Based On The Protein Digests Enrichment Of The Porous Material

With the successful accomplishment of the human genome project, new questions were derived. To further explore the question which cannot be answered by the genomics, the scientists proposed a post-genome project. Proteomics (proteome) study as an component of the post-genome project, has gradually become one of the most important forefront in the biolgical science. In view of their structures and adjustable surface chemistry, porous materials exhibit extraordinary properties in versatile applications. In this paper, the author combined biological mass spectrometry technical and porous materials, and successfully developed some novel methods for proteomics research, including the fast, efficient and convenient enrichment technolgy to effectively solve the loss of low abundance proteins; and a nanopore-based reactor for efficient proteolysis.In chapter 1, we review the recent progress including biological mass spectrometry, proteomics, and nano-materials. We also described the hot or difficult problems in these fields, especially focusing on the enrichment technique in the proteomics research about post-transnational modificatfon, the high efficient proteolysis technology, the imaging mass spectrometry technology, and so on.In chapter 2, we propose a novel strategy for the selective isolation and sensitive analysis of multi-phosphopeptides based on TiO2-gratfed mesoporous materials, in which MCM-41 and SBA-15 were chosen as the hard templates. The commercialized IMAC and TiO2 nanoparticles were further investigated in the phosphopeptide analysis for comparison. The enrichment efficiency was evaluated and measured by MALDI-TOF mass spectrometry. The results indicated that both TiO2-SBA-I5 and TiO2-MCM-41 exhibited the preferential affinity to multi-phosphopeptides compared with the other two widely used strategies. The mesoporous TiO2 based protocol showed highly selective and sensitive properties, where phosphopeptides could be successfully identified when the concentration as low as 3 fmol.In the third part, tryptic digestion of proteins in trypsin loaded porous silica has been presented and showed to be more efficient than standard in-solution digestion. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF). The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the highest proteo lysis efficiency was found to be achieved with MOSF. These results can be well interpreted by a sequential digestion model we proposed taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins solutions with certain amount of interference were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteo lysis as demonstrated by a digest of a real complex sample extracted from the cytoplasm of mouse liver tissue that showed better results than the typical in-solution protocol.In the fourth chapter, we focus on the research of MALDI-imaging mass spectrometry. Direct MALDI analysis in tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. The direct MS analysis obtained from tissue section can be converted into imaging maps,a method now known as MALDI-imaging MS. While this new emerging technology is still in the infant stage, and many difficulties or obstacles are on the road of advance. In the thesis, the rapid proteolysis technology developed above was introduced to the research of MALDI-imaging mass spectrometry, and the initial results was achieved. With the further development and optimization of these techniques, we believe that those bottleneck problems hindering the development of tissue imaging mass spectrometry would be solved, the emerging technology could be fully developed.