Novel Magnetic Nano-affinity Probes Developed For The Application On Enrichment And Separation Of Phosphorylated Proteins/Peptides

Phosphorylation of proteins is one of the most important forms of post-translational modifications of proteins.Many low-abundance endogenous phosphoproteins/phosphopeptides in biological fluids or tissues are biomarkers of high specificity and clinical sensitivity that may provide valuable information for the detection or interpretation of many diseases or pathology.It is still a great challenge to directly detect the phosphorylated proteins/peptides from complex biological matrixs by mass spectrometry(MS)due to their low abundance and the dynamic reversibility of proteins phosphorylation process.Because of their large specific surface area,abundant activity affinity sites and unique physicochemical structures,affinity nano-materials have attracted much attention in the separation and enrichment of phosphoproteins/phosphopeptides and become research hotspots in the current phosphoproteomics separation,enrichment and identification.Therefore,in this research work,contraposing the difficult problems of low stoichiometry and ionization efficiency as well as signal suppression by other high-abundance non-phosphoproteins/non-phosphopeptides in the MS detection of low-abundance phosphoproteins/phosphopeptides,several novel magnetic affinity nano-probes were designed and developed to selectively enrich phosphoproteins/phosphopeptides through efficient,simple,rapid and convenient magnetic separation process.In addition,these magnetic affinity nano probes were applied to separate and enrich phosphoproteins/phosphopeptides from real-world biological samples,and some satisfactory research results were obtained.This thesis is composed of the introduction(Chapter 1)and the research reports(Chapters 2-6).The research reports mainly focused on two types of novel affinity probes,namely,magnetic spinel-like nanomaterials(Chapters 2,3 and 4)and rare earth-based affinity probes(Chapters 5 and 6),which are used to selectively enrich phosphoproteins/phosphopeptides.The summaries of this paper are as follows:1.The development history of biological mass spectrometry technology,including basic structure and working principle of mass spectrometers,was described briefly.The contents of matrix of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),together with choice of matrix were mainly introduced.In addition,the significance of both proteomics/peptidomics and phosphoproteomics/phosphopeptidomics studies was exposited.The application of various magnetic affinity nano-probes in the enrichment of phosphorylated proteins/peptides was reviewed as a key point.This chapter also summarized the application of new methods and technologies of phosphoproteins/phosphopeptides enrichment in recent years.The magnetic materials used in the enrichment process were classified.Finally,the main research ideas of this thesis were briefly introduced.2.The reversible phosphorylation of proteins plays a key role in numerous biological processes,while abnormal phosphorylation is a cause or consequence of many human diseases.Selective enrichment and MALDI-TOF MS or MS/MS characterization and identification of endogenous phosphopeptides fished-out from human saliva sample were achieved using simple and low-cost iron oxide magnetic nanoclusters(Fe3O4 MNCs)affinity probe.The physicochemical properties of the prepared MNCs material was characterized by SEM,TEM,XRD,EDX,SQUID,etc.Based on their merits of high affinity,rapid separation,good biocompatibility,excellent dispersibility and superior specificity for capturing phosphopeptides,the performances of Fe3O4 MNCs were assessed by selectively and efficiently isolating phosphopeptides from standard phosphoprotein and actual non-fat milk tryptic digests,and intricate human saliva,respectively.Moreover,we demonstrated that the probe can be applied for rapid and effective purification and enrichment of phosphoproteins.The Fe3O4 MNCs affinity probe,which possesses the merits of integration of solid Lewis acids function and magnetic property,together with the "double economical"(both time and money)synthesis strategy,should be a promising material for the large scale investigation of endogenous phosphoproteomics.3.The spinel-type magnetic manganese ferrite(MnFe2O4)microspheres synthesized by simple solvothermal method were used as a novel adsorbent for selective enrichment and effective isolation of phosphopeptides.The uniform MnFe2O4 magnetic affinity microspheres(MAMSs)had a narrow particle size distribution and displayed superparamagnetism.Comprehensively,the possible formation mechanism of MnFe2O4 microspheres with ferric and manganous sources as dual precursors was elucidated by comparison with those of Fe3O4 nanoparticles or MnOOH nanosheets respectively with either ferric or manganous source as single precursor.It was suggested that the spherical or sheet nanostructures could be achieved via secondary recrystallization or Ostwald ripening.The MnFe2O4 MAMSs probe exhibited excellent dispersibility in aqueous solution,and rapid magnetic separation,as well as good reusability.More importantly,MnFe2O4 was highly selective for phosphopeptides because of the strong coordination interaction between metal ions(Fe3+ and Mn2+)and phosphate groups of phosphopeptdies.This high specificity was demonstrated by effectively enriching phosphopeptides from digest mixture of ?-casein and bovine serum albumin(BSA)with high content of non-phosphopeptides,and further confirmed in phosphopeptides enrichment from non-fat milk digests and human serum.Consequently,the prepared MnFe2O4 affinity materials are expected to possess great potential in phosphoproteome research.4.A facile solvothermal method for the synthesis of multifunctional magnetic CuFeMnO4 nanospheres affinity probe(NSAP)with controllable morphology and size was developed.The CuFeMnO4 nanospheres combine the brilliant features of Cu2+,Fe3+ and Mn2+,so their multifunction performances were embodied by strong coordination to carboxyl and amine groups of peptides(Cu2+ and Fe3+),special affinity to phosphate groups of phosphopeptides(Fe3+ and Mn2+),and high magnetic responsiveness in a magnetic field.Their potential as affinity probe was evaluated for highly effective enrichment and rapidly magnetic separation of low-abundance peptides(neutral condition)and effectively selective capture of phosphopeptides(acid condition)from various complex biosamples.Notably,the feasibility of CuFeMnO4 NSAP was explored for highly selective capture and isolation of phosphopeptides from A549 cells after exposure to ZnO nanoparticles for different time.Consequently,we put forward a new nano-spinel ferrite-based protocol here to analyze and identify the phosphoproteins/phosphopeptides involved in cellular signaling pathways in response to exogenous stimulation.5.Lichee-like core-shell structured magnetic lutetium phosphate(Fe3O4@LuPOO4)affinity microspheres were successfully synthesized and well characterized.Based on the properties of great specificity and rapid separation,magnetic LuPO4 microspheres were applied to selectively enrich phosphopeptides for MALDI-TOF MS analysis.The enrichment performance of the prepared microspheres was demonstrated by tryptic digests of standard protein(?-casein),complex samples(tryptic digest mixture of ?-casein and bovine serum albumin at different molar ratios of 1:10 to 1:50),and real biological samples(fresh pure milk and human serum),the enrichment ability of magnetic LuPO4 microspheres for phosphopeptides was very satisfying and the captured phosphopeptides with a sharp enhancement in MS signals were reliably identified based on the appearance of metastable ion peaks with poor resolution of the corresponding dephosphorylated peptides in the mass spectra.Enrichment of phosphopeptides by the Fe3O4@LuPO4 affinity microspheres provides a powerful approach for large scale phosphoproteome analysis.6.Two novel Ce-based nanocomposites(P-CCS and CSF)were prepared respectively by co-precipitation and solvothermal methods.Based on the affinity of the two self-designed cerium-based probes for different phosphopeptides,a sequential enrichment strategy was proposed,which not only can effectively separate phosphopeptides from non-phosphopeptides,but can also selectively differentiate mono-and multi-phosphopeptides for direct MALDI-TOF MS analysis.In addition,these two CeO2-doped affinity probes also exhibit the property that catalyzes dephosphorylation process and can act as a catalyst for dephosphorylation.In particular,CSF probe was used to capture multi-phosphorylated peptides,while P-CCF probe assisted in capturing mono-phosphorylated peptides.In this way,effective enrichment of mono-and multi-phosphopeptides has been realized successfully.Thus,this combination strategy can be used as a good alternative to enrichment of all phosphopeptides.