搜索

Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells
代谢重同位素标记法研究培养细胞的甘油磷脂稳态   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope (e.g., deuterium)-labeled head group precursors added to the culture medium incorporate efficiently into glycerophospholipids of mammalian cells, which allows one to determine the rates of synthesis, acyl chain remodeling or turnover of the individual glycerophospholipids using mass spectrometry. This protocol describes how to study the metabolism of the major mammalian glycerophospholipids i.e., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylinositols with this approach.

Keywords: Glycerophospholipid(甘油磷脂), Stable heavy isotope labeling(稳定的重同位素标记), Deuterium-labeled precursor(氘标记前体), Mass spectrometry(质谱法), Lipid metabolism(脂质代谢), Cell culture(细胞培养)

Background

Radiolabeled precursors have been extensively used to study glycerophospholipid (GPL) metabolism in cultured cells. However, this approach has serious drawbacks. First of all, it is unfeasible to study the metabolism of all molecular species of GPLs due to the fact that it is not possible, without reverting to highly complicated and time-consuming protocols, to separate the individual molecular species from each other (Patton et al., 1982), which is obviously necessary to study their metabolism. Second, the radioisotopes needed are quite expensive. Third, for optimal labeling, the unlabeled precursors should be depleted of the medium as much as possible. Fourth, only two different precursors (labeled with 3H or 14C) can be added to the cells simultaneously and even then accurate correction for the overlap between the isotope spectra is necessary upon liquid scintillation counting of the collected fractions. Because of those handicaps, a number of studies have recently introduced an alternative approach to study GPL metabolism (e.g., Heikinheimo and Somerharju, 2002; de Kroon, 2007; Kainu et al., 2008; Postle and Hunt, 2009; Kainu et al., 2013; Hermansson et al., 2016). This approach is based on the use of stable heavy isotope-labeled precursors and mass spectrometric (MS) analysis of GPL labeling. Clearly, this approach is far more convenient and efficient as compared to the traditional methods based on the use of radiolabeled precursors, due to the following facts: (a) multiple labeled precursors can be added simultaneously to the culture medium thus providing information on several GPL classes, (b) labeled and unlabeled GPLs can be conveniently and selectively detected using head group–specific scanning modes, (c) information is obtained on the turnover of individual GPL molecular species and (d) the stable heavy isotope-labeled precursors are generally much cheaper than the radiolabeled ones and can thus be added in amounts which avoids the use of special media depleted of the corresponding unlabeled precursors.

Materials and Reagents

  1. Pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 9400327 , 9401255 , 9401410 )
  2. 35 or 60 mm cell culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 153066 or 150288 )
  3. 12 ml screw-cap glass tubes (Kimble Chase Life Science and Research Products, catalog number: 45066A-16100 )
  4. Pasteur pipettes (BRAND, catalog number: 747720 )
  5. 12 x 32 mm screw-neck vials with caps (WATERS, catalog number: 186002640 )
  6. 11.5 x 75 mm test tubes (VWR, catalog number: ZZ130296772 )
  7. Cell scrapers and lifters (VWR, catalog numbers: 734-2603 and 734-2602 )
  8. HeLa cells or other cultured mammalian cells
  9. Phosphate buffered saline (PBS) (Merck Millipore, catalog number: 524650 )
  10. Methanol (VWR, catalog number: 83638.320 )
  11. Internal standards needed for mass spectrometric analyses:
    1. Di-20:1-phosphatidylcholine (PC) (Avanti Polar Lipids, catalog number: 850396 )
    2. Di-20:1-phosphatidylethanolamine (PE; synthesized in-house from corresponding PC) (Käkelä et al., 2003)
    3. Di-20:1-phosphatidylserine (PS; synthesized from corresponding PC) (Käkelä et al., 2003)
    4. Di-16:0-phosphatidylinositol (PI) (Avanti Polar Lipids, catalog number: 850141 )
  12. Chloroform (Merck Millipore, catalog number: 102445 )
  13. Acetonitrile, LC-MS grade (Fisher Scientific, catalog number: A/0638/17X )
  14. Isopropanol, OptimaTM LC/MS grade (Fisher Scientific, catalog number: A461-212 )
  15. Ammonium formate (Sigma-Aldrich, catalog number: 70221 )
  16. Ammonia solution 25% Suprapur (Merck Millipore, catalog number: 105428 )
  17. Acetic acid, glacial (Fisher Scientific, catalog number: 10171460 )
  18. Deuterium-labeled choline (D9-choline chloride) (C/D/N ISOTOPES, catalog number: D-2142 )
  19. Deuterium-labeled ethanolamine (D4-ethanolamine) (Cambridge Isotope Laboratories, catalog number: DLM-552-1 )
  20. Deuterium-labeled serine (D3-L-serine) (Cambridge Isotope Laboratories, catalog number: DLM-1073-1 )
  21. Deuterium-labeled inositol (D6-myo-inositol) (C/D/N ISOTOPES, catalog number: D-3019 )
  22. Hydroxylamine (Sigma-Aldrich, catalog number: 55460 )
  23. Dulbecco’s modified Eagle medium high glucose (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 ) or another medium appropriate for the cell line of interest
    Note: We grew HeLa cells in DMEM containing 10% fetal calf serum and 100 U/ml penicillin and 100 U/ml streptomycin.
  24. Choline chloride (Sigma-Aldrich, catalog number: C1879 )
  25. Ethanolamine (Merck Millipore, catalog number: 800849 )
  26. Myo-inositol (Sigma-Aldrich, catalog number: I5125 )
  27. L-serine (Sigma-Aldrich, catalog number: S4500 )
  28. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  29. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  30. Milli-Q H2O (Elga Stat Maxima Reverse Osmosis Water Purifier)
  31. Nitrogen gas (Aga, Industrial gases, 99.95%)
  32. Labeling medium (see Recipes)
  33. Chase medium (see Recipes)

Equipment

  1. Pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 4600170 , 4600240 and 4600250 )
  2. Microliter syringe (Hamilton, model: 705 N, catalog number: 80565 )
  3. Vortex mixer (VWR, catalog number: 444-1372 )
  4. Centrifuge (Thermo Fisher Scientific, model: HeraeusTM MegafugeTM 1.0 )
  5. Fume hood
  6. Sample Concentrator with nitrogen evaporation (Cole-Parmer, Techne, catalog number: FSC400D )
  7. Mass spectrometer or analysis of the samples by a service provider
    Note: We used Quattro Micro and Quattro Premier triple-quadrupole mass spectrometers (WATERS, model: Quattro Premier Mass Spectrometers )
  8. Acquity FTN Ultra Performance Liquid Chromatography instrument (WATERS, model: ACQUITY UPLC H-Class System )
  9. 1.0 x 150 mm Acquity BEH C18 column (WATERS, catalog number: 186002347 )

Software

  1. MassLynx 4.1 and QuanLynx software (WATERS)
  2. LIMSA software (Haimi et al., 2009)

Procedure

  1. Metabolic labeling of newly synthesized phospholipids
    To determine the time course of incorporation of deuterium labeled head groups into glycerophospholipids you need several dishes with adherent cells. The cells from a dish are collected at suitable times e.g., after 0, 1, 2, 4, 8, 12, 24, and 48 h of incubation with the labeled head group precursors.
    1. Grow cells until dishes are 70-80% confluent. Adherent cells lines like HeLa, CHO or BHK-21 cells are suitable.
    2. Wash cells once with 1 ml of PBS to remove detached cells.
    3. Add 1 or 1.5 ml of the labeling medium (see Recipes) per 35 or 60 mm dish and incubate the cells for up to 48 h.
    4. Collect cells from a dish at suitable times. Wash the dish thrice with 1 ml of PBS and collect the cells by scraping in 2 ml of ice-cold H2O and move the cells to 12 ml screw-cap tubes.
    5. Vortex briefly, suspend cells with a pipette and collect e.g., 50 µl aliquots of the cell suspension for protein determination, if necessary.
    6. To the remaining suspension add 3 ml of methanol and vortex.
    7. Store at -20 °C until ready for extraction of the lipids.
  2. Pulse-chase experiments
    To determine the turn-over of glycerophospholipids first incubate the cells with the labeled precursors for up to 24 h, wash the cells with PBS and then incubate them in a medium containing the corresponding unlabeled head group precursors. During this chase period, collect cells from a dish at e.g., 0, 1, 2, 4, 8, and 24 h of incubation to determine the rate of turnover.
    1. Label the newly synthesized phospholipids as described in steps 1a-1c for 4-24 h.
    2. Wash thrice with cell culture medium to remove the labeled precursors.
    3. Add 1 or 1.5 ml of the chase medium to each dish and incubate for up to 48 h.
    4. Collect cells (see steps 1d-1g) first at a 1-2 h interval and then with longer intervals.
  3. Extraction of lipids
    1. Add a cocktail of internal GPL standard consisting of di 20:1-PC, di 20:1-PE, di 20:1-PS and di 16:0-PI (10, 5, 2, 2 nmol per 100 nmol of total glycerophospholipid, respectively) for mass spectrometric analyses and then extract the lipids (see Notes). We use the Folch method of lipid extraction (Folch et al., 1957) except that no added salts are included.
    2. Add 6 ml of chloroform, add the caps and turn the tubes upside down and back again rapidly 40-50 times.
    3. Centrifuge at 3,000 x g for 10 min.
    4. Transfer the lower phase with a glass Pasteur pipette to a new 12 ml screw-cap tube, add 4 ml of theoretical upper phase containing chloroform/methanol/H2O (3:48:47, v/v) and mix as described in step 3b.
    5. Centrifuge as in step 3c and collect the lower phase.
    6. Add 0.5 ml of methanol, vortex, and evaporate to dryness under nitrogen stream using mild heating (< 40 °C).
    7. Reconstitute extracted lipids in 20-40 µl of methanol/chloroform (4:1, v/v) in 12 x 32 mm screw-neck vials for LC-MS analysis, or in 250 µl of methanol/chloroform (2:1, v/v) in 11.5 x 75 mm test tubes for MS/MS analysis and store at -20 °C.
  4. Mass spectrometric analysis
    Note: It can be carried out using either LC-MS or MS/MS using direct sample infusion as indicated below.
    1. Liquid chromatography-mass spectrometry
      GPL molecular species were analyzed using an LC-MS system consisting of an Acquity FTN Ultra Performance Liquid Chromatography instrument (WATERS) equipped with a 1.0 x 150 mm Acquity BEH C18 column connected to Quattro Micro or Premier triple-quadrupole mass spectrometer (WATERS). The column was eluted with a gradient of isopropanol/acetonitrile (90:10) into acetonitrile/H2O (60:40) each containing 10 mM ammonium formate and 1% NH4OH (Kainu et al., 2013). The flow rate was 0.13 ml/min. Individual lipid species were detected using Multiple Reaction Monitoring (MRM) as described (Kainu et al., 2013). Mass spectrometer was operated in the positive ion mode and the precursor ions were [M + H]+ for PC, PE, PS and PI. The product ions for the unlabeled species were m/z 184 (PC), 141 (PE), 185 (PS) and 260 (PI) and for the deuterium-labeled species m/z 193, 145, 188 and 266, respectively. Typical m/z transitions are listed in Table 1. Water soluble GPL metabolites were also analyzed with LC-MS as described (Hermansson et al., 2016). Data were collected using the MassLynx 4.1 software (WATERS).
    2. MS/MS analysis with direct sample infusion
      This method does not require LC instrument because the different GPLs in the crude lipid extract can be selectively detected based on their head group–specific fragmentation when the sample is infused to the mass spectrometer with a glass microsyringe at a rate of 10-20 µl/min. The unlabeled PC and PI species are selectively detected by scanning for precursors of m/z +184 and -241, respectively, while the unlabeled PE and PS species are detected by monitoring for a neutral loss (NL) of 141 or 87, respectively. The labeled PC, PI or PE and PS species are selectively detected by scanning for the precursors of 193 (PC) or 247 (PI) or neutral loss of 145 (PE) or 90 (PS).

      Table 1. MRM precursor and product ions used for select GPL molecular species in LC-MS analysis

Data analysis

  1. The analysis method depends on the method used for data acquisition. In case of the LC-MS method, the labeled and unlabeled lipids (and their metabolites) were quantified using the QuanLynx software (WATERS).
  2. When the MS/MS method with direct infusion was used, the head group–specific MS/MS spectral data were copied to Excel and then the LIMSA add-in was run. This provides the concentrations of the individual GPL species based on the included internal standards (Haimi et al., 2009). When needed, GPL concentration vs. time was plotted and the turnover rate was obtained by curve fitting with the Origin software (OriginLab, Northampton, MA).
  3. Typical results
    Figure 1 shows the labeling of one representative PC species from D9-choline added to the growth medium of HeLa cells.


    Figure 1. HeLa cells were incubated in a medium containing D9-choline for 1, 8 or 24 h and the cellular lipids were extracted and the labeled and unlabeled PC species were analyzed by LC-MS using MRM detection. Unlabeled 36:2-PC (m/z 786.5) is shown in purple and the corresponding labeled PC species (m/z 795.5) in green.

Notes

Many GPL standards are available from e.g., Avanti Polar Lipids (Alabaster, AL).
For most accurate quantification, a heavy isotope -labeled internal standard for each lipid species to be analyzed should be used (Krautbauer et al., 2016; Wang et al., 2016), but this is rarely done due to lack of such commercial standards. However, in the present approach labeling is determined from the intensity of a labeled GPL species relative to that of the corresponding unlabeled one and thus no internal standards are necessary.

Recipes

  1. Labeling medium
    1. Adjust pH of the D4-ethanolamine solution to 7.0 with acetic acid and mix it with the other deuterium-labeled precursors and hydroxylamine in H2O to obtain the precursor stock solution (typically 100x). Store at -20 °C
      Note: pH of ethanolamine is high and thus it needs to be neutralized with acetic acid to avoid alkalization of the labeling and chase media.
    2. Prepare the labeling medium by adding stock solution in the growth medium to obtain the following concentrations: D9-choline 100 µg/ml, D4-ethanolamine 100 µg/ml, D3-L-serine 300 µg/ml, D6-myo-inositol 100 µg/ml, and 1 mM hydroxylamine
      Note: Hydroxylamine is an inhibitor of PS decarboxylase that prevents decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) in the cells. The presence of hydroxylamine is necessary to avoid the MS peaks due to the labeled PE species formed via the CDP-ethanolamine pathway from overlapping with the peaks of PE species formed via decarboxylation of labeled PS. However, if the PE species formed from PS via decarboxylation are of interest, hydroxylamine as well as labeled ethanolamine must be omitted from the medium to avoid such overlap.
  2. Chase medium
    DMEM containing unlabeled choline (500 µg/ml), ethanolamine (500 µg/ml), L-serine (1,000 µg/ml), myo-inositol (500 µg/ml) and 1 mM hydroxylamine was stored at 4 °C and used on the same day

Acknowledgments

This protocol was used in the following publications: Kainu et al., 2013; Hermansson et al., 2016; Kainu et al., 2008. This study was supported by grants from Finnish Academy and Sigrid Juselius Foundation to P.S.

References

  1. de Kroon, A. I. (2007). Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae. Biochim Biophys Acta 1771(3): 343-352.
  2. Folch, J., Lees, M. and Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1): 497-509.
  3. Haimi, P., Chaithanya, K., Kainu, V., Hermansson, M. and Somerharju, P. (2009). Instrument-independent software tools for the analysis of MS-MS and LC-MS lipidomics data. Methods Mol Biol 580: 285-294.
  4. Heikinheimo, L. and Somerharju, P. (2002). Translocation of phosphatidylthreonine and -serine to mitochondria diminishes exponentially with increasing molecular hydrophobicity. Traffic 3(5): 367-377.
  5. Hermansson, M., Hanninen, S., Hokynar, K. and Somerharju, P. (2016). The PNPLA-family phospholipases involved in glycerophospholipid homeostasis of HeLa cells. Biochim Biophys Acta 1861(9 Pt A): 1058-1065.
  6. Kainu, V., Hermansson, M., Hanninen, S., Hokynar, K. and Somerharju, P. (2013). Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria. Biochim Biophys Acta 1831(2): 429-437.
  7. Kainu, V., Hermansson, M. and Somerharju, P. (2008). Electrospray ionization mass spectrometry and exogenous heavy isotope-labeled lipid species provide detailed information on aminophospholipid acyl chain remodeling. J Biol Chem 283(6): 3676-3687.
  8. Käkelä, R., Somerharju, P. and Tyynela, J. (2003). Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry. J Neurochem 84(5): 1051-1065.
  9. Koivusalo, M., Haimi, P., Heikinheimo, L., Kostiainen, R. and Somerharju, P. (2001). Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response. J Lipid Res 42(4): 663-672.
  10. Krautbauer, S., Buchler, C. and Liebisch, G. (2016). Relevance in the use of appropriate internal standards for accurate quantification using LC-MS/MS: Tauro-conjugated bile acids as an example. Anal Chem 88: 10957-10961.
  11. Patton, G. M., Fasulo, J. M. and Robins, S. J. (1982). Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography. J Lipid Res 23(1): 190-196.
  12. Postle, A. D. and Hunt, A. N. (2009). Dynamic lipidomics with stable isotope labelling. J Chromatogr B Analyt Technol Biomed Life Sci 877(26): 2716-2721.
  13. Wang, M., Wang, C. and Han, X. (2016). Selection of internal standards for accurate quantification of complex lipid species in biological extracts by electrospray ionization mass spectrometry-what, how and why? Mass Spectrom Rev.

简介

甘油磷脂由甘油磷酸酯骨架组成,酯基化两个酰基链和极性头基。头组(例如胆碱,乙醇胺,丝氨酸或肌醇)限定了甘油磷脂类,而酰基链与头基一起限定了甘油磷脂分子种类。添加到培养基中的稳定的重质同位素(例如,氘)标记的头基前体有效地并入哺乳动物细胞的甘油磷脂中,这允许人们确定合成速率,酰基链重塑或周转使用质谱法测定个体甘油磷脂。该方案描述了如何用这种方法研究主要哺乳动物甘油磷脂的代谢,即磷脂酰胆碱,磷脂酰乙醇胺,磷脂酰丝氨酸和磷脂酰肌醇。


背景 放射性标记的前体已广泛用于研究培养细胞中的甘油磷脂(GPL)代谢。然而,这种方法有严重的缺点。首先,研究GPL的所有分子种类的代谢是不可行的,因为不可能的事实,即没有恢复到高度复杂和耗时的方案来将各个分子种类彼此分离(Patton& em等人,1982),这显然是研究其新陈代谢所必需的。第二,所需的放射性同位素相当昂贵。第三,为了最佳标记,未标记的前体应尽可能耗尽介质。第四,可以同时向细胞中加入两种不同的前体,即使对同位素光谱之间的重叠进行准确校正是必要的对所收集的部分进行液体闪烁计数。由于这些障碍,许多研究最近引入了研究GPL代谢的替代方法(例如,Heikinheimo和Somerharju,2002; de Kroon,2007; Kainu等人2008年; Postle和Hunt,2009; Kainu等人,2013; Hermansson等人,2016)。这种方法是基于使用稳定的重同位素标记的前体和GPL标记的质谱(MS)分析。显然,与基于放射性标记的前体的使用的传统方法相比,这种方法是更方便和有效的,因为以下事实:(a)可以将多个标记的前体同时加入到培养基中,从而提供关于几种GPL的信息类别,(b)标记和未标记的GPL可以使用头组特异性扫描模式方便和有选择地检测,(c)获得关于个体GPL分子种类的更新的信息,和(d)稳定的重同位素标记的前体通常比放射性标记的便宜得多,因此可以以避免使用消耗相应的未标记前体的特殊介质的量加入。

关键字:甘油磷脂, 稳定的重同位素标记, 氘标记前体, 质谱法, 脂质代谢, 细胞培养

材料和试剂

  1. 移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:9400327,9401255,9401410)
  2. 35或60 mm细胞培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:153066或150288)
  3. 12ml螺旋盖玻璃管(Kimble Chase Life Science and Research Products,目录号:45066A-16100)
  4. 巴斯德移液器(BRAND,目录号:747720)
  5. 12 x 32毫米带瓶盖的螺旋瓶(WATERS,目录号:186002640)
  6. 11.5×75mm试管(VWR,目录号:ZZ130296772)
  7. 电池刮刀和升降器(VWR,目录号:734-2603和734-2602)
  8. HeLa细胞或其他培养的哺乳动物细胞
  9. 磷酸盐缓冲盐水(PBS)(Merck Millipore,目录号:524650)
  10. 甲醇(VWR,目录号:83638.320)
  11. 质谱分析所需的内标:
    1. Di-20:1-磷脂酰胆碱(PC)(Avanti Polar Lipids,目录号:850396)
    2. Di-20:1-磷脂酰乙醇胺(PE;由对应PC内部合成)(Käkelä等人,2003)
    3. Di-20:1-磷脂酰丝氨酸(PS;由相应的PC合成)(Käkelä等人,2003)
    4. Di-16:O-磷脂酰肌醇(PI)(Avanti Polar Lipids,目录号:850141)
  12. 氯仿(Merck Millipore,目录号:102445)
  13. 乙腈,LC-MS级(Fisher Scientific,目录号:A/0638/17X)
  14. 异丙醇,Optima TM/LC/MS级(Fisher Scientific,目录号:A461-212)
  15. 甲酸铵(Sigma-Aldrich,目录号:70221)
  16. 氨溶液25%Suprapur(Merck Millipore,目录号:105428)
  17. 乙酸,冰川(Fisher Scientific,目录号:10171460)
  18. 氘标记的胆碱(D 9-胆碱氯化物)(C/D/N同位素,目录号:D-2142)
  19. 氘标记的乙醇胺(D 4 - 乙醇胺)(Cambridge Isotope Laboratories,目录号:DLM-552-1)
  20. 氘标记的丝氨酸(D 3 -L-丝氨酸)(Cambridge Isotope Laboratories,目录号:DLM-1073-1)
  21. 氘标记的肌醇(D 6 - 肌醇 - 肌醇)(C/D/N同位素,目录号:D-3019)
  22. 羟胺(Sigma-Aldrich,目录号:55460)
  23. Dulbecco改良的Eagle培养基高葡萄糖(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:41965039)或适合感兴趣细胞系的其他培养基
    注意:我们在含有10%胎牛血清和100U/ml青霉素和100U/ml链霉素的DMEM中生长HeLa细胞。
  24. 氯化胆碱(Sigma-Aldrich,目录号:C1879)
  25. 乙醇胺(Merck Millipore,目录号:800849)
  26. 肌醇 - 肌醇(Sigma-Aldrich,目录号:I5125)
  27. L-丝氨酸(Sigma-Aldrich,目录号:S4500)
  28. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)
  29. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  30. Milli-Q H 2 O(Elga Stat Maxima反渗透净水器)
  31. 氮气(Aga,工业气体,99.95%)
  32. 标签介质(参见食谱)
  33. 追逐媒介(见食谱)

设备

  1. 移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4600170,46000040和4600250)
  2. 微量注射器(Hamilton,型号:705N,目录号:80565)
  3. 涡街搅拌机(VWR,目录号:444-1372)
  4. 离心机(Thermo Fisher Scientific,型号:Heraeus TM Megafuge TM 1.0)
  5. 通风柜
  6. 带有氮气蒸发的样品浓缩器(Cole-Parmer,Techne,目录号:FSC400D)
  7. 质谱仪或服务提供商分析样品
    注意:我们使用Quattro Micro和Quattro Premier三重四极杆质谱仪(WATERS,型号:Quattro Premier质谱仪)
  8. Acquire FTN超高效液相色谱仪(WATERS,型号:ACQUITY UPLC H-Class系统)
  9. 1.0 x 150 mm Acquity BEH C18色谱柱(WATERS,目录号:186002347)

软件

  1. MassLynx 4.1和QuanLynx软件(WATERS)
  2. LIMSA软件(Haimi等人,2009)

程序

  1. 新合成磷脂的代谢标记
    为了确定将氘标记的头基团掺入甘油磷脂的时间过程,您需要几个具有贴壁细胞的培养皿。在与标记的头基前体孵育的0,1,2,4,8,12,24和48小时之后,适当时候例如在适当时间收集来自培养皿的细胞。
    1. 生长细胞,直到菜肴融入70-80%。粘附细胞系如HeLa,CHO或BHK-21细胞是适合的
    2. 用1ml PBS洗涤细胞一次以除去分离的细胞
    3. 每35或60毫米盘子加入1或1.5毫升标记培养基(参见食谱),孵育细胞长达48小时。
    4. 在适当的时间从盘中收集细胞。用1ml PBS洗涤3次,然后用2ml冰冷的H 2 O 2刮去细胞,并将细胞移至12ml螺旋盖管中。
    5. 短暂地涡旋,用移液管悬挂细胞并收集,例如,如果需要,将50μl等份的细胞悬液用于蛋白质测定。
    6. 向剩余的悬浮液中加入3ml甲醇并涡旋
    7. 储存于-20°C,直至准备好提取脂质。
  2. 脉冲追逐实验
    为了确定甘油磷脂的翻转,首先用标记的前体孵育细胞长达24小时,用PBS洗涤细胞,然后将其在含有相应的未标记头基团前体的培养基中孵育。在这个追逐期间,从例如
    ,0,1,2,4,8和24小时的培养皿中收集细胞以确定转换率。
    1. 按步骤1a-1c所述标记新合成的磷脂4-24 h
    2. 用细胞培养基洗涤三次以除去标记的前体。
    3. 向每个培养皿中加入1或1.5ml的追踪培养基,并孵育长达48小时。
    4. 首先以1-2小时的间隔收集细胞(参见步骤1d-1g),然后以更长的间隔收集细胞。
  3. 脂质的提取
    1. 加入内部GPL标准鸡尾酒,内部GPL标准包括二十:1-PC,di 20:1-PE,di 20:1-PS和di 16:0-PI(10,5,2,2nmol/100nmol甘油磷脂),进行质谱分析,然后提取脂质(见注释)。我们使用Folch脂质提取方法(Folch等人,1957),除了没有添加盐。
    2. 加入6毫升氯仿,加入盖子,将管子反复翻转,再次快速回收40-50次
    3. 以3,000 x g离心10分钟。
    4. 用玻璃巴斯德移液器将下相转移到新的12毫升螺旋盖管中,加入4毫升含有氯仿/甲醇/H 2 O(3:48:47,v/v)并如步骤3b所述混合。
    5. 离心机如步骤3c,并收集较低的相位。
    6. 加入0.5ml甲醇,涡旋,并在氮气流下使用温和加热(<40℃)蒸发至干。
    7. 将20-40μl甲醇/氯仿(4:1,v/v)中的提取的脂质重建在12×32mm螺旋瓶中用于LC-MS分析,或在250μl甲醇/氯仿(2:1,v/v)在11.5 x 75 mm试管中进行MS/MS分析,并在-20°C储存
  4. 质谱分析
    注意:可以使用LC-MS或MS/MS使用直接样品输注进行,如下所示。
    1. 液相色谱 - 质谱
      使用由Acquire FTN超高效液相色谱仪(WATERS)组成的LC-MS系统分析GPL分子种类,该仪器配备有连接到Quattro Micro或Premier三联体的1.0×150mm Acquity BEH C 18 - 反相质谱仪(WATERS)。将柱用异丙醇/乙腈(90:10)的梯度洗脱到各自含有10mM甲酸铵和1%NH 4的乙腈/H 2 O(60:40) (Kainu等人,2013)。流速为0.13ml/min。使用如(Kainu等人,2013)所述的多反应监测(MRM)检测个体脂质物质。质谱仪以正离子模式运行,PC,PE,PS和PI的前体离子为[M + H] + sup。未标记物种的产物离子是184(PC),141(PE),185(PS)和260(PI),氘标记物种m/z分别为193,145,188和266。典型的m/z转换在表1中列出。水溶性GPL代谢物也用LC-MS分析,如(Hermansson等人,2016)所述。使用MassLynx 4.1软件(WATERS)收集数据。
    2. 使用直接样品输注的MS/MS分析 该方法不需要LC仪器,因为当样品用玻璃微量注射器以10-20μl/分钟的速率输入质谱仪时,可以基于其头部基团特异性片段选择性地检测粗脂质提取物中的不同GPL,分钟。通过分别扫描m/z +184和-241的前体来选择性地检测未标记的PC和PI物质,而通过监测中性损失(NL)检测未标记的PE和PS物种,分别为141或87。通过扫描193(PC)或247(PI)的前体或145(PE)或90(PS)的中性损失来选择性地检测标记的PC,PI或PE和PS物种。

      表1. LC-MS分析中用于选择性GPL分子种类的MRM前体和产物离子

数据分析

  1. 分析方法取决于用于数据采集的方法。在LC-MS方法的情况下,使用QuanLynx软件(WATERS)定量标记和未标记的脂质(及其代谢物)。
  2. 当使用直接输注的MS/MS方法时,将头组特异性MS/MS光谱数据复制到Excel,然后运行LIMSA加载项。这提供了基于所包含的内部标准的单个GPL种类的浓度(Haimi等人,2009)。当需要时,绘制GPL浓度对时间,并通过使用Origin软件(OriginLab,Northampton,MA)进行曲线拟合获得更换率。
  3. 典型结果
    图1显示了加入到HeLa细胞生长培养基中的D 9个 - 胆碱的一个代表性PC物种的标记。


    图1. HeLa细胞在含有D 9个 - 胆碱的培养基中培养1,8或24小时,提取细胞脂质,通过LC-PCR分析标记和未标记的PC物质,使用MRM检测的MS。未标记的36:2-PC( m/z 786.5)以紫色显示,并且相应的标记PC种类(em/m/z 795.5 )绿色。

笔记

许多GPL标准可从例如获得,Avanti Polar Lipids(Alabaster,AL)。
对于最准确的定量,应使用要分析的每种脂质物种的重同位素标记的内标(Krautbauer等,2016; Wang等人,2016年) ),但由于缺乏这样的商业标准,很少这样做。然而,在本方法中,标记是根据标记的GPL物种的强度相对于相应的未标记的GPL种类的强度确定的,因此不需要内部标准。

食谱

  1. 贴标介质
    1. 用乙酸将D 4 - 乙醇胺溶液的pH调节至7.0,并与H 2 O 2中的其他氘标记前体和羟胺混合,得到前体原液(通常为100x)。储存于-20°C
      注意:乙醇胺的pH高,因此需要用乙酸中和,以避免标记和追踪培养基碱化。
    2. 通过在生长培养基中加入储备溶液来制备标记培养基以获得以下浓度:D 9 - 胆碱100μg/ml,D 4+乙醇胺100μg/ml, D 3 -L-丝氨酸300μg/ml,D 6 - 肌醇100μg/ml,和1mM羟胺 > 注意:羟胺是PS脱羧酶的抑制剂,其防止磷脂酰丝氨酸(PS)在细胞中磷脂酰乙醇胺(PE)的脱羧。羟胺的存在是避免由于通过CDP-乙醇胺途径形成的标记的PE物质与通过标记的PS的脱羧形成的PE物质的峰重叠而引起的MS峰所必需的。然而,如果由PS经由脱羧形成的PE物质是令人感兴趣的,则必须从介质中省略羟胺以及标记的乙醇胺以避免这种重叠。
  2. 追逐媒介
    含有未标记胆碱(500μg/ml),乙醇胺(500μg/ml),L-丝氨酸(1,000μg/ml),肌醇(500μg/ml)和1mM羟胺的DMEM为储存于4°C并在同一天使用

致谢

该协议用于以下出版物:Kainu等人,2013; Hermansson等人,2016; Kainu等人,2008年。这项研究得到了芬兰学院和Sigrid Juselius基金会的资助。

参考

  1. de Kroon,AI(2007)。  磷脂酰胆碱的代谢和其对酿酒酵母中脂质酰基链组成的影响。 Biochim Biophys Acta 1771(3):343-352。
  2. Folch,J.,Lees,M.and Sloane Stanley,GH(1957)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/13428781" target ="_ blank">用于从动物组织分离和纯化总脂质的简单方法。生物化学226(1):497-509。
  3. Haimi,P.,Chaithanya,K.,Kainu,V.,Hermansson,M.and Somerharju,P。(2009)。用于分析MS-MS和LC-MS脂质组学数据的仪器独立软件工具方法Mol Biol 580 :285-294。
  4. Heikinheimo,L.和Somerharju,P。(2002)。 3(5):367-377。
  5. Hermansson,M.,Hanninen,S.,Hokynar,K。和Somerharju,P。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27317427"target ="_ blank">涉及HeLa细胞的甘油磷脂体内平衡的PNPLA家族磷脂酶。 Biochim Biophys Acta 1861(9 Pt A):1058-1065。 br />
  6. Kainu,V.,Hermansson,M.,Hanninen,S.,Hokynar,K.和Somerharju,P。(2013)。从线粒体进入磷脂酰丝氨酸至磷脂酰乙醇胺分子物质的出口。生物化学Biophys Acta 1831(2):429- 437.
  7. Kainu,V.,Hermansson,M。和Somerharju,P。(2008)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/18056998"目标="_ blank">电喷雾离子化质谱法和外源重同位素标记的脂质物种提供了关于氨基磷脂酰基链重塑的详细信息。 283(6):3676-3687。
  8. Käkelä,R.,Somerharju,P.和Tyynela,J。(2003)。使用液相色谱 - 电喷雾离子化质谱法分析来自患有婴儿和幼年型神经元 - 类脂质脂褐质激素的患者的脑中的磷脂分子种类。 J Neurochem 84(5): 1051-1065。
  9. Koivusalo,M.,Haimi,P.,Heikinheimo,L.,Kostiainen,R。和Somerharju,P.(2001)。通过ESI-MS定量测定磷脂组成:酰基链长度,不饱和度和脂质浓度对仪器响应的影响。 Res 42(4):663-672。
  10. Krautbauer,S.,Buchler,C.和Liebisch,G。(2016)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/27736066"目标="_ blank">使用适当的内部标准,使用LC-MS/MS进行准确定量的相关性:以陶瓷缀合的胆汁酸为例。 88:10957- 10961.
  11. Patton,GM,Fasulo,JM和Robins,SJ(1982)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/7057106"target ="通过高效液相色谱法分离磷脂和磷脂的单个分子种类。 Lipid Res。23(1):190-196。
  12. Postle,AD和Hunt,AN(2009)。动态具有稳定同位素标记的脂质组学。 J Chromatogr B Analyt Technol Biomed Life Sci 877(26):2716-2721。
  13. Wang,M.,Wang,C.and Han,X.(2016)。  选择内部标准,通过电喷雾离子化质谱法精确定量生物提取物中的复杂脂质物质 - 什么,如何和为什么?质谱。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hänninen, S., Somerharju, P. and Hermansson, M. (2017). Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells. Bio-protocol 7(9): e2268. DOI: 10.21769/BioProtoc.2268.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。