Investigation Of New Methodologies In Rapid Protein Digestion And Enrichment Based On Functionalized Magnetic Microspheres

The advent of genomics and proteomics has increased considerably the need for fast,low-cost,and automated tools from protein analysis.Proteolysis is the key step for positive protein sequencing in proteomics research integrated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). Moreover,concentration of phosphopeptides or phosphoproteins is also very crucial in phosphoproteomics research.Therefore,new technologies for rapid and high throughput protein digestion and selective enrichment of phosphopeptides are in great demand.On the other hand,during the past decade,magnetic microspheres with micro-and nanometer size are gaining increasing attention due to their ease of manipulation and recovery.Functionalized magnetic microspheres are extensively applied in cell separation,magnetically assisted drug delivery,and enzyme immobilization.Additionally,magnetic materials are the most commonly selected substrates as affinity probes because of the ease of isolation of the magnetic-material-target conjugate from the sample solution based on their magnetic properties.In this study,we focused on preparing several kinds of novel functionalized magnetic polymer microspheres and developing a series of techniques and methods to resolve current problems in proteolysis digestion and concentration of phosphorylated peptides.This dissertation is divided into five parts.In Chapter 1,advances in proteomics research,current immobilized enzymatic reactor and phosphopeptides enrichment techniques,applications of functionalized magnetic polymer microspheres techniques in proteome research were summarized in details.The intention and meaning of this dissertation were explained.In Chapter 2,we developed two synthesis routes based on mechanisms of metal-ion chelation and covalent bonding,respectively.With these two synthesis routes,we successfully prepared trypsin-immobilized magnetic silica microspheres and applied them for fabrication of on-column/on-chip enzymatic reactor.Fe₃O₄ magnetic microspheres with superparamagnetic property were first synthesized through sovalthermal reaction.After that,Fe₃O₄@SiO₂ magnetic microspheres with narrow size distribution and uniform thickness of silica were synthesized via sol-gel approach.Thereafter,two different strategies for enzyme immobilization were developed:First,immobilization method based on metal-ion chelation.The metal chelating agent of iminodiacetic acid(IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the surface of Fe₃O₄@SiO₂ microspheres. The metal ion of copper and enzyme were subsequently adsorbed onto the surface.Second,immobilization method based on covalent bonding. Aminopropyltriethoxysilane(APTES) and glutaraldehyde(GA) were introduced to functionalize the Fe₃O₄@SiO₂ microspheres in turn for enzyme immobilization. Trypsin was then stably immobilized onto the Fe₃O₄@SiO₂ microspheres through the reaction of primary amines of enzyme with aldehyde groups on the Fe₃O₄@SiO₂ microspheres.The trypsin-immobilized Fe₃O₄@SiO₂ microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-column or on-chip enzymatic microreactor.The digestion efficiency and reproducibility of the microreactors were demonstrated by using model proteins. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach,proteins can be digested by the on-column or on-chip microreactor in several minutes.The microreactors were also successfully applied to the analysis of an RPLC fraction of the rat liver extract.The advantage of the prepared microreactors is that the Fe₃O₄@SiO₂ microspheres(which are retained by a magnet) can be fast and efficiently removed,which makes the microreactors replaceable.Moreover,for microreactors prepared using metal-ion chelation method, the Fe₃O₄@SiO₂ microspheres can be easily regenerated by removing copper ions with EDTA and introducing new copper ions and enzyme,and good reproducibility before and after regeneration was obtained.The results revealed their potential for significant simplification of top-down proteomic analysis protocols,without compromising the quality and specificity of analysis.In Chapter 3,a more convenient method for preparation of trypsin-immobilized magnetic nanospheres was initially developed.At first,amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy.Then,magnetic nanospheres were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanospheres with glutaraldehyde.Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanospheres was achieved through reaction of the aldehyde groups with amine groups of trypsin.The obtained trypsin-immobilized magnetic nanospheres were conveniently applied for protein digestion.The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins.The process of digestion is very facile due to the easy manipulation of magnetic nanospheres.Complete protein digestion was achieved in a short time(5 min),without any complicated reduction and alkylation procedures.Based on the above results,we further applied these trypsin-immobilized magnetic nanospheres for on-plate digestion.After digestion,the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS.The effects of the temperature and incubation time on the digestion efficiency were characterized.Furthermore,RPLC fractions of rat liver extract were also successfully processed using this novel method.These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.In Chapter 4,we employed,for the first time,the Ce⁴⁺-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before.After enrichment,the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet.With the optimized enrichment conditions,the performance of the Ce⁴⁺-chelated magnetic microspheres was compared with the Fe³⁺-chelated microspheres using tryptic digested peptides originating from ovalbumin,a five protein mixture containing phosphoproteins and nonphosphoproteins,as well as a mixture ofβ-casein and BSA with a molar ratio of 1:50.Compared to Fe³⁺,Ce⁴⁺-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures.Even when the amount of the tryptic digest product of BSA is 50 times higher than that ofβ-casein in the sample solution,the trace phosphopeptides derived fromβ-casein can still be concentrated effectively by the Ce⁴⁺-chelated magnetic microspheres in only 30 s.Furthermore,we initially utilized the Ce⁴⁺-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment,which opens up a possibility for their further application in phosphoproteomics.In Chapter 5,a novel strategy for preparation of Fe₃O₄@M_xO_y magnetic microspheres with well-defined core-shell structure was initially developed and the as-prepared microspheres were applied for highly selective enrichment of phosphopeptides from tryptic digest product of proteins.To successfully coat iron oxide microspheres with uniform metal oxide shell,magnetic Fe₃O₄ microspheres were first synthesized via a solvothermal reaction,followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction.Finally,with the use of the Fe₃O₄@C microspheres as templates,metal isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into metal oxide by calcinations.By using this strategy,we successfully prepared a series of Fe₃O₄@M_xO_y magnetic microspheres,including Fe₃O₄@ZrO₂,Fe₃O₄@Al₂O₃,and Fe₃O₄@Ga₂O₃ magnetic microspheres.The as-prepared Fe₃O₄@M_xO_y core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of model proteins to exemplify their selective enrichment ability of phosphopeptides from complex protein samples.In only 0.5 min,phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe₃O₄@M_xO_y microspheres.The performances of these Fe₃O₄@M_xO_y microspheres were further compared with commonly used commercial IMAC resin and TiO₂ beads,and the results proved stronger selective abilities of Fe₃O₄@M_xO_y microspheres over IMAC resin and TiO₂ beads.Finally,the Fe₃O₄@M_xO_y microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum,digests of non-fat milk and rat liver tissue extracts.The results open up a possibility for their further application in phosphoproteome analysis.In summary,the main contributes of this dissertation is that we initially synthesized several functional magnetic materials and successfully utilized them for rapid protein digestion and phosphorylated peptides.We aimed at exploring and finding out new techniques in proteolysis and selective concentration of proteome research fields,so that more breakthroughs can be obtained in the proteome research study.