Category Archives: Resource Library

Molecular Structure and Mass Spectral Data Quality–Driven Processing of High‐Resolution Mass Spectrometry for Quantitative Analysis

February 2025

Fabien Fontaine, Luca Morettoni, Ken Anderson, Bernard Choi, Ismael Zamora, Kevin P. Bateman

Abstract

Rationale

LC-MS-based quantification is traditionally performed using selected or multiple reaction monitoring (SRM/MRM) acquisition functions on triple quadrupole (QQQ) instruments resulting in both high sensitivity and selectivity. This workflow requires a previously identified reaction or transition from a precursor ion to a fragment ion to be monitored to obtain the needed selectivity for the compound of interest. High-resolution mass spectrometry (HRMS) has long sought to be a viable alternative for quantitatipve workflows but has been unable to broadly compete, mainly due to the lack of suitable data processing software.

Methods

The approach we developed agnostically and automatically identifies all ions related to the compound being analyzed in both the MS and MSMS data, acquired with data-dependent or data-independent methods. The algorithm automatically selects optimal parameters (ion extraction window, ions to sum, etc.) to provide the best overall method to meet the acceptance criteria defined by the user (accuracy/precision).

Results

The results obtained are directly compared to QQQ data collected from the same set of samples and show that the automated HRMS approach is as good as and, in some cases, better than the traditional QQQ approach in terms of selectivity, sensitivity, and dynamic range.

Conclusions

This new methodology enables the use of generic methods for data collection for quantitative analysis using high-resolution mass spectrometry. With this approach, data collection is faster, and the processing algorithm provides quality equal to or better than the current QQQ methodology. This enables an overall reduction in cycle time and improved assay performance versus current HRMS-based quantitative analysis as well as traditional QQQ workflows.

Oniro: automation and standardization for workflow definition

The new Workflow definition guides the user through the steps to define experiments, starting from the acquisition sample list. By defining the workflow, new experiments are generated and launched directly on the Oniro server. Oniro directly controls MassMetaSite, MassChemSite, or WebQuant computations, reducing analysis time and minimizing errors from file handling. Once a workflow is defined, it can be saved and automatically applied to new sample lists.”

Customizable spectra signal colors

The user can customize the colors displayed for different types of signals: match (shifted or non-shifted), mismatch, metabolite match, or unassigned m/z. The colors assigned to each signal type are also kept in the report analysis.

WebMetabase & WebChembase: Fragment analysis validation for manual edited compound

The user can draw new metabolites or product chemical structures and validate them by comparing the virtual fragmentation of the compounds with the spectral information extracted from the peak. This functionality can also be applied to manually generated compounds during the new peak generation workflow in the Chromatogram Browser.

Compound Library: automation of Compound set definition

An automatic compound set is defined during the SDF file import process. When chemical structures are imported into the Compound Library, the compound set is established, enabling users to perform various predictions—such as MetaSite fragmentation, and model predictions—directly on the generated compound set. This minimizes the time required to obtain results.