Study On Several Micro - Nano - Technology For Biomass Mass Spectrometry

There are three academic values of this doctoral thesis. First, we have developed three new methods for solving three important problems in proteomics research. Micro-droplets combining with mass spectrometry (MS) were used to solve the problems of loss identification of co-eluted peptides, on-line protein digestion and on-line protein concentration. Second, we have established a novel graphene coated paper-spray MS for drug monitoring in the saliva, which is clinically useful in point of care therapy. Third, we have designed new ion sources and interfaces for MS to improve the functions of MS for sample analysis and some new techniques for sample introducing were also provided.Proteomic was first named by Marc Wilkins in1995, and it developed very fast especially for the past ten years. The development of proteomics relied on the developments of techniques, and MS has been widely recognized as a basic instrument and played an important role in the proteomics research. In MS-based proteomics research, there were still many problems existed and it was very important to solve those problems for promoting the development of proteomics.First, the shot-gun technique was one of important ways for proteomics research. Nano-high performance liquid chromatography (Nano-HPLC) separation and MS detection have been a basic way for proteomics research. For complicated samples, many peptides co-eluted, MS could not have enough time to do all the detail analysis and caused the problem of incomplete identification. The routine methods for solving this problem was to pretreat sample in order to decrease the existence probability of co-eluted peptides or repeat samplings to enhance the probability of identification for co-eluted peptides. None of these methods were good enough from an analytical view. For the pretreatments, sample loss would happen during these processes and pollutions might be introduced; repeat samplings needs more sample and time, this could not be done especially for cherished sample.Second, the top-down technique was the other important way for proteomics research. In the routine analysis, proteins were separated by HPLC and the fractions were collected. Then the fractions were digested by protease and analyzed by HPLC-MS/MS. For the complicated sample, the proteins were separated by HPLC would decrease the complexity and give better result. However, there was a problem during this process that the digestion was finished off-line, the time needed was very long, sample loss and pollutions might happen during these processes. It’s urgent to develop new methods to solve this problem.Third, the low abundance proteins or peptides were very difficult to identify no matter which technique way was used. The different materials which have functions of sample enrichment were used by different labs to enhance the identification of low abundance proteins (peptides), but the efficiency and selectivity were always the key points. The low selectivity may cause big problems for identification and the same problem as described above, the off-line method would cause pollution and sample lose. It’s important to develop new methods to do the on-line concentration and it’s better to suit for all kinds of sample.For the accurate qualify and quantify characters, MS has been used extensively for clinical analysis. Therapy drug monitoring was very important for clinical analysis, and it could supply judgments for medical use and avoid toxic reaction. The routine methods for therapy drug monitoring were very complicated, and pollutions might happen during these processes, the analysis would be affected. It is important to develop new techniques to solve these problems.As a useful tool for sample analysis, MS has many ion sources for different kinds of chemicals. The ion source commercialized has some limitations for special samples for its universality. The low efficiency of electro-spray was taken as an example. For MS based coupling techniques, there were also some limitations like on-line concentration, and with the demanding need for high throughput analysis, sample introducing technique was also need to improve.In this thesis, we introduce our research in six chapters.In the first chapter, the important MS application areas were introduced and the opportunities and challenges were reviewed. For the coupling techniques with MS, the developments and challenges were also introduced, all the backgrounds provide theoretical and applicable support on the following research.In the second chapter, a segment flow protocol has been proposed for coupling HPLC with MSn in peptide-based proteomics research. HPLC effluent was segmented by perfluorodecalin oil and stored as segments in sequence to carry out the following off-line MS" analysis. In this way, incomplete identification of co-eluted peptides resulting from the MS scan rate, relatively slower than the HPLC elution rate, was solved to some extent for on-line coupling HPLC-MS" analysis. The complex proteins from a sample of T.tengcongensis were successfully identified with once run using segment as interface between HPLC and MSn analysis, comparably to the total identified proteins with six runs using routine on-line HPLC-MSn analysis. This segment flow approach could eliminate detection loss and sample cross-contamination, providing a new method for peptide-based proteomic research, especially when the amount of sample is limited.In the third chapter, a platform for on-line digestion proteins combining with microfluidics chip was provided. The effluent from HPLC and protein protease were mixed in the micro-droplet during the micro-channels and the proteins were digested quickly then to do the MS analysis. This platform has been successfully applied for analyzing proteins mixture about lyzosome, cyto-c and myoglobin, supplied a new method for top-down proteomics research.In the fourth chapter, a platform for on-line concentration combing with microfluidics chips was provided. The partial soluble oil was used to produce micro-droplet. During the flow through the micro-channels, the water was lost for the reason that oil absorption. The sample was concentrated and detected by the following MS analysis. This approach has been successfully applied for the analysis of trypic digest of bovine serum albumin (BSA), and the coverage rate was highly enhanced.In the fifth chapter, a graphene coated paper combining MS to do paper-spray was used for therapy drug monitoring in saliva. Sample pretreatment was not needed during this procedure, and the limit of detection was lower than routine paper-spray analysis. The other important advantage was that the linear range for quantification was10times larger than routine analysis.In the sixth chapter, the new developments for ion sources and interface techniques were provided to improve the performance of MS, and the report of self-made MS instrument was also mentioned. These techniques mentioned in this chapter would be very helpful for the future work of MS analysis.