Take a seat, acetylation: analysis of bacteriohopanepolyols streamlined

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  • Published: Oct 3, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Take a seat, acetylation: analysis of bacteriohopanepolyols streamlined

Tools of the trade

Bacteriohopanepolyols are everywhere; in rivers, permafrosts, deep-sea vents, soils, seas, and sediments. Churned out by a select one-in-ten of all prokaryotes, bacteriohopanepolyols (BHPs) are long-chain lipids that bacteria then insert into their membrane.

Bacteriohopanepolyols are everywhere; in rivers, permafrosts, deep-sea vents, soils, seas, and sediments. Churned out by a select one-in-ten of all prokaryotes, bacteriohopanepolyols (BHPs) are long-chain lipids that bacteria then insert into their membrane. They are the lungs of methanotrophs, plucking methane from the atmosphere, from where it can be oxidised for energy.

BHPs come in all shapes and sizes, and invariably carry side chains of sugar and/or amino acid derivatives specific to their species. Furthermore, these lipid signatures are highly stable, which is why their detection has become an invaluable tool in a geologist’s or palaeontologist’s toolbox—some of whom have been able to document the patterns in methane oxidation over the past 1.2 million years.

Anyone who wished to study BHPs hitherto would have to do so indirectly through analysing their derivatives—a step considered unnecessary in the eyes of a handful of scientists, and particularly for Dr Talbot and her team at Newcastle University in the United Kingdom.

Fruitless endeavours

Scientists have toiled away at acetylation-free BHP analyses since 1995, as chronicled by Talbot and colleagues in their paper published in Rapid Communications in Mass Spectrometry. The first attempt was by the United States Department of Agriculture, who successfully resolved three major BHPs with normal-phase HPLC configured with a silica column and a three-component aqueous phase.

Three years later, collaborators at the Universities of Bristol and Newcastle would then successfully identify these three BHPs in lake sediments by taking the American HPLC template and pairing it with a mass spectrometer equipped with an atmospheric pressure chemical ionisation (APCI) source. However, this method was short-lived when others showed it was unsuitable for analysing other major BHPs, particularly those with a terminal amine group at the 35th carbon that would become bottlenecked during separation.

Efforts have since focused on using ultra-performance liquid chromatography (UPLC) paired to some form of mass spectrometer (MS). Most researchers, however, have come up short: either unable to comprehensively profile the major BHPs or complaining that non-acetylated compounds are not as easily ionised as their acetylated cousins.

Streamlined

Taking MS one step further (literally), Talbot and colleagues used tandem mass spectrometry to enhance the selectivity and sensitivity over current, single mass analyses. Actively shunning acetylation, the Newcastle-based scientists streamlined their analyses on cultures of Methylosinus trichosporium and Methylococcus capsulatus as well as sediment procured from the murky estuary where the River Tyne meets the North Sea.

They adjusted the strength of collisional energies to capture a comprehensive picture of ion transitions using a triple quadrupole (QqQ) set to monitor multiple reactions as they disintegrated (MRM), and trialled a range of six LC columns attractive to a range of analyte chemistries.

Meticulously trawling through their reams of data, Talbot observed that the base-deactivated ACE Excel C18 column was ideal for their streamlined assay—including BHPs with terminal amines that troubled past researchers. In combination with MRM tailored to highly specific ion transitions, their new assay shaved 51 minutes off the time for current assays.

What was made unequivocal, however, was that the fragmentation patterns of non-acetylated derivatives differ considerably from their acetylated cousins. ‘Until such time as authentic standards are available for a wide range of non-acetylated BHP structures,’ Talbot warns, ‘it will not be possible to fully test the sensitivity of the system for quantitative analysis.’

Related Links

Rapid Commun. Mass Spectrom., 2016, 30, 2087–2098. Talbot et al. Analysis of non-derivatised bacteriohopanepolyols by ultrahigh-performance liquid chromatography/tandem mass spectrometry.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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