Analysis of non-derivatized bacteriohopanepolyols using UHPLC-HRMS reveals great structural diversity in environmental lipid assemblages

Ellen C Hopmans,Nadine T Smit,Rachel Schwartz-Narbonne,Jaap S Sinninghe Damsté,Darci Rush

doi:10.1016/j.orggeochem.2021.104285

Ellen C Hopmans, Nadine T Smit + Show 3 more

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https://doi.org/10.1016/j.orggeochem.2021.104285

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Abstract

Bacteriohopanepolyols (BHPs) are lipids with great chemotaxonomic potential for microbial populations and biogeochemical processes in the environment. The most commonly used methods for BHP analysis are chemical degradation followed by gas chromatography-mass spectrometry (MS) or derivatization followed by high performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization/MS. Here we report on significant advances in the analysis of non-derivatized BHPs using U(ltra)HPLC-electrospray ionization-high resolution MS2. Fragmentation mass spectra provided information on the BHP core, functionalized side chain, as well as the conjugated moiety of composite BHPs. We successfully identified the common bacteriohopanepolyols and their (di)methylated and (di)unsaturated homologues, aminoBHPs, and composite BHPs (e.g., cyclitol ethers and methylcarbamate-aminoBHPs) in biomass of several known BHP-producing bacteria. To show how the method can be exploited to reveal the diversity of BHPs in the environment, we investigated a soil from an active methane seep, in which we detected ca. 130 individual BHPs, including a complex distribution of adenosylhopanes. We identified the nucleoside base moiety of both adenosylhopane type-2 and type-3. Adenosyl hopane type-3 contains a methylated adenine as its nucleobase, while type-2 appears to contain a deaminated and methylated adenine, or N1-methylinosine. In addition, we detected novel adenosylhopanes. Furthermore, we identified a series of novel composite BHPs comprising of bacteriohopanepolyols conjugated to an ethenolamine moiety. The novel ethenolamineBHPs as well as aminoBHPs were also detected acylated to fatty acids. The analytical approach described allows for simultaneous analysis of the full suite of IPLs, now including BHPs, and represents a further step towards environmental lipidomics.

Highlights

Bacteriohopanepolyols (BHPs) are membrane lipids with great chemotaxonomic potential with regards to microbial populations as well as biogeochemical processes in the environment
Use of the base deactivated Ace Excel C18 column in combination with the mobile phase with pH 4 did not result in improved chromatography and for all experiments described below, a combination of the Acquity BEH C18 column com bined with the pH 4 buffering system was used
We have shown the applicability of ultra high pressure liquid chromatography (UHPLC)-electrospray ionization (ESI)/HRMS2 for the analysis of non-derivatized BHPs in both microbial cultures as well as environmental samples

Summary

Introduction

Bacteriohopanepolyols (BHPs) are membrane lipids with great chemotaxonomic potential with regards to microbial populations as well as biogeochemical processes in the environment. BHPs can include unsaturation in the ring system located at Δ6, Δ11 or both (Talbot et al, 2007b and references therein). A BHP with an unsaturation in the side chain was identified in a methano trophic Methylovulum bacterium (van Winden et al, 2012). The diverse nature of BHP lipid structures has lead to particular BHPs being used as biomarkers for unique bac terial source organisms (van Winden et al, 2012; Kool et al, 2014; Rush et al, 2014), environmental conditions (Ricci et al, 2014; Welander and Summons, 2012), and organic matter origin (Zhu et al, 2011)

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