Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Patrick A. Limbach


There are two prominent problems recurrent during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of oligonucleotides: cation adduction and low molecular ion stability. It has been found that the use of organic base solutions of high gas-phase proton affinity, as used previously in electrospray ionization mass spectrometry (ESI-MS), can significantly reduce the cation adduction problem during MALDI-MS. It is proposed that the organic base solutions compete with alkali metal cations for binding to the phosphodiester backbone of the oligonucleotide upon transfer into the gas phase. A comparison of the utility of adding an organic base solution, such as imidazole, triethylamine and piperidine, as a co-matrix versus the standard addition of cation-exchange resin beads was made. The co-matrices studied were found to be more effective than the cation-exchange resin beads at reducing cation adducts from samples containing a high level of salt. Evidence of improvement in molecular ion stability was seen as well. The role organic base co-matrices play in reducing oligonucleotide fragmentation during MALDI-TOFMS analysis was further investigated. No correlation was found between crystallization or solution pH values and the molecular ion stability of oligonucleotides. Instead, a direct correlation between the co-matrix proton affinity and the oligonucleotide molecular ion stability is seen. A co-matrix whose proton affinity is close to or greater than the proton affinity of the nucleobases can serve as a "proton sink". It is proposed that upon laser desorption/ionization, the co-matrix competes with the nucleobases of the oligonucleotide for additional protons from the matrix. When the mole fraction of the co-matrix approaches that of the matrix, nearly complete elimination of oligonucleotide fragmentation is seen. Results showed that sample preparation could greatly affect ion-molecule reactions between the matrix and oligonucleotides and consequently, spectral results. An extensive study on sample handling was done in which experiments were conducted to determine the most effective oligonucleotide purification method available, and to examine the effects of matrix additives and alternative matrix solvents. It was found that C18 purification tips (ZipTips(TM)) were most effective for purification of low molecular weight oligonucleotides, while minidialysis was most effective for purification of higher molecular weight oligonucleotides. Increased resolution was seen with use of matrix additives and alternative matrix solvents.