Study On The Large Pore-size Mesoporous Materials Used As MALDI Matrix And Carrier For Speccific Enrichment And Proteolysis

The study of this dissertation was focused on the applications of several novel mesoporous materials with large mesopores as inorganic matrix to the analysis of short peptides in MALDI-TOFMS,and as carrier for the highly-selective enrichment of phosphorylated peptides from complex sample and the highly-effective proteolysis on column and on MALDI target.Some meaningful advances have been achieved.MALDI-TOFMS has been established as powerful tools for the identification of proteins by peptide mass fingerprinting in proteomics.In proteomic study,the involved real-world samples are complex,contain thousands of proteins,and especially some post-translational modified proteins,exist with low stoichiometric concentration.Therefore high-throughput MS analysis with rapid speed and enhanced sensitivity is becoming more important and to develop new techniques for the rapid and effective sample analysis based on MS is necessary.Since the discovery of mesoporous material MCM-41 in 1992,the preparation methods of mesoporous material have been kept innovating and developed to be mature,resulting in the successful synthesis of a large amount of novel mesoporous materials.These novel mesoporous materials exhibit large pore size,open mesopore structure,tunable compositions,various morphologies,high mechanical strength and abundant properties.Now the application of mesoporous materials in bio-analysis fields is attracting attention all over the world,which will not only open up a new possibility for the rapid and high-throughput MS analysis but also extend the application of mesoporous material.In this dissertation,the application of several novel mesoporous materials to bio-analysis based on MALDI-TOFMS has been firstly investigated.The research work consists of three parts and is described as follows:In the first part,the mesoporous materials with multi-components were developed to act as matrix for the analysis of short peptides using MALDI-TOFMS.The tested mesoporous materials included WTiO,TiO₂,CeTiO,ZrTiO,TiSiO,ZrO₂,AlTiO, AlSiO,and SiO₂.These materials with different components showed drastically different capacity to ionizing the anlaytes.The results seem to indicate that UV absorption is a pre-requisite for a material to be used as an inorganic matrix,and the stronger the absorption,the higher the ionizing efficiency.Using CeTiO with high UV absorption demonstrated that PPG can be easily detected with reasonable S/N at low pico-mol level,which represents a significant improvement compared with the nano-mol detection level previously reported.This result can be due to the porous structure which enhances the surface area greatly.By using WTiO with different mesopore structures as matrix,effects of characteristic features of mesoporous matrices on laser desorption/ionization process were investigated in detail. Experiments showed that ordered two-dimensional(2D)and three-dimensional(3D) mesoprous matrices displayed a superior detection for analytes.The dramatic enhancement of signal sensitivity by the ordered mesoprous materices can be evidently attributed to the ordered structure,which facilitated the understanding on structure-function relationship in mesoporous cavity for laser desorption process of adsorbed biomolecules.With the ordered mesoporous matrix,the short peptides are successfully detected.The presence of trace alkali metal salt increased effectively the analytical ion yields and the MALDI MS using the inorganic mesoporous matrices displayed a high salt tolerance.The developed technique also showed a satisfactory performance in peptide-mapping and amino-acid sequencing analysis.In the second part,mesopore Fe_2O3 particles with large pores were applied to the separation and enrichment of phosphopeptides.Large mesopore Fe₂O₃ particles were prepared by the assembled Fe(OH)₃ sol-particles with the assembly of urea and formaldehyde as inducement.The experimental conditions for the enrichement of phosphorylated peptides were optimized.Under a more amiable loading/elution condition than the reported,mesoporous Fe₂O₃ particles displayed a highly-selective extraction of phosphor-peptides from the complicated tryptic products of proteins. Especially,the peptides with multi-phosphorylation sites can be directly analyzed from the Fe₂O₃ particles using MALDI-TOFMS,while the multi-phosphorylated peptides were lost in the direct MS analysis of Fe³⁺-IMAC beads.Phosphopeptides including mono- and multi-phosphorylation sites from 100fmol tryptic digests ofβ-casein is still detectable using this new approach and this level is much lower than that which can be obtained using the conventional Fe³⁺-IMAC.The improved singnal sensitivity of phosphopeptides can result from the less strong interaction between the phosphopeptides and Fe₂O₃ surface than that between the phosphopeptides and Fe³⁺. In addition,the process using mesoporous Fe₂O₃ particles to extract phosphopepteds has been dramatically simplified because the metal ion chelating and washing steps required for preparation of IMAC beads were eliminated.In the third part,the use of mesoporous silica with ulta-large pores as the trypsin immobilized host and packed bead of capillary column was studied for the on-column digestion.Protein digestion is one of the key elements in proteomics and immobilization of trypsin on solid supports can increase the digestion speed. Ultra-large mesopore MCFs(21.9nm of window size,32.4nm of pore size)were facilely synthesized and slurry packed into capillary column with the immobilized trypsin in MCFs mesopores.A rapid and highly efficient proteolysis of myoglobin(a protein known to be rather resistant to proteolysis)can be successfully achieved through the trypsin-immobilized MCFs column:sixteen myoglobin tryptic peptides were identified with a residence time of 19s,corresponding to a sequence coverage of 95.4%.This proteolysis efficiency is much higher than that of the conventional in-solution digestion(four peptides corresponding to 26.7%sequence coverage). SBA-15(～8 nm of pore size)was also employed as column substrate for the on-column digestion of myoglobin,resulting in a sequence coverage of 22.2%.The ultra-large pore and window sizes of MCF are expected to facilitate a fast mass transfer.After the adsorption of trypsin onto the inner mesopore surfaces,there is still a large free space left in the ultra-large mesopores of MCFs,ensuring a great myoglobin accessibility to the entrapped trypsin.The trypsin immobilized onto MCFs also showed a good stability and the MCFs column can be efficiently used at least for 30 times.In addition,on-target tryptic digestion was performed to generate peptide-map.The effect of three types of mesoporous particles(FDU-12,SBA-15 and WTiO)on the on-target tryptic digestion was investigated.The experiment showed that the addtion of mesoporous material onto target can effectively improve the digestion efficiency.WTiO demonstrated higher peak intensities of proteolysis products than the siliceous FDU-12 and SBA-15,owing to its desorption/ionization ability to desorb more tryptic peptides out of the mesopores.