SE197:/S02/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	S02
Title	Mouse tissues
Organism - Scientific Name
Organism - ID	NCBI taxonomy 10090
Compound - ID
Compound - Source
Preparation	C57BL/6 mice were purchased from CLEA Japan and maintained according to the Guidelines for Animal Experimentation of Tohoku University. Mouse tissues (approximately 100 mg) were placed in 1.5 ml siliconized sample tubes. Then, nine volumes of acidic methanol (pH 4.0), 1 μM (final concentration) of 17:0-LPA, and 10 μM (final concentration) of 17:0-LPC were added to the tube. The obtained mixture was homogenized for 10 min by Micro Smash MS-100R (TOMY) (3,000 rpm at 4°C). After an initial centrifugation at 1,000 g for 10 min at 4°C, the supernatants were further centrifuged at 21,500 g for 10 min at 4°C. The resulting supernatant was passed through a filter (0.2 μm pore size, 4 mm inner diameter; YMC), and 10 μl of the filtrate was subjected to LC-MS/MS. To obtain a serum sample, a blood sample was collected with a noncoated capillary (910-01-75; Hirschmann Laborgerate), incubated at 37°C for 1 h, allowed to stand at 4°C for 12 h, and centrifuged at 1,500 g for 10 min at 25°C. Plasma was collected with a heparinized capillary (1-040-7500-HC; Drummond), followed by addition of 1 mM of EDTA (final concentration), and centrifuged at 1,500 g for 10 min at 25°C. The plasma and serum samples (10 μl each) were placed in 1.5 ml siliconized sample tubes, deproteinized by mixing with 90 μl acidic methanol (pH 4.0), 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 10 μl of the filtrate was subjected to the LC-MS/MS.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M01
Title	LC-MS/MS analysis
Method Details ID	MS01
Sample Amount
Comment

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.
Comment_of_details