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Jan 2018

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Extraction and Quantification of Sphingolipids from Hemiptera Insects by Ultra-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry
超高效液相色谱-串联质谱联用法提取和定量半翅目昆虫鞘脂质   

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Abstract

Sphingolipids are major structural components of endomembranes and have also been described as an intracellular second messenger involved in various biological functions in all eukaryotes and a few prokaryotes. Ceramides (Cer), the central molecules of sphingolipids, have been depicted in cell growth arrest, cell differentiation, and apoptosis. With the development of lipidomics, the identification of ceramides has been analyzed in many species, mostly in model insects. However, there is still a lack of research in non-model organisms. Here we describe a relatively simple and sensitive method for the extraction, identification, and quantification of ceramides in Hemiptera Insects (brown planthooper), followed by Ultra-Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). C18 is used as the separation column for quantitative detection and analysis on the triple quadruple liquid mass spectrometer. In this protocol, the standard curve method is adopted to confirm the more accurate quantification of ceramides based on the optional detection conditions.

Keywords: Hemipetera Insects (半翅目昆虫), Nilaparvata lugens Stål (褐飞虱), Sphingolipids (鞘脂类), Ceramides (神经酰胺), UPLC-MS/MS (质谱联用), Extraction (提取), Identification (鉴定), Quantification (量化), Standard curve (标准曲线)

Background

Sphingolipids are the second largest group of membrane lipids in living organisms and play an important role in many aspects of cell structure, metabolism, and regulation (Lahiri and Futerman, 2007). At first, it was thought that sphingolipids were a complex family of structurally related molecules, but more and more studies have shown that sphingolipids are involved in numerous cellular processes (Mao and Obeid, 2008). Ceramides (Cer) are essential bioactive lipids implicated in various cell biological processes ranging from cell growth regulation to cell death and senescence (Futerman and Hannun, 2004; Hannun and Obeid, 2008) through influencing of multiple signaling pathways. Although the physiological roles of ceramides are widely reported, few studies have described the extraction, identification, and quantification, thus, analysis of ceramides has gained significant interest in investigating the physiological functions of sphingolipid metabolism in Hemiptera Insects.


Currently, various methods have been described for this purpose, such as Diacylglycerol (DAG) Kinase assay (Preiss et al., 1987), Thin-layer chromatography (TLC) (Gorska et al., 2002), Gas chromatography mass spectrum (GC-MS) (Tserng et al., 2003), High-performance liquid chromatography (HPLC) (Yano et al., 1998; Dobrzyn and Gorski, 2002). In the beginning, DAG kinase assay was commonly used for Cer quantitation, but the specificity has been questioned (Watts et al.,1997). Thin layer chromatography was the method of choice, but the resolution, sensibility, and separation were limited, resulting in inefficient separation of similar molecules (Bielawski et al., 2010). Despite the high sensitivity of chromatographic analysis, this method had some limitations, such as the need for standard substances and derivatization (Dobrzyn et al., 2004). High-performance liquid chromatography (HPLC) was introduced to obtain ceramides separation with higher resolution, but complex samples like tissue extracts, therefore, produced many unspecific signals that did not provide any information concerning the metabolism of molecular species by HPLC (Yano et al., 1998; Bode and Graler, 2012).


Given the ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), it is more efficient, rapid, and sensitive, improving the separation condition of extremely complex samples and reducing matrix interference (Cutignano et al., 2010). Therefore, the current choice method is the analysis of ceramides by Ultra Performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). This protocol provides a relatively rapid and reproducible method. Moreover, this method can be used to profile ceramides from the extraction of the plant sample. It has evolved as the method of choice to detect sphingolipids metabolites due to its high sensitivity and superior specificity. The content of ceramide is determined and a quantitative system of sphingolipids in Hemiptera Insects is established, which lay a foundation for understanding the metabolic process and elucidating the biological function of each component. This method was described and used successfully to extract other sphingolipids in previously published studies (Bielawski et al., 2010; Shi et al., 2018 and 2019).


Materials and Reagents

  1. 1.5 ml Eppendorf tubes (Axygen, catalog number: MCT-150-C)

  2. 1.0 mm Ceramic Beads (Nalgene, catalog number: 150010C)

  3. 2 ml Micro tube (Sarstedt, catalog number: 72.609)

  4. Pipette tips (Axygen, catalog numbers: T-300, T-200-Y, T-1000-B)

  5. Glass Centrifuge Tubes (VWR International, catalog number: 734-4240)

  6. Nitrogen gas (> 99% Purity) (any brand will suffice)

  7. Isopropyl alcohol (Sangon Biotech, catalog number: A503069)

  8. Ethyl acetate (Sangon Biotech, catalog number: A507048)

  9. Liquid nitrogen (any brand will suffice)

  10. HPLC-grade methanol (Sigma-Aldrich, catalog number: 34806)

  11. Formic acid (Sangon Biotech, catalog number: A503066)

  12. MilliQ Water (Millipore, catalog number: Direct-Q3)

  13. Standards (see Table 1)

    Note: All Ceramides standards list is shown in Table 1.

  14. Solvent extraction solution A (see Recipes)

  15. Solvent extraction solution B (see Recipes)

  16. Mobile phase A (see Recipes)

  17. Mobile phase B (see Recipes)

  18. Internal standard (Avanti company) (see Recipes)


    Table 1. Example of ceramides standards list

    Std: Standard; IS: Internal standard

Equipment

  1. Nitrogen evaporator N-EVAP (Organomation, model: HGC-24A)

  2. Centrifuge (Eppendorf, model: 5430R)

  3. Autoclave (SANYO, model: MLS-3780)

  4. UHPLC-Q-TOF-MS/MS system (AB SCIEX, Framingham, MA, USA)

  5. UHPLC column (Zorbax sb-C8, 2.1 × 150 mm, 3.5 μm; Agilent, Palo Alto, CA, USA)

  6. Ivory PTFE/red silicone rubber septa (Agilent Technologies, catalog number: 5182-0731)

  7. 2 ml amber screw vial with patch USP 1 expansion (HAMAG Technologies, catalog number: HM-0716H)

  8. Blue open-topped polypropylene cap and white PTFE/red Scilicone septa (HAMAG Technologies, catalog number: HM-0722)

  9. 250 μl clear glass pulled conical-bottom (HAMAG Technologies, catalog number: HM-2085)

  10. Analytical balance (METTLER TOLEDO, model: XS105)

  11. Tissue homogenizer (MP Biomedicals, USA, FastPrep-24)

  12. Oven (Bluepard, model: BPG-9040A)

  13. Vortexer (Germany, IKA, model: vortex 2)

  14. -80 °C freezer

Software

  1. PeakView (AB Sciex)

  2. Excel software (Microsoft office 2010)

  3. Data Processing software (DPS)

Procedure

  1. Insect samples collection

    The laboratory strain of N. lugens (brown planthopper) used in this study originated from a field population in the Huajiachi campus of Zhejiang University, Hangzhou, China. The BPHs (brown planthopper) were reared on susceptible rice seedlings cv.Taichung Native 1 (TN1) at 27 ± 1 °C, 70% relative humidity and a 16:8 h light:dark photoperiod.

    About 2 g fresh weight of insects was determined and collected at different development stages (e.g., eggs, first-fifth instar nymphs, female and male adults) in labeled 2 ml tissue grinding tubes. Samples were then stored at -80 °C after quickly freezing in liquid nitrogen. The sample was set for three biological repeats.


  2. Total sphingolipids extraction (Figure 1)

    Sphingolipids were extracted from insects according to Bielawski’s method (Bielawski et al., 2010). Details were prepared as follows:

    Note: All following steps were performed at room temperature if not stated otherwise.

    1. The samples were taken out from the -80 °C freezer and dissolved the melted samples in 1 ml solvent extraction solution A (see Recipe 1), to which 10 μl Internal standard had been added (see Recipe 5).

    2. Samples were next grounded twice in tissue homogenizer with grinding beads, each time for 20 s.

      Note: In the case of plant material, clean and pre-cooled mortars and pestles were used to grind the samples in liquid nitrogen, and the powder was then transferred to a 15 ml glass tube. The sample was homogenized into a fine powder and that powder was kept frozen at every homogenization step. The purpose of the griding bead is to facilitate insect tissue grinding, so it is okay just to cover the bottom layer for the amount of griding beads per mass of sample and total volume.

    3. Powdered tissue was vortexed vigorously for 5 min and centrifuged for 5 min at 1,000 × g.

    4. After centrifugation, the solvent from the upper lipid-containing phase was transferred to a 15 ml sterilized glass bottle.

      Note: The vial label was protected with a clear tape to avoid being wiped off by the solution.

    5. The extraction was repeated by adding 1 ml solvent extraction solution A (see Recipe 1) to the remaining aqueous phase, and the organic layers were combined and concentrated under a stream of nitrogen gas.

    6. The dried lipid fractions were re-suspended into 500 μl solvent extraction solution B (see Recipe 2), and immediately sufficient liquid was transferred to glass vials and caped tightly.

    7. The solvent was transferred into a labeled 1.5 ml microcentrifuge tube, centrifuge the samples at 12,000 × g for 5 min at room temperature.

      Note: The tube was caped tightly to avoid leakage of liquid during centrifugation.

    8. The reconstitution solution was finally transferred to a mass spectrometer flask with an internal cannula and stored at -20 °C for machine test.



      Figure 1. Workflow of sphingolipids extraction from Insect sample


  3. Sphingolipids detection and identification

    Sphingolipids were analysed on an AB Seriex 5600+quadrupole Time-of-Flight (Q-TOF) Premier mass spectrometer combined with a Water Acquity Ultra Performance liquid chromatography.

    1. HPLC-program

      1. Solvent A (see Recipe 3)

      2. Solvent B (see Recipe 4)

      3. Constant flow at a rate of 0.3 ml/min stated by solvent A was running in a Waters UPLC (Waters Corp, Milford, MA, USA) coupled with an AB Triple TOF 5600 plus System (AB SCIEX, Framingham, MA, USA).

      4. The changes in gradient were comprised of an increase in solvent B (methanol) from 80 to 99% over 20 min and then from 99 to 100% over 15 min, followed by a reduction back to 80% over 1 min. The percentage of solvent B was then held at 80% for the last 9 min.

    2. The reversed-phase analytical column (Zorbax sb-C8, 2.1 × 150 mm, 3.5 μm; Agilent, Palo Alto, CA, USA) was used to separate ceramides.

    3. 10 μl of the samples were applied to the column.

    4. The column was kept at 35 °C during the whole procedure.

    5. The mass spectrum was acquired with an electrospray ionization (ESI) ion source in the positive ionization mode and following settings (Table 2).


      Table 2. The instrument settings for sphingolipids analysis



  4. Sphingolipids profiling parameters for detection

    The C18 column was used as the separation column on the Aligent 6460+ triple four-pole liquid mass spectrometer to explore the optimal detection conditions of each standard sample. The profiling parameters include precursor ion/targeted ion, retention time, fragmentor, and collision energy. Cer (d18:0/12:0) was used as the internal standard. The scanning parameters for each lipid class were listed in Table 3.


    Table 3. Scanning parameters for sphingolipids detection



    Method optimization notes: For samples containing large amounts of low volatility compounds, the HPLC-program can be made more stringent by increasing the rate flow and by increasing the sampling time. HPLC-program can be adjusted as appropriate for specific analytes and columns by altering holding times. For a few analytes which degrade at high temperatures, it may be desirable to reduce Ion source heater temperatures to 550-580 °C. The timed MS parameters may be used to reduce or eliminate signal from very abundant peaks or contaminants. Those sphingolipids that have different chain lengths, branching, or unsaturation will fragment to yield ions of different m/z. Thus, different precursor ion scans would be necessary to determine the corresponding molecular species. Furthermore, the scan range and collision energy will vary depending on the size and substitution of the various subspecies. All such method adjustments should only be undertaken with the assistance of an experienced MS user.

Data analysis

  1. A standard curve with standards from 0.5 ng/ml to 200 ng/ml was generated for quantitative analysis. Curves consisted of triplicates of one blank sample and five calibration points at a concentration ranging from 0.5 ng/ml to 200 ng/ml (Table 4). The amount of each lipid species was calculated according to the sample peak area compared with the normalized internal standard peak area. We used the fitting curve to show the relationship between the concentration and peak area, followed by the fitting curve equation. Three or more biological repeats were recommended (Figure 2).


    Table 4. The data points of ceramide standard curve




    Figure 2. Example of ceramide standard curve. The following ceramides amounts were used: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml. The x-coordinate is the concentration and the y-coordinate is the Analyte/IS peak area. For the generation of the standard curve, we converted the horizontal and vertical to logarithmic form.


  2. C18 sphingoid bases are the major backbone of most sphingolipids in mammals (Jarne et al., 2018). So we chose the C18-0 as a representative to describe the standard curve (Figure 2). For calibration line measurement, equal amounts and 100 ng Cer (d18:0/12:0) as internal standard were added into different calibration points. The calibration lines from 0.5 ng/ml to 200 ng/ml were converted to a fitted curve, with r values consistently greater than 0.9999 during validation (Table 5). The logistic curve in DPS (Data Processing) software was used to fit the equation.


    Table 5. The fitting value of ceramide standard curve



  3. For acquisition, the multiple reaction monitoring (MRM) mode and the software PeakView were used. The internal standard with the mass transition 482.00 m/z -> 264.40 m/z, the retention time of the internal standard is 13.19 min. The Cer (d18:1/16:0) standard with the mass transition 538.91 m/z -> 264.40 m/z, the retention time of the Cer (d18:1/16:0) standard is 16.45 min. Since the retention times and compound-specific ionization and fragmentation values are highly dependent on the used instrumentation, the given values list in Table 2 may be used as a reference but should be individually determined for different instrument setups (Figure 3; Figure 4).



    Figure 3. Example of internal standard mass spectrometry acquired with ESI ion source in positive mode from Nilaparvata lugens. Representative signal of internal standard Cer (d18:0/12:0) was plotted. Retention time of internal standard was slightly different from the table 2 list (13.19 min vs. 13.28 min).




    Figure 4. Example of Cer (d18:1/16:0) standard mass spectrometry acquired with ESI ion source in positive mode from Nilaparvata lugens. Representative signal of Cer (d18:1/16:0) was plotted. Retention time of internal standard was slightly different from the table 2 list (16.45 min vs. 16.55 min).


  4. Different ceramides were identified by comparing MS/MS ions of analysts with those of sphingolipid standards in ChemSpider base (http://www.chemspider.com/) through the software PeakView (Http://scie.com.cn/products/software/peakview-software). The maximum allowed error for the reliability was set to ± 10 ppm.

Recipes

  1. Solvent extraction solution A

    Ethyl acetate:isopropanol:water, 60:30:10 (vol/vol/vol)

  2. Solvent extraction solution B

    Methanol:0.1% formic acid, 9:1 (vol/vol)

  3. Solution A

    MQ water containing 0.1% formic acid

  4. Solution B

    100% Methanol

  5. Internal standard

    100 μg/ml Cer (d18:0/12:0) dissolved in HPLC-grade MeOH

Acknowledgments

This work was founded by the National Natural Science Foundation of China (31871962), and the Key R & D Plan of Zhejiang Province (2018C04G2011264). We thank Lijuan Mao from the Analysis Center of Agrobiology and Environmental Sciences Zhejiang University for the help in chemical analysis. The protocol was taken from the publication of Bielawski et al. 2010 with minor modified (Bielawski et al., 2010).

Competing interests

The authors declare that no competing financial interest.

References

  1. Bielawski, J., Pierce, J. S., Snider, J., Rembiesa, B., Szulc, Z. M. and Bielawska, A. (2010). Sphingolipid analysis by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS). Adv Exp Med Biol 688: 46-59.
  2. Bode, C. and Graler, M. H. (2012). Quantification of sphingosine-1-phosphate and related sphingolipids by liquid chromatography coupled to tandem mass spectrometry. Methods Mol Biol 874: 33-44.
  3. Cutignano, A., Chiuminatto, U., Petruzziello, F., Vella, F. M. and Fontana, A. (2010). UPLC-MS/MS method for analysis of sphingosine 1-phosphate in biological samples. Prostaglandins Other Lipid Mediat 93(1-2): 25-29.
  4. Dobrzyn, A. and Gorski, J. (2002). Ceramides and sphingomyelins in skeletal muscles of the rat: content and composition. Effect of prolonged exercise. Am J Physiol Endocrinol Metab 282(2): E277-285.
  5. Dobrzyn, A., Knapp, M. and Gorski, J. (2004). Effect of acute exercise and training on metabolism of ceramide in the heart muscle of the rat. Acta Physiol Scand 181(3): 313-319.
  6. Futerman, A. H. and Hannun, Y. A. (2004). The complex life of simple sphingolipids. EMBO Rep 5(8): 777-782.
  7. Gorska, M., Dobrzyn, A., Zendzian-Piotrowska, M. and Namiot, Z. (2002). Concentration and composition of free ceramides in human plasma. Horm Metab Res 34(8): 466-468.
  8. Hannun, Y. A. and Obeid, L. M. (2008). Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9(2): 139-150.
  9. Jarne, C., Saviron, M., Lapieza, M. P., Membrado, L., Orduna, J., Galban, J., Garriga, R., Morlock, G. E. and Cebolla, V. L. (2018). High-Performance Thin-Layer Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry for Identifying Neutral Lipids and Sphingolipids in Complex Samples. J AOAC Int 101(6): 1993-2000.
  10. Lahiri, S. and Futerman, A. H. (2007). The metabolism and function of sphingolipids and glycosphingolipids. Cell Mol Life Sci 64(17): 2270-2284.
  11. Mao, C. and Obeid, L. M. (2008). Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochim Biophys Acta 1781(9): 424-434.
  12. Preiss, J. E., Loomis, C. R., Bell, R. M. and Niedel, J. E. (1987). Quantitative measurement of sn-1,2-diacylglycerols. Methods Enzymol 141: 294-300.
  13. Shi, X.-X., Huang, Y.-J., Begum, M.-A., Zhu, M.-F., Li, F.-Q., Zhang, M.-J., Zhou, W.-W., Mao, C. and Zhu, Z.-R. (2018). A neutral ceramidase, NlnCDase, is involved in the stress responses of brown planthopper, Nilaparvata lugens (Stål). Sci Rep 8(1): 1130.
  14. Shi, X.-X., Zhang, H., Chen, M., Zhang, Y.-D., Zhu, M.-F., Zhang, M.-J., Li, F.-Q., Wratten, S., Zhou, W.-W., Mao, C. and Zhu, Z.-R. (2019). Two sphingomyelin synthase homologues regulate body weight and sphingomyelin synthesis in female brown planthopper, N. lugens (Stål). Insect Molecular Biology 28(2): 253-263.
  15. Tserng, K. Y. and Griffin, R. (2003). Quantitation and molecular species determination of diacylglycerols, phosphatidylcholines, ceramides, and sphingomyelins with gas chromatography. Anal Biochem 323(1): 84-93.
  16. Watts, J. D., Gu, M., Polverino, A. J., Patterson, S. D. and Aebersold, R. (1997). Fas-induced apoptosis of T cells occurs independently of ceramide generation. Proc Natl Acad Sci U S A 94(14): 7292-7296.
  17. Yano, M., Kishida, E., Muneyuki, Y. and Masuzawa, Y. (1998). Quantitative analysis of ceramide molecular species by high performance liquid chromatography. J Lipid Res 39(10): 2091-2098.

简介

[摘要]鞘脂类是endomembranes的主要结构部件和已经也被描述为参与所有真核生物和原核生物几多种生物学功能的细胞内第二信使。 神经酰胺(CER),鞘脂的中心的分子,已经在细胞生长停滞,细胞分化被描述,和细胞凋亡。随着脂质组学的发展,已经在许多物种中对神经酰胺的鉴定进行了分析,其中大多数是模型昆虫。但是,仍然缺乏对非模型生物的研究。在这里,我们描述了用于提取中相对简单和灵敏的方法N,鉴定,并在半翅目昆虫的神经酰胺的定量(棕色planthooper ),随后用超高效液相色谱-串联质谱联用(UPLC-MS / MS)。C18被用作用于对三联四极液体MAS定量检测和分析的分离柱小号光谱仪。在该方案中,采用标准曲线方法以根据可选的检测条件确认更准确的神经酰胺定量。

[背景]鞘脂是膜脂质在活体生物的第二大组和细胞结构,代谢和调节的许多方面发挥了重要作用(拉希里和Futerman,2007)。起初,人们认为鞘脂是结构相关分子的复杂家族,但越来越多的研究表明鞘脂参与了许多细胞过程(Mao和Obeid ,2008 )。神经酰胺(CER)是必不可少的牵连生物活性脂质的各种细胞生物过程从细胞生长调控细胞死亡和衰老(Futerman和Hannun,2004 ; Hannun和奥贝德,2008)通过的多个信号转导途径影响小号。虽然神经酰胺的生理作用广泛报道,很少有研究描述的提取,鉴定,和量化,因此,神经酰胺的分析已经取得了显著的兴趣在investigat荷兰国际集团在鞘脂代谢的生理功能,半翅目昆虫。

当前,已经描述了用于该目的的各种方法,例如二酰基甘油(DAG)激酶测定法(Preiss等,1987),薄层色谱法(TLC)(Gorska等人,2002),气相色谱质谱法(GC)。 -MS)(Tserng等,2003),高效液相色谱法(HPLC)(Yano等,1998; Dobrzyn和Gorski,2002)。在开始时,DAG激酶测定常用的神经酰胺定量,但特异性已受到质疑(瓦特等人。,1997)。薄层色谱法是选择的方法,但是分辨率,š ensibility ,和分离被限制,导致类似的低效率分离分子(Bielaws ķ我等人。,2010)。 尽管色谱分析具有很高的灵敏度,但该方法仍存在一些局限性,例如需要标准物质和衍生化方法(Dobrzyn等,2004)。ħ我GH-高效液相色谱法(HPLC)导入到obtion获得具有较高分辨率的神经酰胺的分离,但像组织提取物的复杂样品,因此,产生了许多非特异性信号即没有不提供关于分子种类通过HPLC代谢的任何信息(矢野等人,1998;Bode and Graler ,2012 )。

鉴于超耦合到串联质谱(UPLC-MS / MS)高效液相色谱法,它是更有效的,快速的,而敏感,即兴荷兰国际集团极其复杂的样品和红眼的分离条件ING矩阵干扰(Cutignano等人。,2010 )。因此,当前的选择方法是通过超高效液相色谱-串联质谱(UPLC-MS / MS)分析神经酰胺。该协议提供了一种相对快速且可重现的方法。此外,该方法可用于从提取轮廓神经酰胺的植物样品。由于它的高灵敏度和优越的特异性,它已发展成为检测鞘脂代谢物的首选方法。神经酰胺的含量被确定,并且在半翅目昆虫鞘脂的定量系统被建立,其中奠定了基础理解代谢过程和阐明biolo每个组件的gical功能。在先前发表的研究中(Bielaws k i等人,2010; Shi等人,2018和2019),已经描述了这种方法并成功地用于提取其他鞘脂。

关键字:半翅目昆虫, 褐飞虱, 鞘脂类, 神经酰胺, 质谱联用, 提取, 鉴定, 量化, 标准曲线

材料和试剂
1. 1.5 ml Eppendorf管(Axygen,目录号:MCT-150-C)     
2. 1.0毫米陶瓷珠(Nalgene ,目录号:150010C)     
3. 2 ml微型管(Sarstedt,目录号:72.609)     
4.移液管头(爱思进,目录号小号:T-300,T-200-Y,T-1000-B)     
5.玻璃离心管(VWR International,目录号:734-4240)     
6.氮气(> 99%纯度)(任何品牌都足够)     
7.异丙醇(Sangon Biotech,目录号:A503069)     
8.乙酸乙酯(Sangon Biotech,目录号:A507048)     
9.液体nitrog恩(一个纽约的品牌就足够了)     
10. HPLC级甲醇(Sigma-Aldrich,目录号:34806) 
11.甲酸(Sangon Biotech,目录号:A503066) 
12. MilliQ Water (Millipore,目录号:Direct-Q3) 
13.标准品(见表1) 
注意:所有神经酰胺标准品清单如表1所示。

14.溶剂萃取溶液A(请参阅配方s ) 
15.溶剂萃取溶液B(请参见配方s ) 
16.流动相甲(见配方小号) 
17.流动相B(见配方小号) 
18.内标(阿凡提公司)(见配方小号) 

表1的实施例Ç eramides标准列表

标准:标准;IS:内标


设备


氮气蒸发器N-EVAP(组织,型号:HGC-24A)
离心机(Eppendorf,型号:5430R)
高压灭菌器(三洋,型号:MLS-3780)
UHPLC-Q-TOF-MS / MS系统(AB SCIEX,美国马萨诸塞州弗雷明汉)
UHPLC色谱柱(Z orbax sb-C8,2.1×150 mm,3.5μm; Agilent,帕洛阿尔托,美国)
象牙PTFE /红色硅橡胶隔垫(Agilent Technologies,目录号:5182-0731)
2毫升琥珀色带刻度USP 1扩展瓶的小瓶(HAMAG Technologies,目录号:HM-0716H)
蓝色开顶聚丙烯帽和白色PTFE /红色Scilicone隔垫(HAMAG Technologies,目录号:HM-0722)
250 μ升透明玻璃拉锥形底部(HAMAG技术,货号:HM-2085)
分析天平(梅特勒-托利多,型号:XS105 )
组织匀浆器(MP Biomedicals,美国,FastPrep-24)
烤箱(Bluepard,型号:BPG-9040A )
Vortexer(德国,IKA ,型号:vortex 2)
-80°C冷冻室

软件


峰V IEW(AB Sciex的)
Excel软件(Microsoft Office 2010)
数据处理软件(DPS)

程序


我NSECT样品采集
实验室株褐飞虱(棕色planthop p ER)在本研究中使用起源于浙江大学,杭州,中国的华家池校区现场人群。所述BPHS (棕色planthop p ER)在27±1℃,相对湿度70%饲养易感稻苗cv.Taichung本机1(TN1),16:8小时光照:黑暗光周期。

约2昆虫克鲜重被确定,并且在不同的发育阶段收集(É 。克。,蛋,第一方第五龄在标签若虫,雌,雄成虫)编2个毫升组织GRI Ñ丁管。然后将样品在液氮中快速冷冻后保存在-80°C 。将样品设置为三个生物学重复。


总鞘脂提取(图1)
鞘脂是为Extrac根据Bielaws从昆虫泰德ķ我的方法(Bielaws ķ我等人。,2010) 。详细情况如下:

注意:除非另有说明,以下所有步骤均在室温下进行。

将样品取出,从-80℃冰箱和渐隐ð的熔融在1毫升溶剂萃取溶液A(见配方样品1),到10 μ升内标已加入(见配方5)。
然后将样品在带有研磨珠的组织均质器中两次接地,每次20 s 。
注意:在的情况下的植物材料,清洁和预冷却迫击炮和研杵都用于研磨样品I Ñ液氮,和POW d ER然后转移到15毫升玻璃管中。Ť他的样品被均匀成细粉末,以及粉末是在每一个同质化冷冻保存的一步。网格珠的目的是促进昆虫组织的研磨,因此可以覆盖每层样品的质量和总体积的网格珠的数量即可。

粉末状组织涡旋剧烈5分钟,并离心d在1000 5分钟×克。
离心后,将来自上层脂质相的溶剂转移至15 ml无菌玻璃瓶中。
注意:小瓶标签用透明胶带保护,以免被溶液擦去。

提取通过加入重复1毫升溶剂萃取溶液A(见配方1)到剩余的水相,和合并,并在氮气流下浓缩有机层。
Ť他干燥的脂质级分重新悬浮到500 μ升溶剂提取液B(见配方2) ,并立即足够的液体被转移到玻璃小瓶中并帽编紧密。
Ť他溶剂转移到标记的1.5毫升microcentrifu克ë管,离心将样品在12000 ×g下在室温下5分钟。
注意:将管盖紧,以免离心过程中液体泄漏。

重构溶液最后转移至质谱仪烧瓶的内部插管并储存在-20℃下机试验。



图1的工作流程ö ˚F小号phingolipids萃取FR OM昆虫样品


鞘脂的检测与鉴定
鞘脂上进行分析的AB Seriex 5600 +四极时间的飞行(Q-TOF)总理质谱仪与水的Acquity超高效液相色谱组合。

HPLC程序
溶剂A (请参见配方3)
                                                                                                                           溶剂B (请参见配方4)
在Waters UPLC(美国马萨诸塞州米尔福德,沃特世公司)和AB Triple TOF 5600 plus系统(AB SCIEX,美国麻萨诸塞州弗雷明汉)结合使用的情况下,溶剂A规定的恒定流量为0.3 m l / min。。
梯度的变化包括:溶剂B(甲醇)在20分钟内从80%增加到99%,然后在15分钟内从99%增加到100%,然后在1分钟内减少到80%。然后在最后9分钟内将溶剂B的百分比保持在80%。
反相分析柱(ZORBAX SB-C8,2.1×150毫米3.5微米;安捷伦,帕洛阿尔托,CA,USA)中的溶液用d来分离神经酰胺。
10 μ样品的升被加到柱上。
该柱被保持在35在整个过程中为°C。
质谱是通过电喷雾电离(ESI)离子源获取的正电离模式和以下设置(表2)。

牛逼能2.将仪器设定š对于SP hingolipids分析


鞘脂分析参数用于检测
C18柱用作Aligent 6460+三重四极杆液体质谱仪上的分离柱,以探索每种标准样品的最佳检测条件。仿形参数包括前体离子/靶向离子,保留时间,˚F ragmentor ,和碰撞能量。Cer (d18:0/12:0)被用作内标。表3列出了每种脂质类别的扫描参数。





表3 。扫描参数为SP hingolipids检测


方法优化说明:对于含有大量低的样品volatilit ÿ化合物中,HPLC-程序可以通过增加提出了更严格速率流动并通过增加采样时间。可以通过更改保留时间来针对特定的分析物和色谱柱适当地调整HPLC程序。对于在高温下降低几个分析物时,可能期望减少离子源加热器温度小号至550-580 ℃。的定时MS参数可以被用于减少或从非常丰富的峰或污染物消除信号。那些具有不同链长,分支或不饱和键的鞘脂将碎裂以产生不同m / z的离子。因此,需要不同的前驱体离子扫描来确定相应的分子种类。此外,扫描范围和碰撞能量将根据各种亚种的大小和替代而变化。所有此类方法的调整应仅在有经验的MS用户的帮助下进行。


数据分析


                                                                                       生成了标准曲线,标准曲线为0.5 ng / ml至200 ng / ml ,用于定量分析。曲线在由一个空白样品和五个校准点的一式三份的一个浓度范围为0.5毫微克/毫升至200毫微克/毫升(表4) 。每个脂质种类的量根据下式计算的相比较样品峰面积的归一化的内标峰面积。我们使用Ð拟合曲线,显示之间的关系concen登记领峰面积,其次是该拟合曲线方程。三个或更多个生物重复进行推荐的(图2)。

表4 。神经酰胺标准曲线的数据点





图2.神经酰胺标准曲线的例子。使用以下神经酰胺量:0 ng / ml,0.5 ng / ml,5 ng / ml,10 ng / ml,100 ng / ml,200 ng / ml。x坐标是浓度,y坐标是分析物/ IS峰面积。对于标准曲线的产生,我们转换编的掘宗塔尔和垂直于对数形式。


                                                                                       C18鞘氨醇碱基是哺乳动物中大多数鞘脂的主要骨架(Jarne等人,2018)。因此,我们选择C1 8-0作为代表来描述标准曲线(图2)。对于校准线测量,将等量的100 ng Cer(d18:0/12:0)作为内标物添加到不同的校准点中。从0.5校准线纳克/ ml至200ng / ml的人转换编到拟合曲线,其中r值始终大于验证期间0.9999 (表5) 。使用DPS(数据处理)软件中的逻辑曲线拟合方程。



表5 。神经酰胺标准曲线的拟合值


                                                                                       用于采集,多反应监测(MRM)模式和软件P EAK V IEW被使用。质量转变为482.00 m / z- > 264.40 m / z的内标,内标的保留时间为13.19分钟。的神经酰胺(D18:1/16:0)与质量标准过渡538.91 M / Z - > 264.40米/ Z,保留时间的神经酰胺(D18:1/16:0)的标准是16.45分钟。由于R etention时间和化合物的具体电离和分裂值是高度依赖于使用的仪器,该给定的值列表中Ť能够2可被用作参考,但应被单独用于不同的仪器设置确定(图3 ;图4 )。

图3.在正模式与ESI离子源获取的内标质谱的实施例从褐升ugens。内标代表信号神经酰胺(D18:0/12:0)被绘制。内标物的保留时间是从表2列出稍有不同(13.19分钟VS 。13.28分钟)。


图4 。È的CER xample(D18:1/16:0)以正模式与ESI离子源获取的标准质谱从褐升ugens 。的代表信号神经酰胺(D18:1/16:0)被绘制。内标物的保留时间是从表2列出稍微不同的(1 6 。45分钟VS 。16.55分钟)。


                                                                                       通过使用软件P eak V iew (http://scie.com.cn/)将分析人员的MS / MS离子与ChemSpider碱(http://www.chemspider.com/)中的鞘脂标准物的MS / MS离子进行比较,鉴定出了不同的神经酰胺。产品/软件/ peakview-software)。可靠性的最大允许误差设置为±10 ppm。

菜谱


溶剂萃取液A
乙酸乙酯:异丙醇:水,60:30:10(vol / vol / vol)

溶剂萃取液B
甲醇:0.1%甲酸,9:1(体积/体积)

解决方案A
MQ水Ç ontaining 0.1%甲酸

解决方案B
100%甲醇

内部标准
100 μ微克/毫升神经酰胺(D18:0/12:0)溶解在HPLC级甲醇


致谢


这项工作由国家自然科学基金(31871962)和浙江省重点研发计划(2018C04G2011264)共同完成。感谢浙江大学农业生物与环境科学分析中心的毛丽娟在化学分析方面的帮助。Ť他协议是从所拍摄的出版物Bielawski等。2010年进行了小幅修改(Bielaws k i等人,2010年)。


利益争夺


作者宣称没有竞争的经济利益。


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引用:Wang, N., Shi, X., Zhang, C., Zhou, W. and ZHU, Z. (2021). Extraction and Quantification of Sphingolipids from Hemiptera Insects by Ultra-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry. Bio-protocol 11(4): e3923. DOI: 10.21769/BioProtoc.3923.
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