Analysis of nucleic acids by on‐line liquid chromatography–Mass spectrometry

Abstract

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single- and double-stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on-line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion-pair reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC-ESI-MS include the characterization of oligodeoxynucleotides synthesized by solid-phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double-stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids.

Typing of single nucleotide polymorphisms by MALDI mass spectrometry: Principles and diagnostic applications

“…Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI -TOF -MS) [13] and electrospray ionization (ESI) mass spectrometry [14] have revolutionized the analysis of nucleic acids and other biomolecules. It has been shown that ESI can be routinely coupled to (liquid) chromatography to provide separation of DNA molecules as well as sample clean-up [15]. ESI can be used to detect large molecules (> 20 kDa) and was applied to analyse small PCR products [16,17].…”

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

“…Another approach is introducing liquid chromatography (LC) for separation. LC provides an on-line separation of the analytes from coexisting substances and does not require a laborious sample preparation prior to ionization 21,22 . Application of LC/ESI-MS genotyping for differentiation of pathogen species, single-nucleotide polymorphisms and short tandem repeats has been reported using a capillary polymer-monolith column that can separate longer oligonucleotides 1,21,[24][25][26][27][28][29][30][31][32][33] .…”

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

“…Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3=-end of primers were corrected and detected within the corresponding amplicons. E lectrospray ionization mass spectrometry (ESI-MS) represents a powerful method for the characterization of nucleic acids [1,2]. Molecular mass determination of intact DNA molecules with lengths up to several hundred base pairs [3][4][5][6] is accomplished with high-performance, which enables the detection of sequence variations directly upon analyzing polymerase chain reaction (PCR) amplified sequences.…”

“…Even moderate concentrations of salts are known to detract from the ionization efficiency of nucleic acids and need to be removed (for a detailed discussion of this problem, please refer to references [1,8,9]. In this context, chromatography was shown to be one of the most efficient nucleic acid purification techniques available [9].…”