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A mass spectrometric method for in-depth profiling phosphoinositide regioisomers and their disease-associated regulation

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. A method allowing the comprehensive analysis of phosphoinositides' phosphorylation status and acyl chain identity has long been awaited. Here, we report a new technique for phosphoinositide regioisomer measurement that employs chiral column chromatography plus mass spectrometry (PRMC-MS). Using this method, we revealed a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our novel approach has revealed previously hidden dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

cell component comparison design

Cell culture and transfection: HEK293T, PC3, LNCap and HeLa cells (ATCC) were maintained in DMEM (high glucose, Nacalai Tesque) supplemented with 100 mg/mL streptomycin, 100 units/mL penicillin and 10% fetal bovine serum (FBS; Hyclone) at 37°C (5% CO2, humidified atmosphere). HEK293T cells (3 x 106) seeded in 100 mm dishes were transfected in 8 mL Opti-MEM (Gibco) using 10 µg plasmid DNA and 30 µL PEI Max (Polysciences) according to the manufacturer's instructions. After 4 hr of transfection, the medium was changed to fresh DMEM containing 10% FBS.
Cell and tissue sample preparation: Cells were harvested using a cell scraper and collected into phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH7.4) transferred to a glass tube, and centrifuged at 1,000 x g for 5 min to pellet cells. Cells (3 x 106) were resuspended in 1.5 mL methanol and subjected to lipid extraction (see below). For mouse prostate tissues, 20 mg tissue was transferred to a Lysing Matrix D tube (MP Bio) in 200 µL methanol. Tissue was homogenized using a FastPrep-24TM 5G (MP Bio), transferred to a glass tube containing 1.5 mL methanol, and subjected to lipid extraction.
Preparation of conditioned medium: Cells were grown to 60-70% confluence, whereupon the culture medium was replaced with serum-free medium (DMEM, 0.1% BSA, 10 mM HEPES, pH7.4). After 20 hr incubation, culture supernatants were collected in 15 mL tubes, centrifuged at 5,000 x g for 5 min, and filtered through 0.45 µm cellulose acetate discs (DISMIC) to remove debris. The filtrate (conditioned medium; CM) was frozen in liquid nitrogen and stored at -80°C. For analysis of extracellular phosphoinositides, filtrate (100 µL) was carefully thawed, mixed with 1.5 mL methanol, and subjected to lipid extraction.
Preparation of mouse plasma: Plasma was prepared from the blood of anaesthetized mice according to a standard protocol3. Briefly, C57BL/6J mice were fasted for 6 hr followed by intraperitoneal injection of PBS (control) or 50 mg/kg lipopolysaccharide (LPS). Blood was collected at 2 hr post-injection and plasma was obtained by centrifugation at 6,000 x g for 10 min. Plasma (100 µL) was mixed with 1.5 mL methanol and subjected to lipid extraction.
Preparation of exosomes: Cells (5 x 106) were seeded in 150-mm culture dishes and cultured for 24 hr. After two washes of the dishes with PBS, RPMI containing 1% exosome-depleted FBS was added. After 48 hr incubation, culture supernatants were centrifuged at 2,000 x g for 10 min at 4°C and then passed through a 0.22 µm filter (Millipore., Bedford, MA, USA) to remove cells and cellular debris. Filtered supernatants were ultracentrifuged at 110,000 x g for 70 min at 4°C (SW41Ti rotor, Beckman Coulter), and the pellets were washed with trehalose-containing HEPES buffer (20 mM HEPES pH7.4, 25 mM trehalose) to prevent the aggregation of exosomes. Resuspended pellets were re-ultracentrifuged at 110,000 x g for 70 min at 4°C, and finally resuspended in HEPES buffer (20 mM HEPES, pH7.4). The size distribution and concentration of exosomes were determined by nanoparticle tracking analysis (NTA) performed using a NanoSight LM10 instrument (Malvern, Worcestershire, UK). Exosomes (1x1010 particles) were mixed with 1.5 mL methanol and subjected to lipid extraction.
Chemical standard: For acquisition of linear correlations between the ion-current peak areas and chemical amounts ranging from sub-fmol to 50 pmol of the phosphoinositide standards (Fig.1d, e and Table S1).

Lipid extraction: A given biological sample in 1.5 mL methanol prepared as described above was mixed with 50 µL of a methanol/chloroform (9/1) solution containing 1 nmol C8:0/C8:0 PI(4,5)P2 (as an absorption inhibitor) and 10 pmol each of synthetic C17:0/C20:4 phosphoinositides [PI(3,4,5)P3, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, PI3P, PI4P, PI5P and PI] as internal standards. Ultrapure water (750 µL), 2 M HCl (750 µL), and 1 M NaCl (200 µL) were added to this mixture. After vigorous vortex-mixing, 3 mL CHCl3 was added followed by further vortexing for 2 min. After centrifugation at 1,200 x g for 4 min at room temperature, the lower organic phase (crude lipid extract) was collected and transferred to a new glass tube.
Pre-concentration of phosphoinositides: DEAE Sepharose Fast Flow (10% slurry) was rinsed twice with an equal volume of water, once with 1M HCl, twice with water, once with 1M NaOH, and twice with water. The resin was resuspended in methanol (50% slurry) and a 0.5 mL bed volume was packed in a Pasteur pipette (IWAKI) plugged with glass wool. The column was then equilibrated with chloroform. Crude lipid extract (2.9 mL) was mixed with 1.5 mL methanol and applied to the column, washed with 3 mL chloroform/methanol (1/1) and 2 mL chloroform/methanol/28% aqueous ammonia/glacial acetic acid (200/100/3/0.9), followed by elution with 1.5 mL chloroform/methanol/12 M hydrochloric acid /ultrapure water (12/12/1/1). The eluate was mixed with 850 µL 120 mM NaCl, centrifuged at 1,200 x g for 4 min at room temperature, and the resultant lower phase (purified phosphoinositides) was collected into a fresh glass tube.
Methylation reaction: Purified phosphoinositides were derivatized by methylation using the method of Clark et al.4. Briefly, 150 µL 0.6 M trimethylsilyl diazomethane was added to the purified phosphoinositide fraction prepared as above at room temperature. After 10 min, the reaction was quenched with 20 µL glacial acetic acid. The samples were mixed with 700 µL methanol/chloroform/ultrapure water (48/47/3) followed by vortexing for 1 min. After centrifugation at 1,200 x g for 4 min, the lower phase was taken to dryness under a stream of nitrogen and redissolved in 100 µL acetonitrile.

Chiral column chromatography of PRMC-MS: PRMC-MS was performed using a Nexera X2 HPLC system (Shimadzu) combined with a PAL HTC-xt (CTC Analytics) autosampler. Lipid sample (10 µL) was injected using the autosampler, and molecules were separated using a CHIRALPAK IC-3 column [cellulose tris(3,5-dichlorophenylcarbamate), 2.1 mmφ x 250 mm, 3 mm, DAICEL] at 22°C. LC was operated at a flow rate of 100 µL/min with a gradient as follows: 40% mobile phase A (methanol/5 mM ammonium acetate) and 60% mobile phase B (acetonitrile/5 mM ammonium acetate) were held for 1 min, linearly increased to 85% mobile phase A over 2 min, and held at 85% mobile phase A for 11 min. The column was re-equilibrated to 40% mobile phase A for 6 min before the next injection.
Reverse-phase column chromatography: LC-MS/MS was performed with an LC system [UltiMate 3000 (Thermo Fisher Scientific) and HTC PAL autosampler (CTC Analytics)] as described previously. Briefly, methylated lipids were separated on a C18 column (GL Sciences) using a solvent gradient as follows: 0-1 min hold 70% A/30% B, 1-3 min constant gradient to reach 90% A/10% B, 3-7.5 min constant at 90% A/10% B, 7.5-13 min 30% A/70% B, where mobile phase A was acetonitrile/water/70% ethylamine (800:200:1.3) and mobile phase B was acetonitrile/isopropanol/ 70% ethylamine (200:800:1.3).

Tandem mass spectrometry components of PRMC-MS: PRMC-MS was performed using a triple quadruple mass spectrometer QTRAP6500 (ABSciex). The mass range of the instrument was set at m/z 5-2000. Spectra were recorded in the positive ion mode as [M+NH4]+ ions, and the scan duration of MS and MS/MS was 0.5 sec. The ion spray voltage was set at 5.5 kV, cone voltage at 30 V, and source block temperature at 100°C. Curtain gas was set at 20 psi, collision gas at 9 psi, ion source gas pressures 1/2 at 50 psi, declustering potential at 100 V, entrance potential at 10 V, and collision cell exit potential at 12 V. Collision energy values for each acyl variant are listed in Table S1.
Tandem mass spectrometry-reverse-phase column chromatography: LC-MS/MS was performed with a triple-stage quadrupole mass spectrometer (TSQ Vantage, Thermo Fisher) as described previously. Multiple reaction monitoring (MRM) was employed in positive ion mode.

Data acquisition: Analyst 1.6.3 (SCIEX) was used for data acquisition and processing. MultiQuant (SCIEX) was used for manual data evaluation for peak integration. No background subtraction was performed. Gaussian smoothing width was 1.0 points. For quality control, the peaks from a sample run in which the cps of the surrogate internal standards (SIS; C37:4 phosphoinositides) from the MRM scan below 2 x 104 were not subjected to quantification analysis. The sample peak area value (equivalent to 1 x 106 cells, 10 mg tissue, 1 x 1010 exosome particles or 1 mL plasma) was divided by the corresponding SIS peak area value (equivalent to 1 pmol) for relative quantification.
Data transformation: MSConverter (ProteoWizard) was used for transformation the acquired .wiff file to the .mzML file.

Table S2 lists the MRM transitions (pairs of m/z values of precursor ions and fragment/diacylglycerol ions) used for the identification and quantification of each phosphoinositide acyl variant. Synthetic phosphoinositides (heptadecanoyl-arachidonyl; C37:4 molecular species) were used as standard analytes to identify retention time.

Chromatography instrument;Autosampler model;Column model;Column type;Guard column

Scan polarity;Scan m/z range;Instrument;Ion source;Mass analyzer

liquid chromatography-mass spectrometry

quadrupole mass spectrometer

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