SE197:/S03/M01

Sample Set Information

ID	SE197
Title	Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS
Description	Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids(GPs ). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.
Authors	Okudaira, M., A. Inoue, A. Shuto, K. Nakanaga, K. Kano, K. Makide, D. Saigusa, Y. Tomioka, and J. Aoki.
Reference	J. Lipid Res. 2014. 55: 2178–2192
Comment

The original paper is available at PubMed.

The original paper is available at The Journal of Lipid Research site.

Sample Information

ID	S03
Title	HEK293 cells
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Preparation	HEK293 cells were maintained in DMEM (Nissui Pharma­ceutical) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin (Sigma-Aldrich), and 100 μg/ml streptomycin (Gibco) at 37°C in the presence of 5% CO2 gas. HEK293 cells (1.0 × 105 cells) were incubated with recombinant PS-PLA1 in 100 μl Opti-MEM in the presence or absence of 0.1% BSA at 37°C for 1 h. The cultures were centrifuged at 1,000 g for 3 min. The supernatants and cell pellets were deproteinized with 160 μl and 200 μl acidic methanol (pH 4.0), respectively, homogenized for 3 min in an ultrasonic bath, and centrifuged at 21,500 g for 10 min at 4°C (ice-cold water). The supernatants were filtered and 20 μl of the filtrate was subjected to LC-MS/MS.
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Analytical Method Information

ID	M01
Title	LC-MS/MS analysis
Method Details ID	MS01
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Analytical Method Details Information

ID	MS01
Title	LC-MS/MS analysis
Instrument	LC, NANOSPACE SI-II HPLC (Shiseido); MS, TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA)
Instrument Type	UPLC-QTOF-MS
Ionization	ESI
Ion Mode	Positive and Negative
Description	The LC-MS/MS system consisted of a NANOSPACE SI-II HPLC (Shiseido) and a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a heated-electrospray ionization-II (HESI-II) source. The positive and negative HESI-II spray voltages were 3,500 and 2,500 V, respectively, the heated capillary temperature was 350°C, the sheath gas pressure was 60 psi, the auxiliary gas setting was 40 psi, and the heated vaporizer temperature was 350°C. Both the sheath gas and auxiliary gas were nitrogen. The collision gas was argon at a pressure of 1.5 mTorr. The LC-MS/MS system was controlled by Xcalibur software (Thermo Fisher Scientific) and data were collected with the same software. LC separation was performed using a reverse-phase column [Capcell Pak ACR (250 mm × 1.5 mm inner diameter, 3 μm particle size; Shiseido)] with a gradient elution of solvent A (5 mM ammonium formate in water, pH 4.0) and solvent B (5 mM ammonium formate in 95% (v/v) acetonitrile, pH 4.0) at 150 μl/min. Solvent A was prepared by mixing 95 ml of Milli-Q (Millipore) water and 5 ml of 100 mM ammonium formate, and adjusting to pH 4.0 with formic acid (approximately 9.6 μl). Similarly, solvent B was prepared by mixing 95 ml of acetonitrile and 5 ml of 100 mM ammonium formate, and adjusting to apparent pH 4.0 with formic acid (approximately 1,160 μl). The initial condition was set at 55% B. The following solvent gradient was applied: maintained 55% B for 10 min and then followed by a linear gradient to 85% B from 10 to 30 min, and hold at 85% B for 7 min. Subsequently, the mobile phase was immediately returned to the initial conditions and maintained for 3 min until the end of the run. Column effluent was introduced into the mass spectrometer between 3 and 37 min after injection.
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