鞘氨醇1-磷酸盐 (S1P)的提取和量化

Christina-Maria Reimann; Markus H. Gräler

doi:10.21769/BioProtoc.1817

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Peer-reviewed

Extraction and Quantification of Sphingosine 1-Phosphate (S1P)

鞘氨醇1-磷酸盐 (S1P)的提取和量化

Christina-Maria Reimann

Markus H. Gräler email

发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1817 浏览次数: 9602

评审: Ivan ZanoniYang FuAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Nature Communications, featuring study using the protocol.

Aug 2015

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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 S1P_1-5. 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

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

Equipment

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 )
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

Analyst 1.6.2 (AB Sciex)

Procedure

English

中文翻译

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如何引用

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.