Sequencing of modified oligonucleotides using in-source fragmentation and delayed pulsed ion extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Abstract

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to sequence modified oligonucelotides (MONs). Under delayed pulsed ion extraction conditions, sequence ions of MONs resulting from fragmentation within the ion source can be observed. In this work, several common types of antisense MONs with sizes up to 25-mer were studied including an oligodeoxynucleotide (ODN) of phosphorothioate-phosphodiester (PS-PO) chimera, an all PS ODN, a partially 2′- O-methylated all PS oligodeoxyribonucleotide-oligoribonucleotide (ODN-ON) chimera, and an ODN of phosphorothioate-methylphosphonate (PS-MP) chimera. The sequence ions observed include ‘w’, ‘d’, as well as hitherto little discussed ‘a’ and ‘z’ ions. While a complete sequence can be constructed from ‘w’ ions for chimeric PS-PO ODN, all PS ODN, and chimeric PS ODN-ON, ‘a’ ions or ‘d’ ions provide useful supplemental information. For the PS-MP ODN, however, ‘d’ ions are critical in filling the gap in the sequence constructed from ‘w’ ions. The method provides a rapid quality control tool in oligonucleotide synthesis allowing sequence verification to be accomplished in minutes rather than hours needed by chemical or enzymatic methods. The observation that the fragmentation pattern in the PS ON region is rather similar to that of PS ODN together with the observation of ‘a’ ions suggests that backbone cleavage pathways may not always involve nucleobases losses. Fragmentation mechanisms alternative to those found in MALDI-TOFMS literature have been proposed.