Analysis of oligonucleotides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS)

This thesis focuses on the use of matrix assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of small nucleic acids. Systematic studies have been conducted to identify experimental factors affecting the efficiency of MALDI method for the generation of intact oligonucleotide molecular ions. Similar to literature findings, proper selection of matrix and co-matrix materials was found to be particularly important. The usefulness of three commonly used matrix materials, namely 2-amino-5-nitropyridine (2,5 ANP), 2,5-dihydroxybezoic acid (2,5-DHB) and 3-hydroxypicolinic acid (3-HPA), for the oligonucleotide analysis were compared. For the analysis of small oligonucleotides (m/z ∼ 1200), these matrices were found to have comparable sensitivity. However, their effectiveness were found to decrease in the order of 2,5-DHB < 2,5-ANP < 3-HPA as the size of the oligonucleotides increases. In all cases, addition of ammonium fluoride as co-matrix improved the homogeneity of the sample crystals and enhanced the signal intensity of the resulting oligonucleotide molecular ions.; It was discovered that both spontaneous and metastable dissociation of the desorbed oligonucleotide molecular ions (M) could be induced using proper selection of matrix materials. 2-Aminobenzoic acid (2-ABA) and 2,5-ANP were found to induce spontaneous decomposition of oligonucleotide molecular ions to form series of b_n−, d_n −, w_n− and y_n −. Only series of y-ion were formed using 2,5-DHB matrix system. Little or no significant prompt fragment ions were detected when 3-HPA matrix was used. Substantial loss of labile base (B), such as adenine (A) and guanine (G), was observed. The extent of base loss was also found to correlate with their positions in the oligonucleotide chain. Labile bases at the two ends were found to cleave much more readily than at the middle of the chain. In addition, 5′-phosphorylated oligonucleotides were found to lose a phosphate group leading to the generation of [M-P]− and [b_n′]− ion series. No similar fragments were observed for 3′-phosphorylated oligonucleotides.; Post-source-decay (PSD) method was employed for the detection and characterization of fragment ions formed by metastable dissociation of the desorbed oligonucleotide molecular ions. 2-ABA and 2,5-ANP were found to be suitable matrices for PSD analysis. Metastable dissociation of negatively-charged oligonucleotide molecular ions led to the formation of intense [a_n-BH]− and w_n− ions ([a_n-BH]+ and w_n+ ions for positive-ion PSD). Some fragment ions formed from double cleavages along the oligonucleotide backbone were also observed. From a systematic study of a number of oligonucleotide isomers, backbone cleavages were found to correlate well with the nature of the base unit at adjacent 5′-position. Thymine (T) was found to inhibit the formation of [M-TH]− ions and the corresponding backbone cleavage. For 3′-phosphorylated oligonucleotides, loss of phosphate group (P) was found to associate exclusively with a simultaneous loss of an adjacent labile base to form [M-P-BH]− ions. For non-phosphorylated and 5′-phosphorylated oligonucleotides, significant [M-BH-H₂O]− ions were observed instead.