| This dissertation is focusing on developing novel methods for proteomics research based on bio-mass spectrometry. The purpose of this research is about how to analyze targeted bio-molecules with a higher sensitivity and higher selectivity. In this dissertation, it demonstrates five newly developed methods which combine some cutting edge tenichques, such as nano material, mesoporous material, surface functionalization and peptide derivatization. In the following part, I will give a brief introduction of the six chapters of this dissertation.In the first chapter, the development of proteomics research was briefly reviewed. Several important methods and tools in this field were introduced. One of the most important soft ionization ion sources, matrix-assisted laser desorption/ionization (MALDI) source was reviewed. The application and major challenges of MALDI-MS was summarized. As the most important part of MALDI process, the MALDI matrix was reviewed in detail including both inorganic matrices and organic matrices. Besides, two of the most important protein post-translational modifications (PTMs), phosphorylation and glycosylation were reviewed. The recent development and several major reseach methods of these two PTMs were discussed in detail. Based on all these backgrounds, the intention and meaning of this dissertation were explained.In the second chapter, a diammoniu citrate doped TiO2 nanoparticles (TiO2DC) was investigated and evaluated as an inorganic MALDI matrix for molecules with masses ranging from 100 to more than 1000 Da. In comparison with traditional organic matrices and other inorganic matrices, the TiO2DC matrix offers several advantages:(a) low cost and simple sample preparation, (b) applicable to several kinds of analytes, (c) high ionization efficiency and low detection limit, (d) quantitative analysis for small molecule (m/z<1200). The limits of detection at a S/N ratio of 3 are down to 5.6 and 9.5 nM for sucrose andβ-CD. However, the sensitivity of DHC is relatively low. When we perform quantitative analysis with glucose, sucrose and P-CD, the linearity of each sample is pretty good (R2>0.98). According to these results, we believe that the TiO2DC matrix could be potentially used for both qualitative and quantitative analysis.In the third chapter, a chemical derivatization method of peptide carboxy groups with 1-(2-pPyrimidyl)piperazine (PP) was introduced to enhance the ionization efficiencies of peptides especially that of phosphopeptides and make them MS detectable without any specific enrichment. Once carboxy groups were derivatized with PP, the hydrophobicities, pI values and gas-phase basicities of phosphopeptides were largely increased so that the ionization efficiencies could be dramatically enhanced accordingly. As PP derivatization neutralizes the extra negative charges and decreases the hydrophilicities brought in by phosphate groups, the ionization efficiencies of phosphopeptides can be increased up to 101-fold. Comparing with other methods, this brand newimproved technique method needs less sample amount, costs less time and lacks any drawback of traditional derivatization methods such as low yields, harsh reaction conditions and unknown side reactions. This new method has been applied to both standard peptides and tryptic protein digests, and was proved to be very efficient and extremely powerful. We also expect it will be very promising in large-scale phosphoproteome and proteome analysis in the near future.In the fourth chapter, a novel di-boronic acid functionalized mesoporous silica (FDU-12-FG) was synthesized to specifically enrich glycopeptides. This developed FDU-12-GA matrix, with high surface area, large pore volume, and well-defined large sizes of pore cavities and entrances, leads to great advantages in glyco-specific enrichment. Firstly, the synthesis of FDU-12-GA is quite simple under moderate temperature and neutral conditions. Secondly, the large specific surface area greatly increases its binding rate, and the entire loading time needs only 15 min. With the plentiful di-boronic acid function groups on the surface, no nonspecific binding is observed in the presence of prominent tryptic BSA, demonstrating specificity of binding. By capturing and concentrating target glycopeptides, the suppression effect from nonglycopeptides can be eliminated, the LOD of glycopeptides is enhanced by close to two orders of magnitude, and the recovery of the enriched glycopeptides is up to 83.5%. Accordingly, with our newly developed method, various kinds of N-glycopeptides could be specifically selected and enriched for further analysis. We also expect it could be further applied to complex biology samples and glycoproteomics.In the fifth chapter, a method usingdi-boronic acid modified gold-coated Si wafer as MALDI target to enrich glycopeptides was introduced. This on-plate enrichment method has several advantages. Firstly, the synthesis procedure is simple and efficient in which no harsh conditions were needed and the whole process was less than 24 hr. Secondly, unlike previously published on-plate enrichment methods, which employed the strategy of direct modifying commercial MALDI plates, small pieces of SiAuB was used instead of commercial MALDI plate in our method. This characteristic makes it suitable for different samples and various purposes. Thirdly, minimizing the sample loss and eliminating the suppression effect have increased the detection limits of glycopeptides by two orders of magnitude. Furthermore, this is a recoverable method and the recovery of glycopeptides is up to 65.8%. At the same time, high concentration of ABC and physiological buffer PBS can also be removed through on-plate enrichment and no additional desalting step was needed. With this method, glycopeptides can be selectively and effectively enriched. It is a potentially powerful tool for high throughput glycoproteome research.In the sixth chapter, a method focusing on O-glycosylated peptide research was introduced and evaluated. Combining the process ofβelimination and Micheal addition, the O-linked glycan could be eliminated and a nucleophilic molecule could be added onto the O-glycosylation site. Thiocholine was adopted as the addition molecule in order to label the O-glycosylation site and improve the ionization efficiency of O-glycopeptide at the same time. Comparing with the former methods, this method gives several advantages. Firstly, the molecule weight of thiocholine is 120, which is large enough to label the O-glycosylation site. This would make the identification of O-glycosylation site much easier. Secondly, thiocholine carries a positive charge which could facilitate the ionization process of glycopeptide at the positive mode. This would dramatically enhance the ionization efficiency of O-linked glycopeptide. Thirdly, thiocholine was prepared through an enzymatic process which is easy to handle and the product is relatively pure. Finally, this method might be able to be applied to quanlitative analysis of O-glycosylation in future.In summary, this dissertation is focusing on proteomics and bio-mass spectrometry related methodology research. Five novel methods about high sensitivity and high selectivity detection of biological samples were demonstrated. We are trying to develop new methods in the analysis of biological samples, so that more breakthroughs can be obtained in the proteome research in the future. |
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