COFs）,and hydrogen-bonding organic frameworks（HOFs）have ordered structures,adjustable pore sizes,higher specific surface areas,and site-modifiable characteristics,exhibiting better separation performance than amorphous materials.Based on the many advantages mentioned above,in the context of the two hotspots of MNPs and organic porous framework materials,the construction of a new type of magnetic nanocomposite material combining organic porous framework materials and MNPs is a new development direction.This paper uses the unique advantages of organic porous materials as functional materials and modifies magnetic particles to enhance their specificity for target adsorbates.A series of novel magnetic adsorbents were synthesized by integrating the advantages of organic porous framework materials and the unique magnetic responsiveness of magnetic particles.Based on these adsorbents,a magnetic solid-phase extraction（MSPE）platform was constructed and coupled with MS technology to achieve the separation and analysis of phosphoproteins and glycoproteins in complex sample systems.The specific experimental content is divided into the following four parts:1.A simple strategy was proposed for the synthesis of magnetic ionic covalent organic frameworks（Fe₃O₄@i COFs）in which ionic guanidinium-based ligand was used as functional unit.The developed Fe₃O₄@i COFs contain a large amount of amino groups and positive charges,as well as owned superparamagnetism.The enrichment of phosphopeptides are based on electrostatic interactions between phosphate groups and guanidine groups,as well as the formation of hydrogen bonds.When combined with MS detection,a high sensitivity（0.4 fmolβ-casein）can be achieved for phosphopeptides.The prepared material shows the ability to selectively enrich phosphopeptides in non-fat milk digests and He La cell lysates,and has great potential for detecting low abundance phosphopeptides in complex real samples.2.We designed a novel magnetic zwitterionic material,glutathione functionalized thioether covalent organic frameworks（Fe₃O₄@Thio-COF@Au@GSH）,holding fast magnetic responsiveness,regular porosity,suitable surface area.Thanks to the hydrophilicity and charge-switchable feature of the GSH,for the first time,both the capture of exosomes from biological fluids and enrichment of the inherent glycoproteins/phosphoproteins in exosomes were achieved with the same material.Furthermore,the high enrichment capacity was validated by theoretical calculations.Low detection limits（0.2/0.4 fmol HRP/β-casein）,high selectivity（1:1000=HRP/β-casein:BSA）,and high exosomal glycoproteomics/phosphoproteomics profiling capability proved the feasibility of the developed method.This work provides a new heuristic strategy to solve the problems of exosomal capture and glycoproteins/phosphoproteins pretreatment in exosomal proteomics.3.Two porous crystalline materials,metal organic framework（MOF）and covalent organic framework（COF）,were covalently integrated on MNPs.The Ti-O groups in the MOF and high hydrophilicity of COF endow the resulting porous hybrid with high-efficiency simultaneous enrichment capacity of glyco-and phospho-proteins from urine samples without additional pretreatment steps.The enrichment mechanism and possible binding modes were validated by theoretical calculations.The synthesized magnetic MOF@COF successfully enriched glyco-and phospho-proteins from urine samples of patients with nephrotic syndrome and healthy controls,indicating that the protocol can be used to distinguish patients with nephrotic syndrome based on the amount of enriched glycoproteins and phosphoproteins.This work not only provides a new idea for the efficient enrichment of glyco-and phospho-proteins with porous hybrid material,but also broadens the research possibilities for biomarker discovery in urine-based liquid biopsy.4.For the first attempt,we reported a hydrogen-bonding organic framework（HOF）constructed from guanidinium cation and borate anion for the simultaneous identification of glycopeptide/phosphopeptide,especially mono-phosphopeptide in human serum.According to theoretical calculations,the HOF functional sites display a synergistic"matching"effect with mono-phosphopeptides,resulting in a stronger enrichment effect for mono-phosphopeptides as compared to multi-phosphopeptides.Due to its high hydrophilicity and boron affinity,this material can efficiently capture glycoproteins from human serum and identify both glycoproteins and phosphoproteins simultaneously using the same material.HOF will become an active research direction in the development of protein enrichment materials,and the powerful H-bonding network will provide a new way to solve the problem of synergistic enrichment of post-translational modified peptides.