Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column

Abstract

In this manuscript, an efficient high resolution reversed phase - ion pairing- liquid chromatography electrospray ionization tandem mass spectrometry (RP-IP-LC-MS/MS) method for separation of isomeric modified oligonucleotides using a polymeric (styrene-divinylbenzene) monolithic capillary column is presented. The effects of different ion pairing reagents (IPR), their concentration, mobile phase additives and conditions were evaluated towards achieving the highest possible resolution and chromatographic separation of isomeric oligonucleotides. Ion pairing reagents and mobile phase conditions were evaluated using as model N-acetylaminofluorene [AAF] adducted ss- oligonucleotides (CCC CGA GCA ATC TCA AT). The optimized mobile phase conditions were then applied for the mapping of sites of base modification of AAF adducted 15 base pair oligonucleotide fragments containing codon 135 of the p53 gene and for profiling a complex synthetic oligonucleotide mixture. The optimized method utilizes a monolithic poly(styrene-divinylbenzene) capillary column, triethylammonium bicarbonate as ion pairing reagent and methanol as organic modifier to perform IP-RPLC-ESI-MS/MS separation. The results show that the method is simultaneously applicable not only to oligonucleotide fragments adducted separately by different carcinogens but also to the analysis of multiple adducts in the same oligonucleotide fragment in a single experiment. The method presents itself as a tool for the identification, characterization and mapping of oligonucleotide adducts as biomarkers for DNA damage from carcinogens.

The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides

“…HFIP can adjust the solution pH and at the same time enhance MS signal intensity of oligonucleotides which explains why it has been used in every LC-MS study since its introduction. There have been several recent studies into alternative alkylamines as ion pairing reagents [20–23]. However, other fluorinated alcohols have never been thoroughly investigated as potential substitutes for HFIP.…”

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene

“…42 TEAB facilitated positional isomer separation of oligonucleotides. A gradient of 15% B for 2 min followed by increasing from 15% B to 20% B over 36 min, then back to 15% B for another 2 min at flow rate 8 μ L/min was used.…”

“…41 Sharma et al used TEAB to separate oligonucleotides adducted by N -acetylaminofluorene, N -hydroxy-4-aminobiphenyl, and (±)- anti -benzo[ a ]pyrene diol epoxide to investigate the site selectivity. 42,43 These papers provide guidance for HPLC separations of isomeric oligonucleotide products.…”

Application of liquid chromatography-mass spectrometry and differential mobility spectrometry-mass spectrometry for detection and quantitation of biomarkers in complex biological matrices

“…The adducts may have site-selectivity or sequence-selectivity, which has been shown by several groups [100][101][102][103][104][105][106]. The mutagenic effects of a carcinogen can be potentially linked with the location of the adducts on the sequence [35].…”

“…Normalization of serum metabolomics results by creatinine level may not be necessary in metabolomics studies of serum, because of serum creatinine homeostasis (60-110 μM male, 45-90 μM female [100]). However, creatinine levels are not stabilized in the same way in urine.…”