Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

Summary

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

Abstract

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS³) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Introduction

Sphingomyelin (SM) is a common sphingolipid in mammalian cells. SM is synthesized intracellularly¹ and present as a precursor for other sphingolipids such as a sphingosine-1-phosphate and a ceramide, which have crucial roles in immune cell trafficking and skin barrier homeostasis, respectively^2,3. Thus, the precise analysis of the SM metabolism is important for elucidating the physiological and pathological roles of sphingolipids.

SM is composed of a ceramide and a phosphorylcholine that is linked to the 1-hydroxy group of the ceramide, which is further compo....

Protocol

Consult all relevant material safety data sheets (MSDS) before use. Wear gloves to minimize sample contamination by skin-derived SM. The present protocol was applied to HeLa cells grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1,000 U/L penicillin, and 100 mg/L streptomycin.

1. Preparation of Lipid Samples

NOTE: It is important that all glassware including test tubes with Teflon-lined screw caps be detergent-free.

1. Extraction of total lipid fraction from cell homogenate using the Bligh & Dyer method⁷ ....

Results

Chemically synthesized d18:1/24:0 SM (Figure 1A) and d18:1/24:0 SM in lipid samples extracted from HeLa cells (Figure 1B) were analyzed by LC-ESI-MS³ employing [M+HCOO]^- and [M-CH₃]^- as first and second precursor ions, respectively. Note that the spectrum intensity of demethylated-sphingosylphosphorylcholine (SPC) (m/z 449) is larger than that .......

Discussion

In the present qualitative method, we obtained MS³ product ions of a SPC and an N-acyl moiety. It is critical to properly assign both a SPC and an N-acyl moiety. To this end, it should be noted that other phosphorylcholine-containing molecules can also be detected as MS³ product ions. Diacyl-phosphatidylcholine (PC) and plasmalogen-PC are abundantly present in mammalian cells, and their hydrophobicity is similar to that of SM. Therefore, diacyl-PC and plasmalogen-PC with an isotope.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	ThermoFisher	10010023
100 mm tissue culture dish	IWAKI	3020-100
Cell scraper	IWAKI	9000-220
Siliconized 2.0 mL tube	Fisher Scientific	02-681-321
Test tube	IWAKI	TST SCR 16-100
Teflon-lined screw cap	IWAKI	9998CAP415-15
Disposable glass tube	IWAKI	9832-1310
CAPCELL PAK C18 ACR 3 µm 1.5 mm I.D. x 100 mm	Shiseido	92223	Guard cartridge is inserted into cartridge holder, and linked to C18 column
CAPCELL C18 MGII S-3 2.0 mm x 10 mm GUARD CARTRIDGE	Shiseido	12197
Cartridge holder	Shiseido	12415
Acetonitrile	Wako	018-19853
2-Propanol (Isopropyl Alcohol)	Wako	161-09163
Methanol	Wako	134-14523
Formic acid	Wako	066-00466
28% Ammonia water	Wako	016-03146
Sonicator (bath type)	SHARP	UT-206H
Vortex mixer for glass test tube	TAITEC	Mix-EVR
1.4 mL glass vial	Tomsic	500-1982	Samples are stored in 1.4 mL glass vial sealed with screw cap and 8 mm septum at -20°C
8 mm septum	Tomsic	200-3322	When samples are analyzed, screw caps are replaced with screw caps with slit septum
Screw cap for 1.4 mm glass vial	Tomsic	500-2762
Screw cap with slit septum	Shimadzu GLC	GLCTV-803
PVDF 0.22 µm filter	Millipore	SLGVR04NL
Triple quadrupole and quadrupole linear ion trap mass spectrometry	SCIEX	QTRAP4500
The software for data acquisition and analysis of product ion spectra	SCIEX	Analyst
The software for data integration in quantitative analysis	SCIEX	MultiQuant
HPLC system	Shimadzu	Nexera
Glass bottle	Sansyo	85-0002
d18:1/24:0 sphingomyelin	Avanti Polar Lipids	860592P
Sphingosylphosphorylcholine	Merck	567735
Fetal bovine serum	ThermoFisher	26140079
L-glutamine	ThermoFisher	25030081
Penicillin and streptomycin	Sigma	P4333
Eagle’s minimum essential medium	Sigma	M4655