Category Archives: Resource Library

LC-MS and High-Throughput Data Processing Solutions for Lipid Metabolic Tracing Using Bioorthogonal Click Chemistry

24 April 2025

Palina Nepachalovich, Stefano Bonciarelli, Gabriele Lombardi Bendoula, Jenny Desantis, Michela Eleuteri, Christoph Thiele, Laura Goracci, Maria Fedorova

Graphical Abstract

This study introduces an integrated analytical and bioinformatics platform for high-throughput tracing of lipid metabolism using bioorthogonal alkyne fatty acids and optimized LC-MS workflow. Applied to human fibrosarcoma cells, the method traced fatty acid metabolism, revealing nuances in sphingolipid routing and metabolic bottlenecks, highlighting its potential for lipidomics research.

Abstract

Tracing lipid metabolism in mammalian cells presents a significant technological challenge due to the vast structural diversity of lipids involved in multiple metabolic routes. Bioorthogonal approaches based on click chemistry have revolutionized analytical performance in lipid tracing. When adapted for mass spectrometry (MS), they enable highly specific and sensitive analyses of lipid transformations at the lipidome scale. Here, we advance this approach by integrating liquid chromatography (LC) prior to MS detection and developing a software-assisted workflow for high-throughput data processing. LC separation resolved labeled and unmodified lipids, enabling qualitative and quantitative analysis of both lipidome fractions, as well as isomeric lipid species. Using synthetic standards and endogenously produced alkyne lipids, we characterized LC-MS behavior, including preferential adduct formation and the extent of in-source fragmentation. Specific fragmentation rules, derived from tandem MS experiments for 23 lipid subclasses, were implemented in Lipostar2 software for high-throughput annotation and quantification of labeled lipids. Applying this platform, we traced metabolic pathways of palmitic and oleic acid alkynes, revealing distinct lipid incorporation patterns and metabolic bottlenecks. Altogether, here we provide an integrated analytical and bioinformatics platform for high-throughput tracing of lipid metabolism using LC-MS workflow.

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.