The use of high-resolution mass spectrometry (HRMS) for the analysis of DNA and other macromolecules: A how-to guide for forensic chemistry

Abstract

High-resolution mass spectrometry (HRMS) is a highly effective technique for the analysis of DNA and other biological macromolecules and has the potential to be a valuable tool for forensic chemists. In this work, we introduce concepts of HRMS, discuss benefits of the technique, and outline the steps involved in developing an application utilizing this platform. We examine the importance of optimizing instrumental conditions, selecting an appropriate solvent, and interpreting mass spectra with considerations to determining charge state, mass accuracy and resolution. Specifically, we present a method for the analysis of short single stranded oligonucleotides (26- and 28-base) using Fourier-transform ion cyclotron mass spectrometry (FT-ICR MS), while exploring the importance of sample matrix. Results of this work indicate that analysis of small oligonucleotides with 50:50 water:acteonitrile + 20 mM imidazole (C3H4N2) produced a mass spectrum with the −5 charge state as the base peak. Charge states of −4 up to −15 were also observed, although at much lower ion intensities. Analysis of the oligonucleotides using water and 20 mM imidazole produced a charge state distribution from −5 to −11 over the mass range m/z 200–2500. These two solvents, paired with the optimized HRMS method, produced multiple charge states, high resolution mass spectra, and high ion signal intensity that allowed for unambiguous peak identification. This work serves as a “how-to guide” for forensics scientists who require efficient and accurate HRMS protocols. Our research supports using nucleic acids for diagnostics to analyze oligonucleotides, short DNA fragments and other biological macromolecules using HRMS.