Free Solution Oligonucleotide Separation by CE-MS in Acidic Buffers and Positive ESI Ionization.

Abstract

This work focuses on the separation of oligonucleotides (ONs) in a free solution by capillary electrophoresis-mass spectrometry (CE-MS). Specifically, we evaluated a combination of separation in acidic background electrolytes (BGEs), nanospray ionization, and time-of-flight mass spectrometry in positive mode. A mixture of synthetic ONs, ranging in length from 15 to 78 nt, was employed as the test compounds. The key to a good separation selectivity of ONs lies in the different protonation of individual nucleobases. To this end, we assessed the acidity constant (pK_a) of the nucleobases in nucleotides experimentally as 3.3 for adenine, 4.4 for cytosine, 2.5 for guanine, and < 2 for thymine. From a set of separations in the 2-9 pH range, it was found that optimum peak shape and resolution are achieved in the interval of acidic pH 2-2.5. The BGE can be conveniently composed of either formic acid (FA) or a combination of ammonium hydroxide and FA. Nanospray ionization provided ions with charge numbers ranging from +4 to +8, proportional to the length of the ON. For short sequences, sheath liquid (SL) comprising 0.5%-1% (v/v) FA + 20% (v/v) methanol was sufficient in order to generate ions in positive mode MS, whereas a stronger SL of 5% (v/v) FA + 20% (v/v) methanol was required for longer ON sequences of approximately > 40 nt.