A Tutorial on Automated Mass Spectral Analysis Using NucleoSpec for Compositional Assignment of Nucleic Acid-Silver Complexes and Nanoclusters

Abstract

Nucleic acid–metal complexes, such as DNA-stabilized silver nanoclusters (DNA-AgN) and Ag(I)-mediated DNA duplexes, show promise for applications ranging from biomedical imaging to bioelectronics and sensing. Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for precisely resolving their compositions, but the data analysis procedure can be challenging and time-consuming. No general-purpose software can automatically assign compositions of nucleic acid–silver complexes, most notably for DNA-AgN, whose masses depend on the oxidation state of the silver core. We address these challenges by developing an automated data analysis workflow for ESI-MS of nucleic acid–silver complexes. This tutorial presents the analysis workflow and introduces NucleoSpec, an open-source, web-based platform that consolidates charge state determination, constraint-based composition enumeration, isotope-pattern matching, and adduct/conjugate handling into a single browser-accessible interface. The platform is benchmarked on published molecular formulas determined by manual analysis for DNA-AgN, xeno-nucleic acid stabilized AgN (XNA-AgN), nucleic acid–Ag(I) complexes, and chemically modified DNA-AgN, reproducing assignments to single-proton precision (median centroid offset of 0.03 m/z) for spectra of adequate signal-to-noise. Low-signal peaks for which adjacent valence electron counts become difficult to distinguish are flagged by NucleoSpec for user inspection. By lowering the barrier to high-resolution compositional analysis and supporting integration into high-throughput pipelines, NucleoSpec addresses a key experimental bottleneck in informatics-guided design of nucleic acid–metal complexes.