Studies On The New Technologies And Methods Of Biological Mass Spectrometry And Their Applications For Proteomics

The main contributions of this dissertation for the degree of doctor of philosophy are listed as followed, Carrying out many exciting discoveries and inventions of the enrichment and desalting of low-abundance proteins and peptides in proteomics research; Novel one-step on-plate desalting and enrichment protocol for low-abundance proteins by using multifunctional hydrophobic polymer micro-contact-printing; On-plate enrichment and desalting method of low-abundance phosphopeptides for direct MALDI-TOF MS analysis by using cationic polymer and above "one-step desalting" system; Fast and effective enrichment of low-abundance proteins based on combined nano-materials; Development of high-resolution ESI-Q-TOF-MS used as the sensitive and efficient detector of HPLC .Proteomics — one of the most important fields in the life sciences during the post-genome era— has burst onto the scientific scene with stunning rapidity over the past few years. Proteomics has also become the frontal discipline of analytical chemistry. Proteomics refers to the analysis of all the proteins expressed in a cell or a tissue. The works on proteomics include the characteristics and abundance studies for proteins to find out their functions and locate the key protein which is related to the disease. By studying global patterns of protein abundance and activity and how these change during development or in response to disease, proteomics research has boosted our understanding of systematical cellular behaviour and mechanism of disease. In addition, proteomics benefits the identification of new drug targets and the development of new diagnostic markers in clinical research. Recently proteomics have been obtained many significant achievements in the studies of generation and evolvement of the diseases.The rapid development of proteomics relies on the advances in the high throughput analytical technologies including biological mass spectrometry (Bio-MS), multi-dimensional separation, and bioinformatics. MALDI and Electrospay —the soft ionizations—have been the key techniques of Bio-MS and applied to the proteomics research. Identification of all proteins in a cell or a tissue and theirlocations in the organism and finding out the key proteins by comparative proteomics has made Bio-MS become the critical tool in proteomics study. Many countries have made a large investment in proteomics and the accomplishment of proteomics would play a significant role in finding out drugs and curing diseases.The work of this doctoral dissertation was mainly accomplished in the Bio-MS laboratory of Department of Chemistry and the Research Center for Proteome of Institutes of Biomedical Sciences of Fudan University. Because the conventional Bio-MS technique platform was difficult for the detection of low-abundance proteins of proteome, the related studies of new technologies and methods become more and more important. Therefore, this dissertation studied on how to enrich and desalt low-abundance proteins effectively and develop some novel strategies: one-step on-plate desalting and enrichment method for low-abundance proteins, on-plate enrichment and desalting of low-abundance phosphopeptides for direct MALDI-TOF MS analysis, fast and effective enrichment of low-abundance proteins depending on combined nano-materials. Since the successful tandem mass spectrometry is very important for the identification of proteins, this dissertation described the whole sequencing process for one synthetic complicated polypeptide composed of 28 amino acids successfully and established a good supplementary method of peptides sequencing for proteomics research. In addition, this dissertation developed high-resolution TOF-MS used as the detector of HPLC.This doctoral dissertation consists of six parts and the contents are summarized as follows:In the first chapter, as the key technique in the proteomics study, Bio-MS and its related techniques and applications in the proteomics have been reviewed. Bio-MS becomes more and more important in proteomics with the development of the two soft ionization techniques namely ESI and MALDI, with the improvement of the mass analyzer, with the hybrid of HPLC and CE, with the development of the micro-electrospay and nano-electrospay, as well as the application of tagging by stable isotope. The Bio-MS technique is a definitely powerful tool for the study of the dynamic process of protein-protein reaction, the structure of genetic product, proteins related to diseases and functional protein. As a fresh discipline, the development of proteomics also depends on the newest technical development such as 2DE, chromatography, protein chip, Yeast two-hybrid system, tandem affinity purification,and so on. The analysis of low abundance proteins has been proved to be a difficult problem since there is no enough technique for the signal amplification of these proteins. Therefore, the isolation and enrichment protocols for low abundance proteins are desirable in proteome research. So a review of the analytical technologies for low-abundance proteins was presented and the investigative direction and purpose of this dissertation were also put forward.The research works described in the second chapter centers on a novel method of one-step on-plate desalting and low-abundance peptides concentration. A novel micro-contact-printing protocol of multifunctional hydrophobic polymer has been developed for 2DE-MALDI-MS, which can achieve one-step on-plate desalting and analyte concentration. The polymers of PMMA, PMMA-C60, PST and PST-C60 are applied successfully as the MALDI coating materials in the first time. The one-step preparation protocol has a unique and ingenious feature which using "outward-flow" for the separation of analytes from salts. The new micro-contact-printing method of hydrophobic polymers permits analyte to be concentrated on the center of MALDI spot, and meanwhile allows salts to be separated and removed toward the outside of MALDI spot. The on-plate desalting process can be accomplished only for one step without any excessive washing procedure such that the new protocol would provide a potential route to solve the identification problem of low-abundance protein. Normally, the process for proteins digested and transformed from a gel spot to the MALDI target needs a number of steps including protein digestion, peptide extraction, desalting and washing, so low-abundance protein might be easily lost during all these steps. It is expected that the new protocol of hydrophobic polymer micro-contact-printing can effectively reduce the losing of peptides because of the on-spot desalting. The high through-put experiment by using this on-spot desalting strategy is currently on the way in our laboratory.For the works mentioned in the third chapter, on-plate enrichment and desalting method of low-abundance phosphopeptides for direct MALDI-TOF MS analysis is well tried. Phosphorylation, the most important and ubiquitous post-translational modification of proteins, is able to regulate almost all aspects of cell life in both prokaryotes and eukaryotes. Widespread interest in protein phosphorylation has led to the development of methods to map phosphorylation sites of proteins. The existing preferred approach for phosphorylation site mapping mainly relies on the use of tandem mass spectrometry to sequence individual peptides after proteolysis. However,this method still remains challenging because the signals for phosphorylated peptides are strongly suppressed by other abundant unphosphorylated peptides contained in protein digests during positive mass spectrometric analysis. Therefore, the separation and enrichment of phosphorylated peptides from unphosphorylated ones is highly desirable. At present, one solution to this problem for proteomics research is to carry out HPLC separation of the phosphopeptides from the other peptides prior to mass spectrometric analysis. Immobilized metal ion affinity chromatography (IMAC) has been often used for the selective enrichment of phosphopeptides from proteolytic digest mixtures. However, chromatography introduces additional sample handling and preparation time prior to MALDI or electrospray mass spectrometry. In this chapter, we reported a solution to the problem of the suppression of phosphorylated peptides during positive ion MALDI-TOF-MS by using the cationic-polymer coating plate combined with the "One-step desalting" system. This cationic-polymer coating can efficiently enriches the phosphopeptides and improves the sensitivity of the MS devices, Meanwhile it also eliminates the interference of the salts on the plate which would simplify the procedure of sample concentration and desalting.In the fourth chapter, the development of a particle-free MALDI and " octopus-like" enrichment protocol is mentioned. In this protocol, we select nanoparticles of calcium carbonate derivatized with poly(methyl methacrylate) (PMMA) because of its ability to adsorb the peptide/protein. The particle denoted as CaCO₃ - PMMA has a destroyable core of CaCO₃, which can be removed later for a particle-free MALDI analysis. When the CaCO₃ - PMMA material is added dropwise to the sample solution, the linear chains of PMMA linked on the CaCO₃ core will be flexible and spread out into the solution like the tentacles of an octopus. Not only the CaCO₃ - PMMA nanoparticles are applied for the first time for the enrichment of low-abundance peptides and proteins, but also the process of enrichment is very quick and efficient. The resulting nanoparticle-adsorbed peptides or proteins can be analyzed by MALDI-TOF MS in the particle-free mode with high reproducibility and good recovery, thereby avoiding sample loss and contamination inside the mass spectrometer. It has been found that CaCO₃ - PMMA nanoparticles exhibit a strong enriching and desalting ability, which could pave way to novel approach to the enrichment of trace-level proteins or peptides, as well as new applications for CaCO₃ - PMMA nanoparticles.One synthetic complicated polypeptide composed of 28 amino acids wassuccessfully sequenced by MALDI-MSMS (CID) and ESI-MSMS (CID) in the fifth study. I obtained the perfect tandem mass spectrum consisting full product ions of b and y by investigating the parameters of mass spectrometry instruments, such as the intensity of Laser, the energy of CID, the difference of sequencing software. et al. Results indicate that this method can effectively solve the difficulty occurring in de novo sequencing, such as dead-response of computer. The established method is a good supplement for peptides sequencing and the discussion of various parameters is important for the establishment of tandem mass spectrometry proocol in proteomics research work.In the last chapter, two ESI-Triple Quadrupole-Orthogonal Acceleration-Reflecting-TOF mass spectrometer analytical systems are successfully developed, which are equipped with electrospray ionization source which could be directly connected with HPLC as an outstanding detector. The ESI-QQQ-TOF mass spectrometers have some promising features, such as an ESI source with heatable nebulizer settled on a smart triaxially moving stage, an excellent ion-optics system composed with triple-quadruple, double-pulse acceleration, and a double stage reflector with homemade grids. The Gramicidin-S sample concentration below to 3 fmol/μL can be identified by these instruments under the optimized conditions. The mass range of these instruments is from 50 to 6000 u with a general ESI interface to the HPLC. The optimal mass resolution exceeds 11000 (Full Width at Half Maximum, FWHM) and the signal/noise ratio of reserpine (10 pg/μL) is greater than 100. We hope the homemade Q-TOF-MS would be widely used in protein chemistry and proteomics research due to its high sensitivity, high resolution and high accuracy.