Evaluation of Different Tandem MS Acquisition Modes to Support Metabolite Annotation in Human Plasma Using Ultra High-Performance Liquid Chromatography High-Resolution Mass Spectrometry for Untargeted Metabolomics.

Julian Pezzatti,Julien Boccard,Davy Guillarme,Serge Rudaz,Víctor González-Ruiz

doi:10.3390/metabo10110464

Abstract

Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.

Highlights

Untargeted metabolomics constitutes a potent approach to assess phenotypic modifications at the molecular level caused by disease, drug efficacy or toxicity, environmental factors, etc
We recently proposed a strategy comprising three complementary LC methods, including reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography with amide and polymeric zwitterionic stationary phases, all of which are coupled with high-resolution mass spectrometry (HRMS) [16]
UHPLC-(IM)-HRMS techniques yield a wealth of information presented as multi-dimensional arrays of measured intensities over m/z, Rt, and collision cross-section (CCS) values/drift times

Summary

Introduction

Untargeted metabolomics constitutes a potent approach to assess phenotypic modifications at the molecular level caused by disease, drug efficacy or toxicity, environmental factors, etc. Metabolites 2020, 10, 464 molecular coverage of untargeted approaches enables the discovery of diverse metabolites involved in different biological pathways and allows researchers to characterize pathologies such as chronic kidney disease, diabetes, or cancer by providing a diagnostic chemical signature of the related metabolic phenotypes [2,5,6,7,8] It is well-recognized within the scientific community that obtaining broad metabolic coverage can only be achieved through a combination of different analytical platforms [9,10,11,12,13,14,15]. Thanks to its hydrophilic partitioning, hydrogen bonding, ion exchange, and dipole–dipole interactions, HILIC is a chromatographic mode that is perfectly adapted for the analysis of very polar compounds, while RPLC is well suited for retaining moderately polar to apolar metabolites [17,18]

Objectives

Methods

Results

Conclusion