A Strategy for Examining Complex Mixtures of Deoxyoligonucleotides Using Ion-Pair–Reverse-Phase High-Performance Liquid Chromatography, Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry, and Informatics

Abstract

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair–reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers.