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Extraction and Quantification of Sphingosine 1-Phosphate (S1P)
鞘氨醇1-磷酸盐 (S1P)的提取和量化

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Abstract

Sphingosine 1-phosphate (S1P) is a lipid metabolite and signaling molecule involved in many different physiological processes including lymphocyte circulation, T cell differentiation, antigen presentation, and maintenance of the vascular endothelial barrier. S1P is a ligand of five different G protein-coupled cell surface receptors designated S1P1-5. It has also been described as an intracellular second messenger. Quantification of S1P in biological samples is therefore an important task to decipher its signaling capabilities in vivo under physiological and pathophysiological conditions in different body fluids and organs. In this protocol, quantification of S1P is performed by liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS).

Keywords: Liquid chromatography(液相色谱法), Mass spectrometry(质谱法), Triple-quadrupole(三重四极), Sphingolipid(鞘脂), Electrospray ionization(电喷雾电离)

Materials and Reagents

  1. Chloroform (CHCl3) (HPLC-Grade) (Carl Roth GmbH + Co., catalog number: 7331.1 )
  2. Methanol (MeOH) (HPLC-Grade) (VWR International, catalog number: 20864.320 )
  3. Formic acid (Carl Roth, catalog number: 4742.1 )
  4. Sphingosine 1-phosphate (S1P) (Sigma-Aldrich, catalog number: S9666 )
  5. C17-S1P (Avanti Polar Lipids, catalog number: 860641P )
  6. Hydrochloric acid (HCl) (37%) (Carl Roth GmbH + Co., catalog number: 9277.1 )
  7. Sodium chloride (NaCl) (Carl Roth GmbH + Co., catalog number: 3957.3 )
  8. Potassium chloride (KCl) (Carl Roth GmbH + Co., catalog number: 6781.3 )
  9. di-Sodium hydrogen phosphate dehydrate (Na2HPO4.2H2O) (Carl Roth GmbH + Co., catalog number: 4984.2 )
  10. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth GmbH + Co., catalog number: 3904.1 )
  11. Phosphate-buffered saline (see Recipes)

Equipment

  1. S1P extraction
    1. VX-2500 vortexer (VWR International, catalog number: 58816-116 )
    2. Pyrex® glass centrifuge vials (VWR International, catalog number: 734-4240 )
    3. RVC 2-25 CD plus vacuum concentrator (Christ)
    4. Autosampler vials (VWR International, catalog number: 548-0029 )
      Note: It is also named “Short thread vials, ND9” on VWR International website.
    5. Inserts for autosampler vials (VWR International, catalog number: 548-3006 )
      Note: It is also named “Screw neck vials, ND10” on VWR International website.
    6. Screw caps for autosampler vials (VWR International, catalog number: 548-0382 )
  2. LC-MS/MS
    1. Binary pump 1100 series HPLC system (Hewlett Packard/Agilent)
    2. 2 x 60 mm MultoHigh C18-RP column, 3 µm particle size (CS Chromatographie-Service GmbH, catalog number: 536201 )
    3. 2000 QTrap LC/MS/MS system (AB Sciex)

Software

  1. Analyst 1.6.2 (AB Sciex)

Procedure

  1. S1P extraction protocol
    Note: All following steps are performed at room temperature if not stated otherwise.
    1. Transfer the sample (plasma, medium, cell suspension) into a glass centrifuge vial and adjust the volume to 1 ml with PBS. Samples were prepared as follows:
      1. 50-200 µl plasma was taken from heparinized blood.
      2. 1 ml medium was directly taken from cell culture.
      3. Cells were trypsinized, washed once in PBS and taken up in 1 ml PBS.
        Note: All samples can be processed directly or stored at -20 °C to -80 °C until use.
    2. Add 10 µl of the internal standard (10 μM C17- S1P in MeOH).
    3. Add 300 µl of 18.5% HCl.
    4. Add 1 ml MeOH and 2 ml CHCl3.
    5. Vortex for 10 min at maximum speed.
    6. Centrifuge the sample for 3 min at 1,900 x g.
    7. Transfer the lower CHCl3 phase into a new glass centrifuge vial by directly placing the pipet into the lower phase (see Figure 1).


      Figure 1. Formation of phases after centrifugation. As an example, S1P extraction from a plasma sample is shown at step A7. The CHCl3-phase is extracted by directly pipetting through the upper aequous phase.

    8. Add 2 ml of CHCl3 to the remaining aqueous phase and repeat vortexing and centrifugation.
    9. Add this CHCl3-phase to the transferred CHCl3-phase of step A7.
    10. Vacuum-dry the CHCl3 in the vacuum rotator at 60 °C for 45 min. Alternatively, the samples can be dried under nitrogen gas flow.
    11. Resuspend the sample in 100 µl MeOH:CHCl3 (4:1,vol/vol).
    12. Vortex the sample for 1 min at maximum speed.
    13. Transfer the sample into an autosampler vial and store it at -20 °C.

  2. MS protocol
    1. HPLC-program
      1. Solution A: ddH2O containing 1% formic acid.
      2. Solution B: MeOH.
      3. Use a flow-rate of 0.3 ml/min.
      4. Equilibrate the column for 5 min with 90% Solution A and 10% solution B.
      5. From 0-0.5 min: Change to 100% solution B.
      6. From 0.5-15 min: Hold 100% solution B.
      7. From 15.1-20 min: Re-equilibrate with 90% solution A and 10% solution B.
    2. 10 µl of the sample is applied onto the column 1 min after starting the HPLC program.
    3. The column is kept at 35 °C during the whole procedure.
    4. The spectrum is acquired with an electrospray ionization (ESI) ion source in the positive mode and following settings:
      1. Ion spray voltage: 4,500
      2. Ion source heater temperature: 450 °C
      3. Collision gas setting: Medium
      4. Ion source gas 1: 30 psi
      5. Ion source gas 2: 60 psi
      6. Curtain gas: 45 psi
    5. For acquisition the multiple reaction monitoring (MRM) mode and the Analyst 1.6.2 software is used. S1P is analyzed with the mass transition 380 m/z -> 264 m/z, and the internal standard C17- S1P with the mass transition 366 m/z -> 250 m/z.
    6. For quantitative analysis a standard curve with S1P amounts of 1 pmol to 100 pmol and 10 pmol C17- S1P as the internal standard is generated.
    7. S1P concentrations are calculated using Analyst 1.6.2 software.

Representative data


Figure 2. Example of S1P standard curve. For the generation of the standard curve, each concentration is measured three times. For this curve the following S1P amounts were used: 1 pmol, 3 pmol, 10 pmol, 30 pmol, 100 pmol.


Figure 3. Example of S1P spectrum acquired with ESI ion source in positive mode. Representative signals of S1P and its respective internal standard C17-S1P in 10 µl of the 10 µM standard are plotted. The total amounts of S1P and C17-S1P are 100 pmol and 10 pmol, respectively. Retention times of S1P and C17-S1P are slightly different (4.93 min vs. 4.70 min).

Notes

  1. If S1P needs to be measured in tissue samples, homogenize up to 50 mg tissue (might need to be adjusted depending on the lipid concentration of the tissue) together with 10 µl of the internal standard (10 µM C17- S1P in MeOH) in 1 ml PBS. Transfer the homogenate into a glass centrifuge vial and proceed with step A3 of the S1P extraction protocol.
  2. The extraction efficiency is close to 100% for S1P (Andréani and Gräler, 2006).

Recipes

  1. Phosphate-buffered saline
    140 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4.2H2O
    1.8 mM KH2PO4
    Adjust pH to 7.4

Acknowledgements

The extraction and measurement method is adapted from Bode and Gräler (2012).

References

  1. Andréani, P. and Gräler, M. H. (2006). Comparative quantification of sphingolipids and analogs in biological samples by high-performance liquid chromatography after chloroform extraction. Anal Biochem 358(2): 239-246.
  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. Sattler, K., Graler, M., Keul, P., Weske, S., Reimann, C. M., Jindrova, H., Kleinbongard, P., Sabbadini, R., Brocker-Preuss, M., Erbel, R., Heusch, G. and Levkau, B. (2015). Defects of high-density lipoproteins in coronary artery disease caused by low sphingosine-1-phosphate content: Correction by sphingosine-1-phosphate-loading. J Am Coll Cardiol 66(13): 1470-1485.
  4. Vettorazzi, S., Bode, C., Dejager, L., Frappart, L., Shelest, E., Klassen, C., Tasdogan, A., Reichardt, H. M., Libert, C., Schneider, M., Weih, F., Henriette Uhlenhaut, N., David, J. P., Graler, M., Kleiman, A. and Tuckermann, J. P. (2015). Glucocorticoids limit acute lung inflammation in concert with inflammatory stimuli by induction of SphK1. Nat Commun 6: 7796.

简介

鞘氨醇1-磷酸(S1P)是参与许多不同生理过程的脂质代谢物和信号分子,所述生理过程包括淋巴细胞循环,T细胞分化,抗原呈递和血管内皮屏障的维持。 S1P是称为S1P 1-5的五种不同的G蛋白偶联的细胞表面受体的配体。 它也被描述为细胞内第二信使。 因此,生物样品中S1P的定量是在不同体液和器官中的生理和病理生理条件下破译其体内信号传导能力的重要任务。 在该方案中,通过耦合到三重四极杆质谱(LC-MS/MS)的液相色谱法进行S1P的定量。

关键字:液相色谱法, 质谱法, 三重四极, 鞘脂, 电喷雾电离

材料和试剂

  1. 氯仿(CHCl 3)(HPLC级)(Carl Roth GmbH + Co.,目录号:7331.1)
  2. 甲醇(MeOH)(HPLC级)(VWR International,目录号:20864.320)
  3. 甲酸(Carl Roth,目录号:4742.1)
  4. 鞘氨醇1-磷酸(S1P)(Sigma-Aldrich,目录号:S9666)
  5. C17-S1P(Avanti Polar Lipids,目录号:860641P)
  6. 盐酸(HCl)(37%)(Carl Roth GmbH + Co.,目录号:9277.1)
  7. 氯化钠(NaCl)(Carl Roth GmbH + Co.,目录号:3957.3)
  8. 氯化钾(KCl)(Carl Roth GmbH + Co.,目录号:6781.3)
  9. 磷酸氢二钠二水合物(Na 2 HPO 4)2·2H 2 O)(Carl Roth GmbH + Co 。,catalog number:4984.2)
  10. 磷酸二氢钾(KH 2 PO 4)(Carl Roth GmbH + Co.,目录号:3904.1)
  11. 磷酸盐缓冲盐水(见配方)

设备

  1. S1P提取
    1. VX-2500涡旋混合器(VWR International,目录号:58816-116)
    2. Pyrex玻璃离心管(VWR International,目录号:734-4240)
    3. RVC 2-25 CD加真空浓缩器(克里斯)
    4. 自动进样器样品瓶(VWR International,目录号:548-0029)
      注意:在VWR国际网站上也称为"短螺纹瓶ND9"。
    5. 用于自动进样器样品瓶(VWR International,目录号:548-3006)的插件
      注意:在VWR国际网站上也称为"螺丝颈瓶,ND10"。
    6. 用于自动进样器样品瓶的螺帽(VWR International,目录号:548-0382)
  2. LC-MS/MS
    1. 二元泵1100系列HPLC系统(Hewlett Packard/Agilent)
    2. 2×60mm MultoHigh C18-RP柱,3μm粒度(CS Chromatographie-Service GmbH,目录号:536201)
    3. 2000 QTrap LC/MS/MS系统(AB Sciex)

软件

  1. 分析员1.6.2(AB Sciex)

程序

  1. S1P提取方案
    注意:如果没有另外说明,所有以下步骤在室温下进行。
    1. 将样品(血浆,培养基,细胞悬液)转移到玻璃杯中 离心管中并用PBS调节体积至1ml。 样品 准备如下:
      1. 从肝素化血液中取50-200μl血浆
      2. 从细胞培养物中直接取出1ml培养基
      3. 将细胞胰蛋白酶化,在PBS中洗涤一次,并吸收在1ml PBS中 注意:所有样品可直接使用或储存于-20°C至-80°C直至使用。
    2. 加入10μl内标(10μMC17-S1P在MeOH中)
    3. 加入300μl的18.5%HCl
    4. 加入1ml MeOH和2ml CHCl 3。
    5. 以最高速度涡旋10分钟。
    6. 以1,900×g离心样品3分钟。
    7. 将较低的CHCl 3相转移到新的玻璃离心管瓶中 直接将移液器置于下层阶段(见图1)

      图1.离心后的相形成。例如,S1P 在步骤A7显示从血浆样品中的提取。 CHCl 3 - 相是 通过直接上移液相萃取。

    8. 向剩余的水相中加入2ml CHCl 3,并重复涡旋和离心。
    9. 将该CHCl 3 - 相加入步骤A7的转移的CHCl 3 - 相。
    10. 在真空旋转器中在60℃真空干燥CHCl 3 3分钟45分钟。 或者,样品可以在氮气流下干燥
    11. 将样品重悬在100μlMeOH:CHCl 3(4:1,体积/体积)中。
    12. 以最大速度涡旋样品1分钟。
    13. 将样品转移到自动进样器小瓶中,并存储在-20°C。

  2. MS协议
    1. HPLC程序
      1. 溶液A:含1%甲酸的ddH 2 O 2
      2. 溶液B:MeOH
      3. 使用0.3ml/min的流速。
      4. 用90%溶液A和10%溶液B平衡该柱5分钟
      5. 从0-0.5分钟:更换为100%溶液B.
      6. 从0.5-15分钟:保持100%溶液B.
      7. 从15.1-20分钟:用90%溶液A和10%溶液B重新平衡。
    2. 在开始HPLC程序后1分钟,将10μl样品加到柱子上
    3. 在整个过程中,色谱柱保持在35℃
    4. 使用电喷雾离子化(ESI)离子源以正模式和以下设置获得光谱:
      1. 离子喷雾电压:4,500
      2. 离子源加热器温度:450℃
      3. 碰撞气体设置:中等
      4. 离子源气体1:30 psi
      5. 离子源气体2:60psi
      6. 幕帘气体:45psi
    5. 用于采集多反应监测(MRM)模式和 使用Analyst 1.6.2软件。 用质量转变分析S1P   380 m/z-> 264 m/z,和内标C17-S1P与质量   跃迁366m/z-> 250 m/z
    6. 定量分析a标准曲线,S1P量为1 pmol至100 pmol和10 pmol C17- 生成S1P作为内部标准
    7. 使用Analyst 1.6.2软件计算S1P浓度。

代表数据


图2. S1P标准曲线的示例。 为了生成标准曲线,每个浓度测量三次。 对于该曲线,使用以下S1P量:1pmol,3pmol,10pmol,30pmol,100pmol。

图3.以ESI模式以正离子模式获得的S1P光谱的实施例。绘制10μl10μM标准品中S1P和其相应的内标C17-S1P的代表性信号。 总金额S1P和C17-S1P分别为100pmol和10pmol。 S1P和C17-S1P的保留时间略有不同(4.93分钟对4.70分钟)。

笔记

  1. 如果S1P需要在组织样品中测量,与10μl的内标(10μMC17-S1P在MeOH中)一起匀化高达50mg组织(可能需要根据组织的脂质浓度进行调整) ml PBS。 将匀浆转移到玻璃离心管中,并进行S1P提取方案的步骤A3
  2. S1P的提取效率接近100%(Andréani和Gräler,2006)。

食谱

  1. 磷酸盐缓冲盐水
    140mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4 SubO 2·2H 2 O 1.8mM KH 2 PO 4 sub/
    将pH调节至7.4

致谢

提取和测量方法改编自Bode和Gräler(2012)。

参考文献

  1. Andréani,P.和Gräler,M.H。(2006)。 氯仿提取后,通过高效液相色谱对生物样品中的鞘脂和类似物进行比较定量。 a Anal 358(2):239-246。
  2. Bode,C。和Graler,M.H。(2012)。 通过与串联质谱联用的液相色谱法定量鞘氨醇-1-磷酸和相关鞘脂类。 a> Methods Mol Biol 874:33-44
  3. Sattler,K.,Graler,M.,Keul,P.,Weske,S.,Reimann,CM,Jindrova,H.,Kleinbongard,P.,Sabbadini,R.,Brocker-Preuss,M.,Erbel, ,Heusch,G。和Levkau,B。(2015)。 由低鞘氨醇-1-磷酸含量引起的冠状动脉疾病高密度脂蛋白缺陷:修正通过鞘氨醇-1-磷酸负载。 J Am Coll Cardiol 66(13):1470-1485。
  4. Vettorazzi,S.,Bode,C.,Dejager,L.,Frappart,L.,Shelest,E.,Klassen,C.,Tasdogan,A.,Reichardt,HM,Libert,C.,Schneider, ,F.,Henriette Uhlenhaut,N.,David,JP,Graler,M.,Kleiman,A.and Tuckermann,JP(2015)。 糖皮质激素通过诱导SphK1限制急性肺部炎症与炎症刺激一致。 Nat Commun 6:7796。
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引用:Reimann, C. and Gräler, M. H. (2016). Extraction and Quantification of Sphingosine 1-Phosphate (S1P). Bio-protocol 6(10): e1817. DOI: 10.21769/BioProtoc.1817.
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