New capabilities in tandem mass spectrometry. Applications to chemically modified deoxyribonucleosides and ionic fragmentation mechanisms
Abstract
The capabilities of tandem mass spectrometry (MS/MS) are extended through the study of chemically modified DNA and gas phase ion fragmentation mechanisms. Deoxyribonucleosides, modified by methylating agents of known activity and selectivity, are investigated. High performance liquid chromatography (HPLC) effects separation of structural isomers. A desorption chemical ionization technique is employed on a triple stage quadrupole (TSQ) tandem mass spectrometer. Collision-induced dissociation of the protonated molecular ion of the targeted nucleoside produces abundant fragment ions. Multiple reaction monitoring of the modified nucleoside and an isotopically-labeled analog permits quantitation by comparison of ion intensity ratios. The limits of detection of 3-methylthymidine and O$\sp6$-methyl-2$\sp\prime$-deoxyguanosine, both minor modifications, approach those encountered in studies of in vivo methylation. Various instrumental and data system parameters were found to have a critical role in overall sensitivity and precision. The capabilities of a novel scan mode on a prototype hybrid tandem mass spectrometer of BEQQ geometry (B = magnetic sector, E = electric sector, Q = quadrupole) to both confirm suspected ionic fragmentation mechanisms and reveal unexpected sequences are demonstrated using a series of simple organic compounds. The scan detects those fragment ions formed in the high-energy (3 keV) collision-induced dissociation of a selected parent ion that are themselves precursors to a selected product ion generated by low-energy (30 eV) collision-induced dissociation. The scan is therefore designated as the reaction intermediate scan. The isotopic selectivity of this scan as well as its excellent signal-to-noise characteristics demonstrate its analytical potential.
Degree
Ph.D.
Advisors
Cooks, Purdue University.
Subject Area
Analytical chemistry|Biochemistry
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