酸水解法提取古生菌核心脂质及HPLC-MS分析

Maxime Tourte; Philippe Schaeffer; Vincent Grossi; Philippe M. Oger

doi:10.21769/BioProtoc.4118

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Peer-reviewed

Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis

酸水解法提取古生菌核心脂质及HPLC-MS分析

MT Maxime Tourte

PS Philippe Schaeffer

VG Vincent Grossi

PO Philippe M. Oger email

发布: 2021年08月20日第11卷第16期 DOI: 10.21769/BioProtoc.4118 浏览次数: 1965

评审: Alba BlesaKevin BeckerAnonymous reviewer(s)

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Abstract

Lipid membranes are essential cellular elements as they provide cellular integrity and selective permeability under a broad range of environmental settings upon cell growth. In particular, Archaea are commonly recognized for their tolerance to extreme conditions, which is now widely accepted to stem from the unique structure of their lipids. While enhancing the stability of the archaeal cell membrane, the exceptional properties of archaeal lipids also hinder their extraction using regular procedures initially developed for bacterial and eukaryotic lipids. The protocol described here circumvents these issues by directly hydrolyzing the polar head group(s) of archaeal lipids and extracting the resulting core lipids. Although leading to a loss of information on the nature of polar heads, this procedure allows the quantitative extraction of core lipids for most types of archaeal cells in an efficient, reproducible, and rapid manner.

Keywords: Archaea (古生菌)

Cell membrane (细胞膜)

Core lipids (核心脂)

Acid hydrolysis (酸水解)

HPLC-MS (HPLC-MS)

Extremophiles (极端微生物)

Background

Although found in all biotopes on Earth, Archaea are known as the main inhabitants of the most extreme environments, be it due to temperature, pH, salinity, hydrostatic pressure, or other environmental stressors (Komatsu and Chong, 1998; Baba et al., 1999). This ability to cope with multiple extreme conditions has been associated with one of their most diagnostic features, i.e., the unique structure of their membrane lipids. Indeed, instead of the typical lipids commonly found in Bacteria and Eukarya, which are built upon straight fatty-acyl chains ester-linked to a glycerol backbone in a sn-1,2 configuration, archaeal lipids contain polyisoprenoid alkyl chains that are ether-linked to a glycerol in a sn-2,3 configuration (De Rosa and Gambacorta, 1988). While polyisoprenoid alkyl chains provide greater membrane packing and impermeability compared to fatty acyl chains due to their heavy branching (Komatsu and Chong, 1998), ether bonds are more chemically and thermally resistant than ester linkages and less polar (Baba et al., 1999), both contributing to the enhanced stability of archaeal membranes. In addition, most Archaea are also capable of synthesizing both bilayer-forming diether lipids (one polar moiety) and membrane-spanning tetraether lipids (two polar moieties), the latter generating membrane monolayers that are even more rigid and impermeable than archaeal bilayers (Chong, 2010). In contrast to their core lipids, the polar head groups of archaeal lipids remain very similar to those of Bacteria and Eukarya, with phospho- and glyco-lipids deriving from sugars (glycerol, inositol, glucose, and N-acetylhexosamine) (Jensen et al., 2015), aminoacids (serine and ethanolamine) (Koga et al., 1993), or combinations of both (Koga et al., 1993). Although substantial lipidomes, e.g., containing up to 100 structures, have been described for Archaea (e.g., Elling et al., 2017; Bale et al., 2019), those remain less diverse than the lipidomes of Bacteria and Eukarya, which comprise hundreds to thousands of different lipid structures (Gerl et al., 2012). In addition to this large difference between archaeal and other typical lipidomes, several studies have reported biases in archaeal lipid extraction and detection (Huguet et al., 2010; Cario et al., 2015), suggesting that a significant part of the archaeal lipidome might remain inaccessible to current methodologies. The absence of procedures to completely extract and analyze archaeal lipids currently hinders our understanding of their membrane physiology and adaptation. However, partial comprehension of these phenomena can easily and reproducibly be obtained by the removal of the lipids’ polar head groups, granting access to exhaustive, and thus precise, archaeal core lipid compositions (De Rosa et al., 1980; Trincone et al., 1992; Hopmans et al., 2000; Cario et al., 2015).

Materials and Reagents

Pyrex^TM tubes with PTFE caps (Fisher Scientific, catalog number: 10004654)
Glass pipettes and hand pump (Fisher Scientific, catalog number: 11546963)
1 ml-glass syringe (gastight Series 1000; Hamilton, catalog number: 81320)
PTFE glass vial caps (Agilent Technologies, catalog number: 5182-0719)
Glass bottles (Dutscher, catalog number: 090971)
50-ml round bottomed flasks (Fisher Scientific, catalog number: CG150689)
Glass funnels (DWK Life Sciences, catalog number: 213514609)
2 ml-glass vials (Sodipro, catalog number: 2108081)
Freeze-dried archaeal biomass
Sand (VWR, catalog number: 27460.364)
Glass wool (Fisher Scientific, catalog number: 12373866)
Celite^TM (ACROS Organics, catalog number: 349675000)
Silica gel Geduran Si 60^® (Merck, catalog number 1.11567)
Hydrochloric acid 37% (HCl; Fisher Scientific, catalog number: 10294190)
Deionized water (H₂O)
Methanol (MeOH; ACROS Organics, catalog number: 364390025)
Dichloromethane (DCM; ACROS Organics, catalog number: 326850025)
Acetone (ACROS Organics, catalog number: 444150050)
n-Heptane (Hept; ACROS Organics, catalog number: 364360025)
Isopropyl alcohol (or propan-2-ol; IPA; ACROS Organics, catalog number: 184130250)

Equipment

Oven (up to 450°C)
Vacuum pump (Edwards Vacuum, catalog number: A34317984)
Note: Here, we used a vacuum pump at 50 Hz with a maximum displacement of 20.5 m³ h^-1, a maximum pumping speed of 17.0 m³ h^-1, and an ultimate vacuum of 2 × 10^-2 mbar. Although a strong suction power allows for a faster procedure, any vacuum pump able to reach an ultimate vacuum below 20 mbar can be used.
Rotary evaporator (Büchi, models: Rotavapor RE 111; heating bath, B-491)
Note: Any rotary evaporator compatible with your vacuum pump and heating bath up to 50°C can be used.
N₂ drying system
Note: Here, we connected our building N₂ supply (Air Products, CryoEase^® supply system) to a distributing system consisting of polycarbonate tubes (Cole-Parmer, catalog number: FV-30526-18) and splitting valves (Cole-Parmer, catalog number: FV-30600-02) ending on needles (BD, catalog number: 300700) or glass pipettes. Refer to Figure 3 for a depiction of the system.
High performance liquid chromatography (HPLC) instrument
Note: Here, we used a HP 1100 series LC system equipped with binary pumps (Agilent G1312A), a solvent degasser (Agilent G1379A), an autoinjector (Agilent G1913A), a thermostated column compartment (Agilent G1316A), and Chemstation chromatography manager software (Agilent Technologies Rev. A.09.03). However, any HPLC system that can function with a solvent gradient can be used.
Prevail Cyano 3 microns column (150 mm × 2.1 mm; Grace Davison Discovery Sciences, VWR, catalog number: HICH99243)
Note: When not used, our column was stored in Hept/IPA (95:5, v/v). However, the solvent mixture allows for both the storage and equilibration of the column and thus depends on the separation gradient (see Step D4). It might hence be adjusted to your requirements.
Mass spectrometer equipped with an ion trap and an atmospheric pressure chemical ionization (APCI) source
Note: Here, we used an Ion Trap MS Esquire 3000Plus (Bruker Daltonics, GE0100-G552) and an Agilent APCI source (Agilent G1947A), but any Mass spectrometry (MS) instrument equipped with an APCI source can be used.

Software

Mass spectrum and chromatogram processing software.

Note: Here, we used the Compass Data Analysis software (version 5.0, 2017) provided by Bruker Daltonics, but any mass spectrum and chromatogram processing software, especially the one provided by your HPLC-MS instrument manufacturer, can be used.

Procedure

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Tourte, M., Schaeffer, P., Grossi, V. and Oger, P. M. (2021). Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis. Bio-protocol 11(16): e4118. DOI: 10.21769/BioProtoc.4118.