Oligonucleotides Of Biological Mass Spectrometry Research

The order of base losing and fragmentation of oligonucleotide during electrospray ionization and MS/MS process has been studied using ESI-Q-TOF and described in chapter 1. It is found that base losing is favored in the order of A>OG>T and the fragmentation process is related to the number of charges that oligonucleotide ions hold. The more charges they hold, the easier they lose bases. Influence of base localization is not obvious to the process. The order we observed is not the same as the one reported during MALDI process. It maybe respondent to the energy of the covalent bond linked the base with DNA strand. These ions losing different base were chosen as the parent ions and their MS/MS process were studied. We found that the ions losing one base can lose the other base continuously and this process could occur spontaneously. It was also found that base losing has no direct correlation with the cleavage of the 3'C-O bond adjacent to the deoxyribose unit. A new fragmentation mechanism of oligonucleotide during MS/MS process is put forward based on the observed phenomena and mechanisms reported before. In the meantime, the process of base losing and fragmentation of phosphorothioate oligonucleotide during electrospray ionization and MS/MS has also been studied. The same mechanism as oligonucleotide was observed and ten base sequences could be read out based on MS/MS spectrum easily.A robust high-throughput single nucleotide polymorphism (SNPs) genotyping method was reported in chapter 2, which applied allele-specific extension to achieve allelic discrimination and used MALDI-TOF-MS to measure the natural molecular weight difference of oligonucleotides for determination of the base in a single nucleotide polymorphic location. Each procedure in the method was optimized and discussed in detail. ESI mass spectrometry was also attempt to replace MALDI-TOF as a detecting method. The advantages and disadvantages by using MALDI-TOF mass spectrometry and ESI mass spectrometry in large-scale and highthrough-put SNP genotyping are discussed.The complex of anti-sense drug and BSA was studied using ESI-Q-TOF massspectrometry and described in the last chapter. The stoichiometry and concentrations of all components in the system were calculated based on the molecular weight of the complex and the peaks intensity in the mass spectrum. Then dissociation constants are deduced. One molecular BSA can bind two molecular anti-sense drugs. Km equals to 0.5μM and KD2 equals to 4μM...