Abstract
Mass spectrometry is now widely used in many areas of biomolecule analysis and this diversity is well represented by the analysis of oligonucleotides, carbohydrates and steroids. In this paper an overview of the common techniques used for analysis of these biomolecules is presented, including electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), desorption/ionization on silicon (DIOS), and negative chemical ionization gas chromatography mass spectrometry (NCI GC/MS). Additionally, important aspects of biomolecule analysis, such as molecular weight determination, sequencing using tandem mass spectrometry and enzymes, precursor ion scanning, noncovalent interactions, and sensitivity, will be discussed.
Highlights
Oligonucleotides are routinely assembled using automated solid phase DNA synthesis
The most common technique for oligonucleotide analysis is matrix-assisted laser desorption/ionization (MALDI)-MS [1,2,3], given that it is comparatively tolerant of mixtures and impurities, electrospray ionization (ESI)-MS and LC ESI-MS are employed
MALDI-MS ladder sequencing of a 50mer is shown in Fig. 2, which illustrates that approximately 5 residues from either the 5 or 3 end of the molecule were generated and observed
Summary
Oligonucleotides are routinely assembled using automated solid phase DNA synthesis. This technique is highly efficient, resulting in stepwise coupling yields of nucleoside phosphoramidite monomers as high as 99%. Even with such efficient coupling reactions the theoretical yield for a 20mer is less than 90%. Synthetic oligonucleotides require confirmation of purity and identity, which can be troublesome since the impurities frequently coelute on HPLC. The most common technique for oligonucleotide analysis is MALDI-MS [1,2,3], given that it is comparatively tolerant of mixtures and impurities, ESI-MS and LC ESI-MS are employed. In addition to structure conformation, mass spectrometry is being developed as a diagnostic tool for single nucleotide polymorphorisms (SNP’s) analysis
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