MSdeCIpher: A Tool to Link Data from Complementary Ionization Techniques in High-Resolution GC-MS to Identify Molecular Ions

Abstract

Electron ionization (EI) and molecular ion-generating techniques like chemical ionization (CI) are complementary ionization methods in gas chromatography (GC)-mass spectrometry (MS). However, manual curation effort and expert knowledge are required to correctly assign molecular ions to fragment spectra. MSdeCIpher is a software tool that enables the combination of two separate datasets from fragment-rich spectra, like EI-spectra, and soft ionization spectra containing molecular ion candidates. Using high-resolution GC-MS data, it identifies and assigns molecular ions based on retention time matching, user-defined adduct/neutral loss criteria, and sum formula matching. To our knowledge, no other freely available or vendor tool is currently capable of combining fragment-rich and soft ionization datasets in this manner. The tool's performance was evaluated on three test datasets. When molecular ions are present, MSdeCIpher consistently ranks the correct molecular ion for each fragment spectrum in one of the top positions, with average ranks of 1.5, 1, and 1.2 in the three datasets, respectively. MSdeCIpher effectively reduces candidate molecular ions for each fragment spectrum and thus enables the usage of compound identification tools that require molecular masses as input. It paves the way towards rapid annotations in untargeted analysis with high-resolution GC-MS.

Keywords: analyte identification; gas chromatography (GC); high-resolution mass spectrometry (HRMS); ionization technique; molecular ion; software tool.

Figure 1

Graphical user interface (screenshot) of MSdeCIpher. All user input, as well as output evaluation, is provided graphically, making MSdeCIpher suitable even for users unfamiliar with R programming.

Scheme 1

Flowchart of the MSdeCIpher workflow. Dotted arrows describe optional steps the user can add to the workflow when needed. Deconvoluted spectral data of all compounds contained in both chromatographic runs, as they can be obtained from any deconvolution algorithm, serve as the input for MSdeCIpher. To connect molecular ion candidates from one dataset to fragments in the other one, deconvoluted pseudospectra are matched based on their retention time. The lists of molecular ion candidates are refined based on user input criteria, for example, a specific adduct/fragment series that molecular ions display in the ionization method used. Then, the sum formulas of all ions are calculated and refined, based on the sum formulas of smaller fragments in the chain and several heuristics. Lastly, molecular ion candidates for each fragment pseudospectrum are scored based on how much of the fragment pseudospectrum fits the molecular ion candidate when considering previously calculated sum formulas.

Figure 2

Identification of molecular ion candidates. MSdeCIpher searches for specific adduct and neutral loss patterns, customizable by user input. Shown here is the molecular ion [M + H]⁺ of TMS and MeOX-derivatized glucose with the pattern [M − CH₃]⁺; [M + C₂H₅]⁺ and [M + C₃H₅]⁺ that is common to TMS-derivatized compounds in methane-positive CI [30,31]. The red numbers indicate the changes in mass compared to the molecular ion.

Figure 3

Methane-positive CI spectrum of TMS- and MeOX-derivatized glucose. Marked with arrows are all putative [M + H]⁺ ions that display the adduct/neutral loss pattern [M − CH₃]⁺; [M + C₂H₅]⁺, and [M + C₃H₅]⁺ that thus represents molecular ion candidates. Only candidates with the highest m/z are retained for further processing.

Figure 4

Scoring of molecular ion candidates. Displayed here is the extracted fragment pseudospectrum of TMS-derivatized L-proline (molecular weight 259.1424 Da). Two putative molecular ions from two different CI pseudospectra (260.1499 m/z and 253.0980 m/z) are compared as molecular ion candidates for this pseudospectrum. MSdeCIpher calculates the sum formula of each fragment and compares it to the proposed sum formulas of molecular ion candidates. The log-scaled intensity of each fitting fragment (coloured) contributes to the score. Despite similar m/z, the true molecular ion fits a larger fraction of the fragments.