Novel Isolation And Identification Approaches For N-termini Analysis Of Proteins Using Bio-Mass Spectrometry And Protein Chemistry

Proteomics has become a frontial discipline of life sciences. Proteomics refers to the analysis of all the proteins expressed in a cell or tissue, and then the discovery of key proteins with potentials to be used as markers for diagnosis or targets for meditation by functional or comparative proteomics.N-termini of proteins play very important roles in biological processes. First, the characteristic three-dimensional structure is vital for the biological function of the protein, while the native folded structure of a protein is dictated by its covalent structure, that is, its primary amino acid sequence. So the primary amino acid sequence of a protein has been an important topic in the life sciences as a means of understanding the complicated biological processes in living cells, and the N-terminal sequencing is one of the most important content in it. In all living cells, protein synthesis is initiated from the N-terminal with either methionine (in the cytosol of eukaryotes), or formylmethionine (in prokaryotes, mitochondria, and chloroplasts).N-terminal area provides the original and important site for protein processing and has great effect on the biological functions of proteins. For example, in vivo half-life of a protein is a function of its amino-terminal residue (the N-end rule). And the myristoylation, a saturated C14 fatty acid linked with proteins through an amide bond to N-terminal glycine residue was observed to participate in intracellular growth factor signaling pathways and the increased hydrophobicity provided by a long-chain fatty acid might be expected to facilitate interaction with the lipid bilayer and/or other membrane proteins. In addition, the terminal sequence of a protein is important in identification of protein. Analysis of the theoretical N-terminal peptides of a large number of proteins in the SWISS-PROT databases found that N-terminal sequences of five amino acids in length were unique for the protein identification from 74% to 97%, depending on the species studied. This remarkable specificity reveals that the terminal peptides are ideal sequence tags for rapid protein identification; especially for those entries for which complete genome sequencing has been finished.To characterize mature target proteins from living cells, candidate proteins are often analyzed by biomass spectrometry through characteristics of proteins such as peptide mass fingerprinting (PMF) and tandem mass spectrometry (include peptide sequence tag (PST) and de novo sequencing), combind with database searching by computational algorithms. Because of a great amount of information contained in terminal sequences and potentially high throughput of the method not requiring very high sequence coverage to be achieved by extensive sequencing, proteomics based on the analysis of N- and C-terminal amino acid sequences (terminal proteomics) should render higher fidelity results. With the success of proteomics and in light of the importance of protein modifications, there is a need to the specifically fine characterization of N-termini with various long standing and brand-new protein chemistry methods, but a simple method to enrich and sequence terminal peptides from a protein mixture has not yet been well established. The aim of this study was to develop some new methods to enrich and identify terminal peptides of proteins based on some chemical modifications and mass spectrometry.This dissertation consists of four chapters and the contents are summarized as follows:In the first chapter, the key techniques in the proteomics study and the progress of N-termini research of proteins have been reviewed.In chapter 2, a new type of functional magnetic nanoparticles coated with DITC was synthesized and applied to the isolation and identification of N-terminal blocked peptides. Fe3O4 magnetic particles were synthesized by the chemical coprecipitation method, and then were coated with tetraethyl-orthsilicate onto the surfaces of the nanoparticles. And finaly, phenylene diisothiocyanate-coupled magnetic nanoparticles (DITC-coupled MNPs) were prepared through a linker reagent. A quick isolation and identification of N-blocked peptides from protein digests was achieved by using this newly synthesized type of DITC-coupled MNPs. The new method combines the selective isolation of residual N-blocked peptides with direct sequencing of the peptides by mass spectrometry. After the tryptic products of proteins were guanidinated to prevent side-reactions and to improve the signal intensities of the lysine-terminal peptides in PMF, the peptide mixture was mixed with the DITC-coupled MNPs, and the unmodified N-terminal peptides which were covalently bound to the functional magnetic nanoparticles under alkaline conditions were selectively removed from solution when the nanoparticles were placed in a magnetic field. Thus, the residual N-blocked peptides remaining in solution could be isolated and then analyzed by MALDI mass spectrometry. The covalent interaction between the DITC-MNPs and the amino groups is stronger than an affinity-based interaction, thus permitting a good isolation of N-blocked peptides. Their extremely small size and large surface area confer a higher trapping capacity for the free amino groups of unblocked peptides to these nanoparticles than to micro-scale beads. Moreover, the DITC-coupled MNPs are low-cost and easily prepared, and the process of isolation of N-terminal blocked peptides is very easily carried out due to using the magnetic particles.In chapter 3, a new method for quantifying proteins and identifying their N-termini by using homolog labeling coupled with MALDI-TOF mass spectrometry were proposed. First, guanidination of the lysine side-chain was performed to prevent multiple labeling. Then acetic anhydride and its homolog, propionic anhydride were used to label the N-termini of proteins at room temperature. Two equimolar parts of differently homolog labeled proteins were combined and then separated through SDS-PAGE. After in-gel digestion with trypsin, peptides were extracted and analyzed by using a MALDI-TOF mass spectrometer. Peak pairs with a mass difference of 14 Da were identified as N-terminal peptides and the ratios of their peak intensity were calculated for relative quantification of proteins. The results obtained from 3-protein mixture demonstrate that this homolog labeling method enables the identification of N-terminal fragment ions from other fragments and quantitation of proteins simultaneously. Homolog tags are more readily available for a bigger mass shift than isotopic tags, whichcan avoid peak overlapping and make the interpretation of peaks easier and cheaper in price. These make the method potential for application in quantitative proteome study.In chapter 4, we applied a new method to enrich and identify terminal peptides of a RP-fraction of proteins from Ecoli by sulfonation and strong cation exchange (SCX) chromatography and electraspray ionization tandem mass spectrometry (ESI MS/MS). Protein databases provide only a small amount of sometimes inaccurate N-terminal data, and more detailed information is required except only depending on database searching. The N-terminal sulfonated derivative is reported to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment y-ion series, which facilitates the de novo sequence analysis of peptides. After proteins were successively sulfonated, reduced, and digested with trypsin, the digest of proteins were separated using SCX chromatography and the sulfonated terminal peptides were effectively collected and sequenced using ESI MS/MS. Terminal peptides and their parent proteins were successfully analyzed by de novo sequencing and protein database search. And some peptides with Met cleavage and a peptide with 23-length signal peptide removed were found in the proteins from Ecoli through this method. In summary, three new methods were developed based on protein chemistry and mass spectrometry, and experimental results demonstrated the potential of these methods for isolating and confirming amino acid sequence of N-terminal peptides from complex mixtures.