Proteomics Efficient Enzymatic Hydrolysis Of New Technologies

In chapter 1,a bird view of proteomics advancement and achievements were given.Instruments and methods used in proteomics were summarized,along with the impacts of proteomics on modern biological science.And the proteolysis techniques ever developed was summarized with two parts:1) the new types of enzyme reactor,2) microwave and ultrasound techniques.Enrichment techniques used in proteomics was also briefly introduced.The new strategies of high efficient proteolysis in this thesis were briefed at the end of this chapter,demonstrating their importance and usefulness. This dissertation contributes a series of new strategies of high efficient proteolysis for proteomic analysis.And a short study on nanomaterial for phosphopeptides enrichment was described.The main contents are:In Chapter 2,a four-component nanocomposite,trypsin-immobilized polyaniline-coated Fe₃O₄/carbon nanotube composite,was synthesized for highly efficient protein digestion.Fe₃O₄ was deposited by the chemical coprecipitation of Fe²⁺ and Fe³⁺ in an alkaline solution containing carbon nanotubes(CNTs) to prepare nano-Fe₃O₄/CNT composite.Subsequently,polyaniline(PA) was assembled on the Fe₃O₄/CNT composite by the in situ polymerization of aniline in the presence of trypsin to obtain trypsin-immobilized PA/Fe₃O₄/CNT nanocomposite.The novel 1D superparamagnetic biomaterial has been characterized by TEM,SEM,XRD,and magnetometric analysis.The feasibility and performance of the unique magnetic biomaterial have been demonstrated by the tryptic digestion of bovine serum albumin, myoglobin,and lysozyme within 5 min.The digests were identified by MALDI-TOF MS with sequence coverages that were comparable to those obtained from the conventional in-solution tryptic digestion.The present biocomposite offers considerable promise for protein analysis due to its high magnetic responsivity and excellent dispersibility.It can be easily isolated from the digests with the aid of an external magnetic field.Because the enzyme-immobilized nanocomposite can be prepared by a simple two-step deposition approach with low cost,it may find a wide range of biological applications including proteome research.In Chapter 3,a core-changeable needle enzymatic reactor was developed for highly efficient proteolysis.Self-assembled layers of CTS and SA were first constructed on the surface of a glass fiber;this glass fiber was then immersed in trypsin solution to immobilize trypsin.A piece of enzyme-immobilized fiber core was inserted into the needle of a syringe pump to form a flow-through bioreactor.The novel in-needle bioreactor could be regenerated by changing its fiber core.The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin and lysozyme,and the digestion time was significantly reduced to less than 5 s.The digests were identified by MALDI-TOF-MS with sequence coverage comparable to those obtained by the conventional in-solution tryptic digestion.The significantly enhanced digestion efficiency of the in-needle fiber bioreactor can be attributed to the high concentration of trypsin in the modified layer on the fiber core and the higher surface area of the fiber core in the needle,increasing the interaction frequency between trypsin and proteins.In addition,the biocompatibility of CTS and SA(two naturally occurring polymers) may provide milder environmental conditions so that the denaturation of the immobilized trypsin was minimized.Besides the high digestion efficiency,an additional advantage of the present approach is its simplicity,which is promising for the automatic high-throughput protein analysis using a platform containing multiple in-needle fiber bioreactors.In Chapter 4,Sinusoidal alternating voltages(typically 5V) were employed to enhance the efficiency of proteolysis for peptide mapping.Protein solutions containing trypsin were allowed to digest with the assistance of alternating electric fields(AEFs) between a pair of platinum wire electrodes in Eppendorf tubes.The feasibility and performance of the novel proteolysis approach were investigated by the digestion of several standard proteins.It was demonstrated that AEFs significantly accelerated in-solution proteolysis and the digestion time was substantially reduced to 5 min.The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) with sequence coverages that were comparable to those obtained by using conventional 12-h in-solution proteolysis. The suitability of AEF-assisted proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by sodium dodecyl sulphate/polyacrylamide gel electrophoresis(SDS-PAGE). The present proteolysis strategy is simple and efficient and will find a wide range of applications in protein identification.In Chapter 5,alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping.Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity.Alternating current(AC) was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c(Cyt-c).It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min.The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) with sequence coverages of 42%(BSA) and 77%(Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion.The present proteolysis strategy is simple and efficient,offering great promise for MALDI-TOF-MS peptide mapping.In Chapter 6,infrared(IR) radiation was employed to enhance the efficiency of tryptic proteolysis for peptide mapping.The ease,simplicity,efficiency,and low cost of the novel proteolysis approach indicate great promise for the high-throughput protein identification.Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under IR lamp radiation at 37℃.The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of bovine serum albumin(BSA) and myoglobin(MYO) and the digestion time was significantly reduced to 5 min.The obtained digests were identified by MALDI-TOF MS with the sequence coverage of 69%(BSA) and 90% (MYO) that were much better than those obtained by conventional in-solution tryptic digestion.The present IR-assisted proteolysis strategy is simple and efficient,offering great promise for high-throughput protein identification.Infrared(IR) radiation was also employed to enhance the efficiency of chymotryptic proteolysis for peptide mapping.The suitability of IR-assisted chymotryptic proteolysis to complex proteins was demonstrated by digesting human serum.The present proteolysis strategy is simple and efficient,offering great promise for high-throughput protein identification.The significantly enhanced digestion efficiency of the present proteolysis approach can be attributed to IR radiation,which increases the energy level of vibrations of peptide bonds so as to decrease the activation energy of proteolysis reaction,which might lead to a great increase in the reaction speed.In addition,the IR-induced vibrations of the chains in proteins might also lead to more cleavage sites exposed to enzymes,resulting in easier cleavage of peptide bonds.It might be the reason why there were more matched peptides in the PMF spectra of the digests obtained by using IR-assisted digestion.In Chapter 7,infrared(IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping.Protein solutions containing trypsin were allowed to digest directly on the spots of MALDI plates under IR radiation.The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin(BSA) and cytochrome c(Cyt-c).It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min.The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) with sequence coverages of 55%(BSA) and 75%(Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion.The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample.The present proteolysis strategy is simple and efficient,offering great promise for high-throughput protein identification.In Chapter 8,to faciliate the enrichment of phosphopeptide or phosphoprotein several kinds of metal ions Ga(â…¢),Al(â…¢),Zr(â…£),Zn(â…¡),Fe(â…¢),Ni(â…¡),were trapped on the surface of Fe₃O₄ magnetic nanoparticles with phosphate group binding. Amine functioned Fe₃O₄ nanoparticles were synthesized first,then the amine groups on the surface was reacted with POCl₃,resulting in a phosphate group functioned surface.Then the nanoparticles were immersed in solutions of different kinds of metal ions to immobilize metal ions on its surface.And the ability of those magnetic particles in isolating phosphopeptides was studied withβ-casein tryptic digest.Results show that only Fe(â…¢) modified magnetic nanoparticles work best.When the magnetic nanoparticle enrichment was coupled with MALDI TOF mass analysis,a detection limit lower than 20 fmol/μL was observed.And good specificity was demonstrated whenβ-casein digest was mixed with BSA in the ration of 1:1000.