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Mar 2020
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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   

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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

  1. PyrexTM tubes with PTFE caps (Fisher Scientific, catalog number: 10004654)

  2. Glass pipettes and hand pump (Fisher Scientific, catalog number: 11546963)

  3. 1 ml-glass syringe (gastight Series 1000; Hamilton, catalog number: 81320)

  4. PTFE glass vial caps (Agilent Technologies, catalog number: 5182-0719)

  5. Glass bottles (Dutscher, catalog number: 090971)

  6. 50-ml round bottomed flasks (Fisher Scientific, catalog number: CG150689)

  7. Glass funnels (DWK Life Sciences, catalog number: 213514609)

  8. 2 ml-glass vials (Sodipro, catalog number: 2108081)

  9. Freeze-dried archaeal biomass

  10. Sand (VWR, catalog number: 27460.364)

  11. Glass wool (Fisher Scientific, catalog number: 12373866)

  12. CeliteTM (ACROS Organics, catalog number: 349675000)

  13. Silica gel Geduran Si 60® (Merck, catalog number 1.11567)

  14. Hydrochloric acid 37% (HCl; Fisher Scientific, catalog number: 10294190)

  15. Deionized water (H2O)

  16. Methanol (MeOH; ACROS Organics, catalog number: 364390025)

  17. Dichloromethane (DCM; ACROS Organics, catalog number: 326850025)

  18. Acetone (ACROS Organics, catalog number: 444150050)

  19. n-Heptane (Hept; ACROS Organics, catalog number: 364360025)

  20. Isopropyl alcohol (or propan-2-ol; IPA; ACROS Organics, catalog number: 184130250)

Equipment

  1. Oven (up to 450°C)

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

Material preparation

  1. Archaeal biomass must be freeze-dried beforehand.

    Note: As for every lipid extraction procedure, the growth medium may contain a significant amount of lipids unrelated to the cells to be analyzed. To avoid extracting these lipids from the culture medium, it is important to rinse the cells twice with an appropriate isotonic solution prior to freeze-drying.

  2. Remove all traces of potential organic contaminants by combusting all glassware, sand, celite, and glass wool at 450°C for 4 h before use. Rinse the glassware with deionized water, acetone, and DCM (in that order). Let it dry before use.

  3. Silica gel is pre-extracted with DCM in a Soxhlet apparatus.

  4. All reagents and solvents (except Hept) must be high grade, i.e., >99% purity, to reduce the risk of contamination.

    Note: High purity solvent can also be obtained by distillation if possible.

  5. Hept is cleaned from organic contaminants by filtration over a column filled with silica gel.

  6. Prepare 100 ml of 1.2 N HCl in MeOH by adding 10 ml of HCl 37% to 90 ml of MeOH.

    Note: The solution can be stored for several weeks in a capped glass bottle at room temperature.

  7. Prepare 100 ml of MeOH/DCM (1:1, v/v) by adding 50 ml of DCM into 50 ml of MeOH.

    Note: The solution can be stored in a capped glass bottle at room temperature for several weeks.

  8. Prepare 50 ml of Hept/IPA (99:1, v/v) by adding 0.5 ml of IPA to 49.5 ml of Hept.

    Note: The solution can be stored for several weeks in a capped glass flask at room temperature.

  9. Prepare 1,000 ml of Hept/IPA (95:5, v/v) by adding 50 ml of IPA to 950 of Hept.

    Note: The solution can be stored for several weeks in a glass bottle at room temperature.

  10. Prepare a 1,000-ml glass bottle filled with Hept.

    Note: The solution can be stored for several weeks at room temperature.


  1. Archaeal cell hydrolysis (Figure 1)

    1. Transfer the archaeal cell pellet to a PyrexTM glass tube using a small glass funnel.

    2. Use 4 ml of 1.2 N HCl in MeOH to resuspend the archaeal cell pellet and rinse the glass funnel using glass pipettes and a hand pump.

      Note: The volume at this step may be modified depending on the size of the cell pellet. However, do not fill the PyrexTM tube above half of its total volume.

    3. Tightly close the PyrexTM tube with a cap containing a PTFE seal.

      Note: Double check that the cap is closed to avoid any evaporation that could lead to the destruction of the sample.

    4. To avoid any cross-contamination of samples and contamination of the oven in case of leakage, put 5 cm of sand in a glass bottle, place your PyrexTM tubes in the sand, and close the bottle with a stopper.

    5. Heat your samples at 110°C for 4 h.

      Note: This procedure is strong enough to remove most of the archaeal polar head groups without further degrading archaeal lipids, i.e., ether linkages remain unharmed.

    6. Take your samples out of the oven and let them cool down to room temperature.

      Note: At this step, if your tubes were not sealed tightly enough, your sample might have dried out and completely burned (black-brown color of the sample), which might result in the destruction of core lipids. As a quality control of the procedure, check that tubes still contain solvent and that cell pellets are almost completely disrupted and colorless.



      Figure 1. Schematic of the workflow for the direct acid methanolysis of archaeal dried biomass. Circled numbers refer to the steps in the text. Abbreviations: MeOH, methanol; PTFE, polytetrafluoroethylene.


  2. Core lipid extraction (Figure 2)

    1. Once your sample has cooled down, carefully open the PyrexTM tube.

      Warning: Due to the gas generated during the heating step, hot gas and liquid may gush out from the tube when opening it.

    2. Add 4 ml of DCM into the PyrexTM tube (final MeOH/DCM 1:1, v/v).

      Note: The volume at this step may be modified depending on the size of the cell pellet.

    3. Create a filter by placing glass wool and then celite into a glass funnel. Place the glass funnel onto a 50 ml-round-bottomed flask suited for the rotary evaporator.

    4. Transfer the sample onto the filter and filter it through the funnel into the flask to remove the solid particles (i.e., cell debris) from the lipid extract.

    5. Rinse the PyrexTM tube twice with 4 ml of MeOH/DCM (1:1, v/v) and filter it into the same flask as above.

    6. Rinse the filter into the same flask twice with 4 ml of MeOH/DCM (1:1, v/v).



      Figure 2. Schematic of the workflow for the extraction of core lipids from the hydrolyzed biomass. Circled numbers refer to the steps in the text. Abbreviations: MeOH, methanol; DCM, dichloromethane.


  3. Sample preparation for HPLC analysis (Figure 3)

    1. Dry the sample under reduced pressure using a rotary evaporator set to 40°C.

    2. Rinse the flask with MeOH and dry again under reduced pressure to allow for the complete evaporation of the remaining HCl/H2O.

    3. Once dried, carefully dissolve the content of the flask with 0.5 ml of Hept/IPA (99:1, v/v) to recover core lipids and transfer into a 2 ml-vial using a glass syringe.

    4. Rinse the flask three more times and transfer into the 2 ml-vial.

    5. Dry the sample under a steam of N2.

    6. Resuspend the sample into 1 ml of Hept/IPA (99:1, v/v) prior to HPLC analysis.

      Note: The lipid extract can be stored at -20°C for long periods of time (i.e., several years).



      Figure 3. Schematic of the workflow for the core lipid extract preparation for HPLC analysis. Circled numbers refer to the steps in the text. Abbreviations: MeOH, methanol; Hept, heptane; IPA, isopropanol.


  4. HPLC-MS core lipid separation and analysis

    Note: This section is based on Tourte et al. (2020), but the nature and volume of solvents can be adjusted to fit the requirements of your chromatographic separation, i.e., nature and size of the column.

    1. Start your chromatography instrument.

    2. Set the temperature of the column thermostat to 30°C.

    3. Empty the trash collectors and fill the vial used for rinsing (Hept/IPA 95:5, v/v).

    4. Feed the chromatography manager software with the gradient used for HPLC chromatography.

      Note: Here, we used the following gradient with A = Hept/IPA (95:5, v/v) and B = Hept, and a flow rate of 0.2 ml min-1: 95% B (5 min isocratic) to 65% B in 30 min (5 min isocratic), then to 0% B in 1 min (10 min isocratic), and back to 95% B in 1 min (5 min isocratic). The total run time per sample is 57 min.

    5. Double-check for stable pressure in the system to ensure proper separation. Flush your system if necessary.

    6. Start the MS instrument.

    7. Feed the MS manager software with the conditions for the MS analysis.

      Note: Here, we used the following MS conditions: nebulizer pressure, 50 psi; APCI temperature, 420°C; drying temperature, 350°C; drying gas (N2) flow, 5 L min-1; capillary voltage, -2 kV; corona, 4 µA; and scan range m/z, 600-2,200.

    8. Double-check that all parameters properly reach the set values.

    9. Place the 2 ml-vials containing your samples dissolved in 1 ml of Hept/IPA (99:1, v/v; see Step C6) onto the HPLC autosampler.

    10. Fill the chromatography manager software file with your sample information and sequence.

      Note: The injection volume is set to 10 µl. To enhance reproducibility, adjust the concentration of your samples (Step C6) rather than change the injection volume.

    11. Run your sequence.

      Note: Run a control sample every 30-40 analyses to check for appropriate separation and intensity. Reproducibility is evaluated by running selected samples in duplicate or triplicate and a reference sample. For instance, as a reference sample, we used a core lipid extract from Pyrococcus furiosus, which contains diphytanyl glycerol diethers (DGD) and multiple isomers of tetraether lipids, i.e., glycerol mono-, di-, and trialkyl glycerol tetraethers (GMGT, GDGT, and GTGT, respectively) with 0 to 4 cyclopentane rings, to assess the performance of the HPLC column. Additionally, run a standard mixture (synthetic archaeal-like lipids can be purchased from Avanti) to assess the relative response factors of the compounds detected. As a standard mixture, we used a solution of DGD/GDGT0 at 2:1 molar ratio which gave a relative response factor of DGD ca. 10 times lower than that of GDGT0 under our analytical conditions (Tourte et al., 2020).


  5. Chromatogram and mass spectrum analysis

    Note: Relatively few examples of archaeal core lipid mass spectra have been published, but identification can be aided by referring to, e.g., Hopmans et al. (2000).

    1. Draw the total ion current (TIC) of your analysis using mass spectrum and chromatogram processing software.

    2. Major compounds, i.e., the most abundant on the TIC, are tentatively identified based on their mass spectra, fragmentation patterns, retention time in LC, and comparison with published data (where possible). Use an average of several spectra of a given core lipid peak to ensure proper identification (Figure 1).

      Note: Very few ions besides the protonated adduct of the intact compound are generated by our APCI procedure, and compounds are thus identified based on their protonated masses. To further ascertain the structures of identified core lipids, MS/MS can be performed.

    3. Draw the extracted ion chromatogram (EIC) by extracting the masses of all compounds identified (Figure 4). For each compound, select a mass range suited to your MS system.

      Note: Considering our system, we usually use a mass range of ±0.5.

    4. Integrate each peak on your EIC to determine the relative proportion of each core lipid and correct with the response factor of the compounds under your analytical conditions (see Step D11).



      Figure 4. Core lipid analysis of Thermococcus acidaminovorans. Cells were grown under optimal conditions (85°C, pH 9.0, 3.0% NaCl (w/v); Dirmeier et al., 1998). The chromatogram was drawn by extracting the following masses: 653.5, 1302.5, and 1304.5. Inserts show the mass spectrum and the resulting core structure for each colored peak: green, diphytanyl glycerol diether (DGD); blue, glycerol trialkyl glycerol tetraether with no cyclopentane ring (GTGT0); and purple, glycerol dibiphytanyl glycerol tetraether with no cyclopentane ring (GDGT0). The indicated masses correspond to protonated adducts. Correcting for the response factors observed under our analytical conditions, DGD, GTGT0, and GDGT0 had relative proportions of 46, 1, and 53%, respectively.

Acknowledgments

The authors would like to thank the French National Research Agency for funding the ArchaeoMembranes project (ANR-17-CE11-0012-01) and the CNRS interdisciplinary program 'Origines' for funding the ReseArch project.

Competing interests

The authors declare no conflicts of interest.

References

  1. Baba, T., Toshima, Y., Minamikawa, H., Hato, M., Suzuki, K. and Kamo, N. (1999). Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids. Biochim Biophys Acta-Biomembr 1421: 91-102.
  2. Bale, N. J., Sorokin, D. Y., Hopmans, E. C., Koenen, M., Rijpstra, W. I. C., Villanueva, L., Wienk, H. and Damsté, J. S. S. (2019). New insights into the polar lipid composition of extremely halo (alkali) philic euryarchaea from hypersaline lakes. Front Microbiol 10: 377.
  3. De Rosa, M., Gambacorta, A., Nicolaus, B. and Bu'Lock, J. D. (1980). Complex lipids of Caldariella acidophila, a thermoacidophile archaebacterium. Phytochemistry 19: 821-825.
  4. Trincone, A., Nicolaus, B., Palmieri, G., De Rosa, M., Huber, R., Huber, G., Stetter, K.O. and Gambacorta, A. (1992). Distribution of complex and core lipids within new hyperthermophilic members of the Archaea domain. Syst Appl Microbiol 15: 11-17.
  5. Hopmans, E. C., Schouten, S., Pancost, R. D., van der Meer, M. T. and Sinninghe Damste, J. S. (2000). Analysis of intact tetraether lipids in archaeal cell material and sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Rapid Commun Mass Spectrom 14(7): 585-589.
  6. Cario, A., Grossi, V., Schaeffer, P. and Oger, P. M. (2015). Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus. Front Microbiol 6:1152.
  7. Chong, P. L.-G. (2010). Archaebacterial bipolar tetraether lipids: physico-chemical and membrane properties. Chem Phys Lipids 163: 253-265.
  8. De Rosa, M. and Gambacorta, A. (1988). The lipids of archaebacteria. Prog Lipid Res 27: 153-175.
  9. Dirmeier, R., Keller, M., Hafenbradl D., Braun, F-J., Rachel, R., Burggraf, S. and Stetter K. O. (1998). Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids. Extremophiles 2: 109-114.
  10. Elling, F. J., Könneke, M., Nicol, G. W., Stieglmeier, M., Bayer, B., Spieck, E., de la Torre, J. R., Becker, K. W., Thomm, M., Prosser, J. I., Herndl, G. J., Schleper, C. and Hinrichs, K.-U. (2017). Chemotaxonomic characterisation of the thaumarchaeal lipidome. Environ Microbiol 19: 2681-2700.
  11. Gerl, M. J., Sampaio, J. L., Urban, S., Kalvodova, L., Verbavatz, J.-M., Binnington, B., Lindemann, D., Lingwood, C. A., Shevchenko, A., Schroeder, C. and Simons, K. (2012). Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane. J Cell Biol 196: 213-221.
  12. Huguet, C., Martens-Habbena, W., Urakawa, H., Stahl, D. A. and Ingalls, A. E. (2010). Comparison of extraction methods for quantitative analysis of core and intact polar glycerol dialkyl glycerol tetraethers (GDGTs) in environmental samples. Limnol Oceanogr Meth 8: 127-145.
  13. Jensen, S. M., Brandl, M., Treusch, A. H. and Ejsing, C. S. (2015). Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics. J Mass Spectrom 50: 476-487.
  14. Koga, Y., Akagawa-Matsushita, M., Ohga, M. and Nishihara, M. (1993). Taxonomic significance of the distribution of component parts of polar ether lipids in methanogens.Syst Appl Microbiol 16: 342-351.
  15. Komatsu, H. and Chong, P. L.-G. (1998). Low permeability of liposomal membranes composed of bipolar tetraether lipids from thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochemistry 37: 107-115.
  16. Tourte, M., Schaeffer, P., Grossi, V. and Oger, P. M. (2020). Functionalized membrane domains: an ancestral feature of Archaea? Front Microbiol 11: 526.

简介

[摘要]脂质膜是必不可少的细胞元件,因为它们在细胞生长时在广泛的环境设置下提供细胞完整性和选择性渗透性。特别是,古生菌因其对极端条件的耐受性而广为人知,现在人们普遍认为这种耐受性源于其脂质的独特结构。在增强古细菌细胞膜稳定性的同时,古细菌脂质的特殊性质也阻碍了使用最初为细菌和真核生物脂质开发的常规程序进行提取。此处描述的协议通过直接水解古菌脂质的极性头部基团并提取产生的核心脂质来规避这些问题。尽管会导致有关极头性质的信息丢失,但该过程允许以高效、可重复和快速的方式定量提取大多数类型的古细菌细胞的核心脂质。


[背景]虽然在地球上的所有生物小区发现,古菌是已知的一个S中的最极端的环境的主要居民,它是由于温度,pH,盐度,流体静压力,或其他环境应激(小松和Chong,1998;巴巴等al. , 1999)。这种应对多种极端条件的能力与其最具诊断性的特征之一有关,即,其膜脂的独特结构。事实上,与细菌和真核生物中常见的典型脂质不同,它们建立在以sn -1,2 构型与甘油骨架酯连接的直脂肪酰基链上,古细菌脂质包含聚异戊二烯烷基链,该链是醚-以sn -2,3 构型与甘油相连(De Rosa 和 Gambacorta, 1988)。虽然聚异戊二烯烷基链由于其重支化而比脂肪酰基链提供更大的膜堆积和不渗透性(Komatsu 和 Chong,1998),但醚键比酯键更具化学性和耐热性,并且极性更小(Baba等,1999) ,两者都有助于增强古菌膜的稳定性。此外,大多数古细菌还能够合成双层形成的二醚脂质(一个极性部分)和跨膜四醚脂质(两个极性部分),后者生成的膜单层比古菌双层(Chong , 2010)。与其核心脂质相反,古细菌脂质的极性头部基团仍然与细菌和真核生物的极性头部基团非常相似,磷酸脂和糖脂源自糖类(甘油、肌醇、葡萄糖和N-乙酰己糖胺)(Jensen等人. , 2015)、氨基酸(丝氨酸和乙醇胺)(Koga等人,1993 年),或两者的组合(Koga等人,1993 年)。虽然实质lipidomes,例如,。含有多达100层的结构,已为古细菌描述(例如,埃林。等人。,2017;罢了。等人,2019),这些保持小于多样的lipidomes细菌和真核生物,其包含的成百上千种不同的脂质结构(Gerl等,2012)。除了古细菌和其他典型脂质组之间的巨大差异外,一些研究报告了古细菌脂质提取和检测的偏差(Huguet等人,2010 年;Cario等人,2015 年),这表明古细菌脂质组的很大一部分可能仍然无法使用当前的方法。目前缺乏完全提取和分析古细菌脂质的程序阻碍了我们对其膜生理学和适应的理解。然而,通过去除脂质的极性头部基团,可以轻松且可重复地获得对这些现象的部分理解,从而获得详尽且精确的古细菌核心脂质成分(De Rosa等人,1980 年;Trincone等人. ,1992;Hopmans等人,2000 年;Cario等人,2015 年)。

关键字:古生菌, 细胞膜, 核心脂, 酸水解, HPLC-MS, 极端微生物

材料和试剂
 
1. 带 PTFE 帽的Pyrex TM管(Fisher Scientific,目录号:10004654)
2. 玻璃移液管和手动泵(Fisher Scientific,目录号:11546963)
3. 1毫升玻璃注射器(气密系列 1000 ; Hamilton,目录号:81320)
4. PTFE 玻璃瓶盖(Agilent Technologies,目录号:5182-0719)
5. 玻璃瓶(Dutscher,目录号:090971)
6. 50毫升圆底烧瓶(Fisher Scientific,目录号:CG150689)
7. 玻璃漏斗(DWK Life Sciences,目录号:213514609)
8. 2毫升玻璃小瓶(Sodipro,目录号:2108081)
9. 冷冻干燥的生物质古
10. 沙子(VWR,目录号:27460.364)
11. 玻璃棉(Fisher Scientific,目录号:12373866)
12. Celite TM (ACROS Organics ,目录号:349675000)
13. 硅胶Geduran的Si 60 ® (Merck公司,目录号1.11567)
14. 盐酸37%(盐酸; Fisher Scientific公司,目录号:10294190)
15. 将去离子水(H 2 O)
16. 甲醇(MeOH ;ACROS Organics ,目录号:364390025)
17. 二氯甲烷(DCM;ACROS Organics,目录号:326850025)
18. 丙酮(ACROS Organics,目录号:444150050)
19. 正庚烷(Hept;ACROS Organics,目录号:364360025)
20. 异丙醇(或丙-2-醇;IPA;ACROS Organics,目录号:184130250)
 
设备
 
烤箱(高达 450°C)
真空泵(爱德华兹V acuum,目录号码:A34317984 )
注意:在这里,我们使用了一个真空泵在50赫兹与20.5 m的最大位移3 ħ -1 ,17.0 m的最大排气速度3 ħ -1 ,和2×10的极限真空-2毫巴。虽然强大的吸力可以加快程序,但可以使用任何能够达到低于 20 毫巴的极限真空的真空泵。
旋转蒸发器(Büchi公司,型号:旋转蒸发器RE 111;加热浴,B-491)
注意:可以使用任何与您的真空泵和加热浴兼容的旋转蒸发仪,最高可达 50°C。
N 2干燥系统
注意:在这里,我们将建筑物 N 2供应(空气产品公司,CryoEase ®供应系统)连接到由聚碳酸酯管(Cole-Parmer,目录号:FV-30526-18)和分流阀(Cole-Parmer,目录号:FV-30600-02)以针(BD,目录号:300700)或玻璃移液管结尾。有关系统的描述,请参阅图 3。
高效液相色谱仪
注:在这里,我们使用配备有二元泵(安捷伦G1312A),溶剂脱气装置(安捷伦G1379A),自动注射器(安捷伦G1913A),恒温的柱室(安捷伦G1316A)一台HP 1100系列液相色谱系统,和化学工作站色谱管理器软件(安捷伦科技修订版 A.09.03)。然而,任何HPLC系统可以用于用溶剂梯度的功能。
Prevail Cyano 3 微米色谱柱(150 mm × 2.1 mm;Grace Davison Discovery Sciences,VWR ,目录号:HICH99243)
注意:不使用时,我们的色谱柱储存在 Hept/IPA (95:5, v/v) 中。然而,溶剂混合物可用于存储和柱的平衡两者并因此依赖于分离梯度(见小号TEP d 4)。因此,它可能会根据您的要求进行调整。
配备离子阱和大气压化学电离 (APCI) 源的质谱仪
注意:这里,我们使用了离子阱 MS Esquire 3000Plus(Bruker Daltonics,GE0100-G552)和安捷伦 APCI离子源(Agilent G1947A),但任何配备 APCI 离子源的MS 仪器都可以使用。
 
软件
 
质谱和色谱处理软件。
注:在这里,我们采用了布鲁克·道尔顿提供的指南针数据分析软件(版本5.0,2017年),但任何质谱和色谱处理软件,尤其是你的HPLC-MS仪器制造商提供的一个,都可以使用。
程序
 
材料准备
古菌生物质必须事先冷冻干燥。
注意:甲S表示每脂质提取过程中,生长培养基中可以包含脂质无关,待分析的细胞的量显著。为了避免从培养基中提取这些脂质,在冷冻干燥之前用适当的等渗溶液冲洗细胞两次是很重要的。
使用前将所有玻璃器皿、沙子、硅藻土和玻璃棉在 450°C 下燃烧 4 小时,以去除所有潜在有机污染物的痕迹。用去离子水,丙酮,和DCM(按该顺序)的玻璃器皿。使用前让其干燥。
硅胶在索氏装置中用 DCM 预萃取。
所有试剂和溶剂(除庚烷)必须是高品位,即。, > 99% 纯度,以降低污染风险。
注意:高纯度溶剂也可以通过蒸馏如果可能获得。
Hept 通过在填充有硅胶的柱子上过滤来清除有机污染物。
制备100毫升,加入10的1.2N HCl的MeOH溶液的毫升的盐酸37%至90毫升MeOH中。
注意:该溶液可以在室温下在带盖的玻璃瓶中储存数周。
制备100 ml的的MeOH / DCM(1:1,V / V)加入50 ml的DCM中于50 ml的MeOH中。
注意:该溶液可以在室温下储存在带盖的玻璃瓶中数周。
将 0.5 ml IPA 添加到 49.5 ml Hept 中,制备 50 ml Hept/IPA (99:1, v/v)。
注意:该溶液可以在室温下在带盖的玻璃烧瓶中储存数周。
通过将 50 毫升 IPA 添加到 950 毫升 Hept ,制备 1 , 000 毫升 Hept/IPA(95:5,v/v)。
注意:该溶液可以在室温下在玻璃瓶中储存数周。
准备一个装有 Hept的 1 , 000 毫升玻璃瓶。
注意:该溶液可在室温下保存数周。
 
古菌细胞水解(图1)
转移古细菌细胞沉淀到在Pyrex TM使用小玻璃漏斗玻璃管中。
使用 4 ml 1.2 N HCl 的 MeOH 重新悬浮古菌细胞沉淀,并使用玻璃移液管和手动泵冲洗玻璃漏斗。
注意:这一步的体积可能会根据细胞颗粒的大小进行修改。但是,不要将 Pyrex TM管填充到其总体积的一半以上。
用带有 PTFE 密封件的盖子紧紧关闭 Pyrex TM管。
注意:仔细检查盖子是否关闭,以避免任何可能导致样品破坏的蒸发。
为了避免样品和污染的任何交叉污染烘箱中泄漏的情况下,放5厘米砂的在玻璃瓶中,将您的Pyrex TM管中的砂,并关闭瓶用塞子。
在 110°C 下加热样品 4 小时。
注意:此方法是强大到足以除去大部分古极性头组没有进一步恶化古脂质,即,醚键仍然安然无恙。
将样品从烤箱中取出,让它们冷却至室温。
注意:在这一步,如果您的试管密封不够严密,您的样品可能已经变干并完全燃烧(样品呈黑褐色),这可能会导致核心脂质的破坏。甲S上的过程的质量控制,Ç赫克该管还含有溶剂和细胞沉淀几乎完全破坏和无色的。
 
 
图 1. 古菌干生物质直接酸甲醇分解的工作流程示意图。带圆圈的数字是指文本中的步骤。缩写:MeOH,甲醇;聚四氟乙烯,聚四氟乙烯。
 
核心脂质提取(图2)
样品冷却后,小心地打开 Pyrex TM管。
警告:由于在加热步骤中产生的气体,热气体和液体可以在打开时它从管涌出。
将 4 ml DCM 添加到 Pyrex TM管中(最终 MeOH/DCM 1:1,v/v)。
注意:这一步的体积可能会根据细胞颗粒的大小进行修改。
通过将玻璃棉和硅藻土放入玻璃漏斗中来创建过滤器。将玻璃漏斗放在适合旋转蒸发仪的 50 ml 圆底烧瓶上。
将样品转移到过滤器上,并通过漏斗将其过滤到烧瓶中,以去除脂质提取物中的固体颗粒(即细胞碎片)。
冲洗的Pyrex TM (:1,1用4ml的MeOH / DCM的管两次V / V ),并将其过滤到同一烧瓶如上。
用4ml的MeOH / DCM的冲洗过滤器进入相同的烧瓶两次(1:1,V / V )。
 
 
图 2. 从水解生物质中提取核心脂质的工作流程示意图。带圆圈的数字是指文本中的步骤。缩写:MeOH,甲醇;DCM、二氯甲烷。
 
HPLC 分析的样品制备(图 3)
使用设置为40°C的旋转蒸发器在减压下干燥样品。
冲洗烧瓶用MeOH并在减压下再次干燥以允许剩余的HCl / H的完全蒸发2 O.
干燥后,用 0.5 ml Hept/IPA (99:1, v/v )小心溶解烧瓶中的内容物,以回收核心脂质并使用玻璃注射器转移到 2 ml 小瓶中。
再冲洗烧瓶 3 次,然后转移到 2 ml 小瓶中。
干燥的N蒸汽下的样品2 。
在HPLC 分析之前,将样品重悬到 1 ml Hept/IPA (99:1, v/v ) 中。
注意:脂质提取物可以在 -20°C 下长时间(即几年)储存。
 
 
图3.工作流用于HPLC分析的核心脂质提取物制备的示意图。带圆圈的数字是指文本中的步骤。缩写:MeOH,甲醇;庚,庚烷;异丙醇,异丙醇。
 
HPLC-MS 核心脂质分离和分析
注意:本节基于 Tourte 等人,2020 年,但可以调整溶剂的性质和体积以适应您的色谱分离要求,即色谱柱的性质和大小。
启动您的色谱仪。
将色谱柱恒温器的温度设置为 30°C。
清空垃圾收集器并填充用于冲洗的小瓶 (Hept/IPA 95:5, v/v)。
使用用于 HPLC 色谱的梯度馈送色谱管理器软件。
注意:这里,我们使用了以下梯度,A = Hept/IPA (95:5, v/v) 和 B = Hept,流速为 0.2 ml min -1 :95% B(5 分钟等度)在 30 分钟内(5 分钟等度)升至 65% B,然后在 1 分钟内升至 0% B(10 分钟等度),然后在 1 分钟内升至 95% B(5分钟等度洗脱)。每个样品的总运行时间为 57 分钟。
仔细检查系统中的压力是否稳定,以确保正确分离。如有必要,请刷新您的系统。
启动 MS 仪器。
为 MS 管理器软件提供 MS 分析条件。
注意:这里,我们使用了以下 MS 条件:雾化器压力 50 psi,APCI 温度 420°C,干燥温度 350°C,干燥气体 (N 2 ) 流量 5 L min -1 ,毛细管电压-2 kV,电晕 4 µA,扫描范围 m/z 600-2 , 200。             
仔细检查所有参数是否正确达到设定值。
将包含溶解于1ml庚烷/ IPA的您的样品2毫升小瓶(99:1,V / V;参见小号TEP C6 )到HPLC自动进样器。
用您的样品信息和序列填充色谱管理器软件文件。
注意:进样量设置为 10 µl。为了提高可重复性,调整样品(浓度小号TEP C6 ),而不是改变的注射体积。
运行您的程序。
笔记: 每 30-40 次分析运行一个控制样本,以检查适当的分离和强度。通过运行一式两份或三份选定的样品和参考样品来评估再现性。例如,作为参考样品,我们使用了来自 Pyrococcus furiosus 的核心脂质提取物,其中包含二植烷基甘油二醚 (DGD) 和四醚脂质的多种异构体,即甘油单、二和三烷基甘油四醚 (GMGT、GDGT、和 GTGT,分别具有 0 到 4 个环戊烷环,以评估 HPLC 色谱柱的性能。 此外,运行标准混合物(合成古菌类脂质可以从 Avanti 购买)以评估检测到的化合物的相对响应因子。作为标准混合物,我们使用摩尔比为2:1的 DGD/GDGT0 溶液,这给出了 DGD 的相对响应因子约。10倍低于我们的分析条件下GDGT0的(Tourte等人,2020)。              
 
色谱图和质谱分析
注:古核心脂质质量的例子相对较少光谱已公布,但标识可以帮助参照例如,Hopmans等。( 2000 ) 。
使用质谱和色谱处理软件绘制分析的总离子流 (TIC)。
主要化合物,即TIC 上丰度最高的化合物,根据其质谱图、碎裂模式、LC 中的保留时间以及与已公布数据的比较(如果可能)进行初步鉴定。使用给定核心脂质峰的几个光谱的平均值,以确保正确识别(图 1)。
注:非常FE瓦特除了完整化合物的质子化加合离子是由我们APCI过程生成的化合物,和因此被鉴定基于其质子化质量。为了进一步确定已鉴定的核心脂质的结构,可以执行 MS/MS。
绘制的提取离子色谱图(EIC)通过提取识别的所有化合物的质量(图4)。对于每种化合物,选择适合您的 MS 系统的质量范围。
注意:考虑到我们的系统,我们通常使用 ±0.5 的质量范围。
在您的EIC整合各峰,以确定你的分析条件下每个核心的脂质的相对比例和与正确的化合物的响应系数(参照小号TEP D11 )。
 
 
图 4.嗜酸嗜热球菌的核心脂质分析。细胞在最佳条件下生长(85°C,pH 9.0,3.0% NaCl (w/v);Dirmeier等,1998)。通过提取以下质量数绘制色谱图:653.5、1302.5 和 1304.5。插入物示出了质谱和每个彩色峰所得到的核心结构:绿色,diphytanyl甘油二醚(DGD); 蓝色,无环戊烷环的甘油三烷基甘油四醚 (GTGT0);紫色,不含环戊烷环的甘油二联植烷基甘油四醚 (GDGT0)。指示的质量对应于质子化加合物。校正在我们的分析条件下观察到的响应因子,DGD、GTGT0 和 GDGT0 的相对比例分别为46%、1% 和 53%。
 
致谢
 
笔者想感谢法国国家研究署资助的项目ArchaeoMembranes(ANR-17-CE11-0012-01)和CNRS我nterdisciplinary程序“ORIGINES”资助的研究项目。
 
利益争夺
 
作者宣称没有利益冲突。
 
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引用: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.
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