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Quantitative analysis of a novel glucosylated phospholipid by liquid chromatography–mass spectrometry
Analytical Biochemistry 376 (2008) 252–257
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Quantitative analysis of a novel glucosylated phospholipid by liquid chromatography–mass spectrometry Shinya Ito a,b, Takuji Nabetani b, Yoko Shinoda b, Yasuko Nagatsuka b, Yoshio Hirabayashi b,1,* a b
Hitachi High-Technologies Corp., 1-24-14 Nishi-shinbashi, Minato-ku, Tokyo 105-8717, Japan Hirabayashi Research Unit, Brain Science Institute, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e
i n f o
Article history: Received 28 December 2007 Available online 13 February 2008 Keywords: Phospholipid Phosphatidylglucoside LC-MS MS/MS Quantitative analysis C6 glioma cell
a b s t r a c t Building upon the demonstrated presence of a new glyceroglycolipid, phosphatidylglucoside (PtdGlc), in rat embryonic brain tissues, we have developed a method to identify minute amounts of PtdGlc in cultured cells by using nano-flow high-performance liquid chromatography and negative-ion-mode electrospray linear-ion trap time-of-flight mass spectrometry (LC-MS). A normal-phase silica gel-based column enabled us to separate PtdGlc from other lipid classes. PtdGlc was identified from its tandem mass spectrometry spectrum and from its retention time in the column. Using an internal standard collection and LC-MS, we obtained the linearity of PtdGlc at a range of 6.3–800 fmol per injection. We applied this method to analyze quantitative changes in PtdGlc in C6 glioma cells after cellular differentiation into GFAP-positive glial cells. PtdGlc in C6 glioma cells consisted exclusively of C18:0/C20:0 fatty acyl chains. Differentiation induced by the addition of anti-PtdGlc antibody plus cAMP in culture medium significantly increased the glycolipid content. Ó 2008 Elsevier Inc. All rights reserved.
The identification and comprehensive characterization of the molecular species within a phospholipid has been challenging because glycerophospholipids contain diverse fatty acid chains that are present with limited abundance (for review, see [1–3]). Highperformance liquid chromatography (HPLC) with UV detection allows the separation and quantification of many phospholipid classes. [4] However HPLC-UV for analysis of phospholipids is suffering from the lack of specificity and differential responses depending on double bond composition. Moreover, it is difficult to identify minor constituents such as phosphatidylglucoside (PtdGlc)2, which was recently isolated in rodent brain tissues [5– 8] by conventional analytical methods. In recent years, HPLC combined with mass spectrometry (LCMS) has been used to analyze a variety of lipids [9,10]. Tandem mass spectrometry, in particular, is a powerful tool for identification and quantitation of lipids [11–15]. Combining tandem mass spectrometry with normal-phase liquid chromatography enables
* Corresponding author. Fax: +81 48 467 6372. E-mail address: [email protected] (Y. Hirabayashi). 1 Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency (JST). 2 Abbreviations used: PtdGlc, phosphatidylglucoside; ESI, electrospray ionization; PtdGly, phosphatidylglycerol; CID, collision-induced dissociation; FT-ICR, Fourier transform ion cyclotron resonance; MRM, multiple-reaction monitoring; FBS, fetal bovine serum. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.02.007
the separation of phospholipids into several major subclasses [16,17]. Its application to the analysis of lipids was a breakthrough for lipid biochemistry. Compared to other ionization methods, electrospray ionization (ESI) is very useful for analyzing polar lipids, because polar lipids such as phospholipids are sensitive to being ionized without a derivatization procedure. Phosphatidylcholine, for example, can be detected as protonated molecules in positive-ion mode, and phosphatidylglycerol (PtdGly) can be detected as deprotonated molecules in negative-ion mode [12,18– 20]. Furthermore, ESI can be coupled with nano-flow HPLC easily. This analytical method has been successfully used for the identification and characterization of phospholipids from biological samples [10,18,19]. Because PtdGlc has a unique structure consisting of a single molecular species of C18:0 and C20:0 of diacylglycerol and is a minor constituent in developing rodent brains, PtdGlc has never been studied extensively. To elucidate the biological significance of PtdGlc, it is essential to determine its distribution in mammalian tissues and cells. To this end, we sought to develop a straightforward method for identifying and quantifying PtdGlc. In this paper, we describe a procedure for identifying and quantifying PtdGlc by nano-flow HPLC coupled on-line to ESI linear ion-trap time-of-flight mass spectrometry. This analytical method was reproducible and sensitive enough to identify and measure PtdGlc, a minor membrane component in C6 rat glioma cells.
Quantitative analysis of a novel phospholipid / S. Ito et al. / Anal. Biochem. 376 (2008) 252–257
A
253
C18:0/C20:0 PtdGlc O
OH O
O
HO HO
OH
H O
O P O OH
O O
B
O
C17:0/C17:0 PtdGly O
OH HO
O P O OH
H O O O
Fig. 1. Molecular structures of glycerophospholipids. (A): C18:0/C20:0 PtdGlc. (B): C17:0/C17:0 PtdGly.
Materials and methods
Lipid extraction
Chemicals and standard lipids
The harvested cells were suspended in a small amount of methanol and placed into a ODS cartridge column (Oro-Sep C18 600 mg, OROCHEM CRC 8600) prewashed with methanol. ODS-unbound compounds were washed out with 10 mL of water, and then ODS-retained compounds were eluted with 1 mL of C:M (2:1, v/ v). Extracts were dried under a stream of N2 gas, dissolved in C:M (4:1, v/v), and placed into an Iatrobeads (Iatron Laboratories, Inc.) Si column (5.3u 4 cm). The column was washed with a 5column volume of C:M (4:1 and 3:1, v/v), and the PtdGlc-containing fraction was eluted with a 5-column volume of C:M (2:1, v/v). The PtdGlc fraction was dried under nitrogen gas stream and resolved into 200 lL of C:M (2:1, v/v) for stock solution. The lipid solution was diluted 10 fold with C:M (9:1, v/v) and added PtdGly as an internal standard for LC-MS analysis. The concentration of PtdGly in the sample solution was 250 nmol/L.
Methanol (LC-MS grade), chloroform (HPLC grade), 2-propanol (HPLC grade), and ammonia hydroxide (grade for analysis of poisonous metals) were purchased from Wako Pure Chemical (Osaka, Japan). Ammonium acetate for mass spectrometry was purchased from Sigma-Aldrich (St. Louis, MO). The internal standard, 1,2-diheptadecanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (C17:0/C17:0 PtdGly) and other phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). The calibration standard, 2-arachidoyl 1-stearoyl-sn-glycerol-3-phosphorylb-D-glucopyranoside (C18:0/C20:0 PtdGlc), was extracted and purified from lyophilized rat fetal brains in our laboratory [6]. The amount of isolated PtdGlc was calculated from its phosphorus content, which was determined by the method of Chen et al. [21] with modification. Briefly, 10 lL of standard phosphate solution containing 10–80 nmol phosphorus or samples were oxidized by 70% perchloric acid at 180 °C for 1 h and then phosphorus contents were quantified by hexaammonium heptamolybdate reagent containing ascorbic acid. The molecular structures of PtdGlc and PtdGly used for the internal standard are shown in Fig. 1. PtdGlc standard stock solution was prepared by dissolving PtdGlc in chloroform:methanol (C:M) (3:1, v/v). Calibration standards containing the internal standard (C17:0/ C17:0 PtdGly) were prepared by adding C17:0/C17:0 PtdGly to the stock solution. The stock solution and calibration standards were stored below 20 °C. Culture and differentiation of C6 glioma cells The C6 rat glioma cell line (ATCC number CCL 107) used in this study was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 lg/mL streptomycin. To induce differentiation, the cells were plated in 6-well culture plates at a starting density of 8.3 x 104 cells/well and cultured for 2 days. The cells were serum starved for 1 h by replacing the culture medium with serum-free or conventional (control) fresh medium. Afterward, the cells were treated with 1 mM dibutyryl cAMP plus 0.25 mM theophylline [22] with 10 lg/mL of monoclonal anti-PtdGlc antibody (mAb DIM21) [23], and then cultured further for 2 days. The cells were harvested by treating the cultures with trypsin, washed with Hanks’ balanced salt solution (HBSS) and subjected to lipid extraction for LC-MS analysis.
Two-dimensional TLC immunostaining analysis The lipid solutions were applied onto POLYGRAM SIL G TLC plates (Macherey-Nagel, Germany), and developed with a solution of C:M:acetic acid:water (65:35:4:4, v/v/v/v) for the first dimension, then developed with C:M:2.5 N NH4OH (65:35:8, v/v/v) for the second dimension. Developed TLC plates were soaked in 1% ovalbumin (OVA) in Tris-buffered saline for 1 h. Plates were then incubated overnight with 40 lg/ml mAb DIM21 dissolved in Can Get Signal solution 1 (TOYOBO Co., Ltd.). After three 5-min washes with TBS, the TLC plates were incubated for 4 h with HRP-conjugated anti-mouse IgG (1:500; ICN/Cappel) dissolved in Can Get Signal solution 2. Immunoreactive spots were visualized with 4-chloro-1-naphthol/H2O2 reagent [24]. LC-MS analysis LC-MS was performed with a NanoFrontier L (Hitachi HighTechnologies, Tokyo, Japan) consisting of a nano-flow HPLC equipped with an AT10PV nano-flow gradient generator [25] and a LIT-TOF MS with an electrospray ion source. The AT10PV nanoflow gradient generator consists of a conventional microflow gradient pump with low-pressure gradient capability, a nano-flow isocratic pump, and a 10-port switching valve with two injection loops. The sample injector was an Upchurch M-435 microinjection valve (Upchurch Scientific, Oak Harbor, WA). The HPLC column was an Inertsil SIL-100A (3 lm, 0.075 mm i.d., and 150 mm length) Si column from GL Science (Tokyo, Japan). One hundred nanoliters
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we found that adding small amounts of water to the eluents effectively promoted the chromatographic separation of PtdGlc and PtdGly. A normal-phase column is useful for the class separation of phospholipids, but it is hard to separate each lipid class based upon its molecular species. In the current study, PtdGlc existed exclusively as a single molecular species in rat brain and could be isolated from other eluents only by using a Si column. Mass spectra and CID product ion spectra data on PtdGlc and PtdGly are shown in Fig. 3. PtdGlc gave a deprotonated ion at m/z 893.6 (Fig. 3a). The product ion in the CID spectrum of PtdGlc, a characteristic product ion of arachidic acid (C20:0), was detected at m/z 311.3 (Fig. 3b). Because arachidic acid rarely exists in mammalian membrane lipids, we used the CID spectrum of arachidate anion as an indicator for PtdGlc. The CID spectrum of PtdGlc was similar to the SORI-CID mass spectrum of PtdGlc obtained through FT-ICR MS. C17:0/C17:0 PtdGly, used as internal standard, gave a deprotonated ion at m/z 749.6 (Fig. 3c) and the CID spectrum is shown in Fig. 3d.
of calibration standard or extracted rat brain sample solution was injected into the Si column. Solvent A (C:M:2-propanol:water [80:12.5:7:0.5, v/v/v/v] in 5 mmol/L ammonium acetate, pH 7.5) and Solvent B (methanol:2-propanol:water [92.5:7:0.5, v/v/v] in 5 mmol/L ammonium acetate, pH 7.5) were used as eluents. The gradient pump delivered solvents A and B at a flow rate of 100 lL/min. The following gradient profile was used: The composition was 100% of solvent A, then increased linearly to 70% of B in 30 min, and maintained for 10 min until the column was washed. After washing, the column, gradient composition was changed to that of initial conditions to equilibrate the column for 40 min. The flow rate of the nano-flow pump was set to 200 nL/min and the 10-port switching valve was switched every 1 min. The ESI spray potential was -4000 V in negative-ion mode, curtain gas flow was 0.8 L/min, Ex lens potential was 120 V, and AP1 temperature was 140 °C. The scan mass range was m/z 200 to 2000, an accumulation time was 20 ms, and a microsequence was 52 times. In the MS/MS experiment, the data-dependent MS/MS scan mode was used. Isolation time was 10 ms, collision-induced dissociation (CID) time was 10 ms, and CID gain energy was determined automatically for the target m/z.
Quantitation of PtdGlc MS analysis is expected to facilitate the estimation of the amount of the very minor lipid, PtdGlc. For the quantification of PtdGlc, we first examined the internal standard. Isotopically labeled compounds have been used extensively to improve the quantitation performance of LC-MS analysis [26,27]. Indeed, such compounds are useful to correct for variation because they possess essentially the same chemical properties and retention times as their nonlabeled counterparts. At present, because it is difficult to synthesize isotopically labeled PtdGlc, we decided to use commercially available C17:0/C17:0 PtdGly as an internal standard. C17:0/C17:0 PtdGly was useful because its retention behavior is similar to that of PtdGlc in normal-phase liquid chromatography and it was sensitively detected in negative ion mode electrospray mass spectrometry. To determine the detection limits (s/n = 3) of our system, we extracted the ion chromatographic peak of the deprotonated molecule of PtdGlc by injecting 100 nL of the standard solution. MS/ MS chromatogram data were not used for quantitation because
Results and discussion Detection and identification of PtdGlc We first tested detection sensitivity of PtdGlc in mass spectrometry in both positive- and negative-ion modes and found that detection of PtdGlc by negative-ion mode gave better sensitivity than positive-ion mode. Therefore, in this communication, all mass spectra were obtained in negative-ion mode. Parameters such as curtain gas flow, spray potential, and AP1 temperature were optimized by monitoring the base peak ion of PtdGlc at m/z 893.6 as a standard sample. Next, we applied a mixture of PtdGlc and other standard phospholipids to a Si column and found that the major phospholipids were easily separable from one another as shown in Fig. 2. We previously reported that the retention of PtdGlc by Si column HPLC was almost the same as that of PtdGly [8]. In the present study,
a TIC
100 0 100
b
XIC; m/z 749.5
c
XIC; m/z 893.6
C17:0/C17:0 PtdGly
Relative Intensity
0 100
C18:0/C20:0 PtdGlc
0 100
d XIC; m/z 746.5
C18:0/C18:0 PtdEth
0 100
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XIC; m/z 809.5
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XIC; m/z 772.6
C16:0/C16:0 PtdIns
0 100 0
0
12
C18:0/C18:1 PtdCho
24
36
48
60
Retention Time (min) Fig. 2. Total ion chromatogram and extracted ion chromatograms of the glycerophospholipids standard mixture (each 1 lM). A 100-nL sample was injected into a 150 0.075-mm I.D. Si column and eluted at a flow rate of 200 nL/min.
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C18:0/C20:0 PtdGlc
1.0
b
[M-H]893.6
Relative Intensity
Relative Intensity
a
0.8 0.6 0.4 0.2 0
200
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840
749.6
0.8 0.6 0.4 0.2 0
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m/z [R1COO]283.3
1.0 0.8
d [M-H-Glc-R2COOH]419.3
[R2COO] -
0.6
[M-H-Glc-R1COOH]-
311.3
[M-H-R2COOH]-
0.4 447.3
0.2
599.3 0
200
360
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680
840
1000
840
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m/z
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Relative Intensity
c
[M-H]-
C17:0/C17:0 PtdGly
1.0
[M-H-Glc]731.6 680
840
1000
m/z
[RCOO]269.3
1.0 0.8
[M-H-Gly-RCOOH]-
0.6 [M-H-RCOOH]-
0.4 405.3
0.2 0
200
360
497.3 520
680
m/z
Fig. 3. Mass spectra of C18:0/C20:0 PtdGlc (a) and C17:0/C17:0 PtdGly (c) and their respective product ion spectra at m/z 893.6 (b) and m/z 749.6 (d) obtained by collisioninduced dissociation, injecting 100 nL of the standard solution (each 1 lM).
insufficient chromatogram data were obtained in the data-dependent MS/MS scan mode. The detection limit of PtdGlc was 10 nmol/ L; therefore, the detection limit of the injection amount was 1.0 fmol. The PtdGlc calibration curve obtained through LC-MS analysis is shown in Fig. 4. Linearity for solutions containing 6.3–800 fmol of PtdGlc was found to be good for this range (correlation coefficient, R2>0.995). The intercept of the calibration curve was 0.025 relative response units. However, the linearity was obtained from highly purified sample. The crude or partially purified sample is considered to contain causative compounds for signal suppression or interference effects by other ions. To check the applicability to real samples, the control
5.0 y = 0.0053x + 0.0131
4.5
2
Peak Area (PtdGlc/PtdGly)
R = 0.9956
4.0 3.5 3.0
2.5 2.0
5
1.5 1.0 0.5 0.0 0
100
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400
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PtdGlc amount (fmol) Fig. 4. PtdGlc calibration curve. A given amount of PtdGlc was applied to a Si column (150 0.075-mm I.D.) and eluted at a flow rate of 200 nL/min. Peak intensities obtained at m/z 893.6 were plotted against the amounts of PtdGlc applied to the column. The amount of the internal standard (PtdGly) applied to the column was 25 fmol.
C6 glioma cell extract was spiked with a given amount of PtdGlc and PtdGly (as an internal standard) solutions and the calibration was checked again. When PtdGlc amounts spiked into the control cell extracts were 20 and 200 fmol/injection, respectively, quantitative amounts of PtdGlc by this LC-MS method were 19.0 ± 3.0 and 191 ± 15 fmol/injection (mean ± SD, n=3), respectively. Consequently, signal suppressions and interferences effects caused by the C6 glioma cell solutions were negligibly small in the extract samples. Quantitative analysis of PtdGlc in C6 rat glioma cells We identified PtdGlc to be a novel cell-surface marker of radial astroglial cells in developing brains [5]. C6 glioma cells have been used as a model cell line to study the molecular mechanisms underlying the c-AMP/theophylline-induced differentiation of neural cells into GFAP-positive glia (astroglia) [22]. Thus, we examined whether PtdGlc in C6 glioma cells was involved in glial differentiation. We found that treatment of C6 glioma cells with DIM21 caused increased expression of GFAP protein (data not shown). To study further the role of PtdGlc in glial cells, we examined whether PtdGlc levels change during C6 glioma cell differentiation. First, we used the TLC-immunostaining technique to examine the presence of PtdGlc in C6 glioma cells. Although PtdGlc in control cells was undetectable, we detected a significant increase of DIM21 antigen when C6 glioma cells were treated with DIM21 in the presence of cAMP (data not shown). Next, we used our LC-MS method to analyze the PtdGlc content in C6 glioma cells. As shown in Fig. 5 and Table 1, mass spectrometric analysis did not detect PtdGlc in control cells. However, PtdGlc was found in cells cultured in serum-free conditions at 0.51 nmol/ 106 cells. Similar levels of PtdGlc were also detected in cells treated with DIM21. In cAMP/theophylline-plus-DIM21-treated cells, PtdGlc levels increased significantly to 4.61 nmol/106 cells. These findings supported the results obtained by the TLC-immunostaining method.
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100
a
10% FBS (control) m/z 893.6
0
Relative Intensity
100
b
Serum-free medium m/z 893.6
0 100
c
DIM21 m/z 893.6
d
DIM21 + cAMP m/z 893.6
0 100
0 0
12
24
36
48
60
Retention Time (min) Fig. 5. Representative extracted ion chromatograms of PtdGlc in C6 rat glioma cells. y axes were normalized by the chromatogram of (d). LC conditions were same as those in Fig. 2.
Table 1 Quantitative estimate of PtdGlc in C6 glioma cells using an internal standard for calibration Culture conditions
Amount of PtdGlca (nmol/106 cells)
10% FBS (control) Serum-free medium cAMP DIM21 DIM21 + cAMP
N.D.b 0.51 ± 0.16 N.D. 0.51 ± 0.14 4.61 ± 0.11
Lipids extracted from one million cells were analyzed. a Mean ± SD; n=3. b N.D., not detected.
Previously, we demonstrated that in HL60 cells, stimulation of the cell-surface PtdGlc-rich microdomain with anti-PtdGlc antibody causes granulocytic differentiation accompanied by an increase of PtdGlc content [5]. The present results from C6 glioma cells indicated that PtdGlc also forms lipid signaling domains or lipid rafts involved in astroglial differentiation. We have succeeded in developing a method for rapid identification and quantification of minor glucosylated phospholipids, such as PtdGlc, using a LC-MS system. Recently the multiple-reaction monitoring (MRM) approach has been widely used for quantitative analysis. However, the instrument used in the present experiment does not equip the detector for MRM mode. In this communication we showed that PtdGlc could be quantified using the present system even when the mixture of lipid extract was analyzed. The amounts of PtdGlc obtained with differentiated C6 glioma cells is very similar to the TLC-immunostaining results. Our novel method does not require the time-consuming step of purifying phospholipids prior to LC-MS analysis. In cells and tissues thus far examined, PtdGlc possesses C18:0 and C20:0 as its sole fatty acids, indicating that PtdGlc has very important biological functions and that its biosynthesis is critically controlled in cells and tissues. The present analytical method should elucidate the biological importance of PtdGlc in biological membranes.
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Quantitative analysis of a novel glucosylated phospholipid by liquid chromatography–mass spectrometry Shinya Ito a,b, Takuji Nabetani b, Yoko Shinoda b, Yasuko Nagatsuka b, Yoshio Hirabayashi b,1,* a b
Hitachi High-Technologies Corp., 1-24-14 Nishi-shinbashi, Minato-ku, Tokyo 105-8717, Japan Hirabayashi Research Unit, Brain Science Institute, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e
i n f o
Article history: Received 28 December 2007 Available online 13 February 2008 Keywords: Phospholipid Phosphatidylglucoside LC-MS MS/MS Quantitative analysis C6 glioma cell
a b s t r a c t Building upon the demonstrated presence of a new glyceroglycolipid, phosphatidylglucoside (PtdGlc), in rat embryonic brain tissues, we have developed a method to identify minute amounts of PtdGlc in cultured cells by using nano-flow high-performance liquid chromatography and negative-ion-mode electrospray linear-ion trap time-of-flight mass spectrometry (LC-MS). A normal-phase silica gel-based column enabled us to separate PtdGlc from other lipid classes. PtdGlc was identified from its tandem mass spectrometry spectrum and from its retention time in the column. Using an internal standard collection and LC-MS, we obtained the linearity of PtdGlc at a range of 6.3–800 fmol per injection. We applied this method to analyze quantitative changes in PtdGlc in C6 glioma cells after cellular differentiation into GFAP-positive glial cells. PtdGlc in C6 glioma cells consisted exclusively of C18:0/C20:0 fatty acyl chains. Differentiation induced by the addition of anti-PtdGlc antibody plus cAMP in culture medium significantly increased the glycolipid content. Ó 2008 Elsevier Inc. All rights reserved.
The identification and comprehensive characterization of the molecular species within a phospholipid has been challenging because glycerophospholipids contain diverse fatty acid chains that are present with limited abundance (for review, see [1–3]). Highperformance liquid chromatography (HPLC) with UV detection allows the separation and quantification of many phospholipid classes. [4] However HPLC-UV for analysis of phospholipids is suffering from the lack of specificity and differential responses depending on double bond composition. Moreover, it is difficult to identify minor constituents such as phosphatidylglucoside (PtdGlc)2, which was recently isolated in rodent brain tissues [5– 8] by conventional analytical methods. In recent years, HPLC combined with mass spectrometry (LCMS) has been used to analyze a variety of lipids [9,10]. Tandem mass spectrometry, in particular, is a powerful tool for identification and quantitation of lipids [11–15]. Combining tandem mass spectrometry with normal-phase liquid chromatography enables
* Corresponding author. Fax: +81 48 467 6372. E-mail address: [email protected] (Y. Hirabayashi). 1 Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency (JST). 2 Abbreviations used: PtdGlc, phosphatidylglucoside; ESI, electrospray ionization; PtdGly, phosphatidylglycerol; CID, collision-induced dissociation; FT-ICR, Fourier transform ion cyclotron resonance; MRM, multiple-reaction monitoring; FBS, fetal bovine serum. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.02.007
the separation of phospholipids into several major subclasses [16,17]. Its application to the analysis of lipids was a breakthrough for lipid biochemistry. Compared to other ionization methods, electrospray ionization (ESI) is very useful for analyzing polar lipids, because polar lipids such as phospholipids are sensitive to being ionized without a derivatization procedure. Phosphatidylcholine, for example, can be detected as protonated molecules in positive-ion mode, and phosphatidylglycerol (PtdGly) can be detected as deprotonated molecules in negative-ion mode [12,18– 20]. Furthermore, ESI can be coupled with nano-flow HPLC easily. This analytical method has been successfully used for the identification and characterization of phospholipids from biological samples [10,18,19]. Because PtdGlc has a unique structure consisting of a single molecular species of C18:0 and C20:0 of diacylglycerol and is a minor constituent in developing rodent brains, PtdGlc has never been studied extensively. To elucidate the biological significance of PtdGlc, it is essential to determine its distribution in mammalian tissues and cells. To this end, we sought to develop a straightforward method for identifying and quantifying PtdGlc. In this paper, we describe a procedure for identifying and quantifying PtdGlc by nano-flow HPLC coupled on-line to ESI linear ion-trap time-of-flight mass spectrometry. This analytical method was reproducible and sensitive enough to identify and measure PtdGlc, a minor membrane component in C6 rat glioma cells.
Quantitative analysis of a novel phospholipid / S. Ito et al. / Anal. Biochem. 376 (2008) 252–257
A
253
C18:0/C20:0 PtdGlc O
OH O
O
HO HO
OH
H O
O P O OH
O O
B
O
C17:0/C17:0 PtdGly O
OH HO
O P O OH
H O O O
Fig. 1. Molecular structures of glycerophospholipids. (A): C18:0/C20:0 PtdGlc. (B): C17:0/C17:0 PtdGly.
Materials and methods
Lipid extraction
Chemicals and standard lipids
The harvested cells were suspended in a small amount of methanol and placed into a ODS cartridge column (Oro-Sep C18 600 mg, OROCHEM CRC 8600) prewashed with methanol. ODS-unbound compounds were washed out with 10 mL of water, and then ODS-retained compounds were eluted with 1 mL of C:M (2:1, v/ v). Extracts were dried under a stream of N2 gas, dissolved in C:M (4:1, v/v), and placed into an Iatrobeads (Iatron Laboratories, Inc.) Si column (5.3u 4 cm). The column was washed with a 5column volume of C:M (4:1 and 3:1, v/v), and the PtdGlc-containing fraction was eluted with a 5-column volume of C:M (2:1, v/v). The PtdGlc fraction was dried under nitrogen gas stream and resolved into 200 lL of C:M (2:1, v/v) for stock solution. The lipid solution was diluted 10 fold with C:M (9:1, v/v) and added PtdGly as an internal standard for LC-MS analysis. The concentration of PtdGly in the sample solution was 250 nmol/L.
Methanol (LC-MS grade), chloroform (HPLC grade), 2-propanol (HPLC grade), and ammonia hydroxide (grade for analysis of poisonous metals) were purchased from Wako Pure Chemical (Osaka, Japan). Ammonium acetate for mass spectrometry was purchased from Sigma-Aldrich (St. Louis, MO). The internal standard, 1,2-diheptadecanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (C17:0/C17:0 PtdGly) and other phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). The calibration standard, 2-arachidoyl 1-stearoyl-sn-glycerol-3-phosphorylb-D-glucopyranoside (C18:0/C20:0 PtdGlc), was extracted and purified from lyophilized rat fetal brains in our laboratory [6]. The amount of isolated PtdGlc was calculated from its phosphorus content, which was determined by the method of Chen et al. [21] with modification. Briefly, 10 lL of standard phosphate solution containing 10–80 nmol phosphorus or samples were oxidized by 70% perchloric acid at 180 °C for 1 h and then phosphorus contents were quantified by hexaammonium heptamolybdate reagent containing ascorbic acid. The molecular structures of PtdGlc and PtdGly used for the internal standard are shown in Fig. 1. PtdGlc standard stock solution was prepared by dissolving PtdGlc in chloroform:methanol (C:M) (3:1, v/v). Calibration standards containing the internal standard (C17:0/ C17:0 PtdGly) were prepared by adding C17:0/C17:0 PtdGly to the stock solution. The stock solution and calibration standards were stored below 20 °C. Culture and differentiation of C6 glioma cells The C6 rat glioma cell line (ATCC number CCL 107) used in this study was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 lg/mL streptomycin. To induce differentiation, the cells were plated in 6-well culture plates at a starting density of 8.3 x 104 cells/well and cultured for 2 days. The cells were serum starved for 1 h by replacing the culture medium with serum-free or conventional (control) fresh medium. Afterward, the cells were treated with 1 mM dibutyryl cAMP plus 0.25 mM theophylline [22] with 10 lg/mL of monoclonal anti-PtdGlc antibody (mAb DIM21) [23], and then cultured further for 2 days. The cells were harvested by treating the cultures with trypsin, washed with Hanks’ balanced salt solution (HBSS) and subjected to lipid extraction for LC-MS analysis.
Two-dimensional TLC immunostaining analysis The lipid solutions were applied onto POLYGRAM SIL G TLC plates (Macherey-Nagel, Germany), and developed with a solution of C:M:acetic acid:water (65:35:4:4, v/v/v/v) for the first dimension, then developed with C:M:2.5 N NH4OH (65:35:8, v/v/v) for the second dimension. Developed TLC plates were soaked in 1% ovalbumin (OVA) in Tris-buffered saline for 1 h. Plates were then incubated overnight with 40 lg/ml mAb DIM21 dissolved in Can Get Signal solution 1 (TOYOBO Co., Ltd.). After three 5-min washes with TBS, the TLC plates were incubated for 4 h with HRP-conjugated anti-mouse IgG (1:500; ICN/Cappel) dissolved in Can Get Signal solution 2. Immunoreactive spots were visualized with 4-chloro-1-naphthol/H2O2 reagent [24]. LC-MS analysis LC-MS was performed with a NanoFrontier L (Hitachi HighTechnologies, Tokyo, Japan) consisting of a nano-flow HPLC equipped with an AT10PV nano-flow gradient generator [25] and a LIT-TOF MS with an electrospray ion source. The AT10PV nanoflow gradient generator consists of a conventional microflow gradient pump with low-pressure gradient capability, a nano-flow isocratic pump, and a 10-port switching valve with two injection loops. The sample injector was an Upchurch M-435 microinjection valve (Upchurch Scientific, Oak Harbor, WA). The HPLC column was an Inertsil SIL-100A (3 lm, 0.075 mm i.d., and 150 mm length) Si column from GL Science (Tokyo, Japan). One hundred nanoliters
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we found that adding small amounts of water to the eluents effectively promoted the chromatographic separation of PtdGlc and PtdGly. A normal-phase column is useful for the class separation of phospholipids, but it is hard to separate each lipid class based upon its molecular species. In the current study, PtdGlc existed exclusively as a single molecular species in rat brain and could be isolated from other eluents only by using a Si column. Mass spectra and CID product ion spectra data on PtdGlc and PtdGly are shown in Fig. 3. PtdGlc gave a deprotonated ion at m/z 893.6 (Fig. 3a). The product ion in the CID spectrum of PtdGlc, a characteristic product ion of arachidic acid (C20:0), was detected at m/z 311.3 (Fig. 3b). Because arachidic acid rarely exists in mammalian membrane lipids, we used the CID spectrum of arachidate anion as an indicator for PtdGlc. The CID spectrum of PtdGlc was similar to the SORI-CID mass spectrum of PtdGlc obtained through FT-ICR MS. C17:0/C17:0 PtdGly, used as internal standard, gave a deprotonated ion at m/z 749.6 (Fig. 3c) and the CID spectrum is shown in Fig. 3d.
of calibration standard or extracted rat brain sample solution was injected into the Si column. Solvent A (C:M:2-propanol:water [80:12.5:7:0.5, v/v/v/v] in 5 mmol/L ammonium acetate, pH 7.5) and Solvent B (methanol:2-propanol:water [92.5:7:0.5, v/v/v] in 5 mmol/L ammonium acetate, pH 7.5) were used as eluents. The gradient pump delivered solvents A and B at a flow rate of 100 lL/min. The following gradient profile was used: The composition was 100% of solvent A, then increased linearly to 70% of B in 30 min, and maintained for 10 min until the column was washed. After washing, the column, gradient composition was changed to that of initial conditions to equilibrate the column for 40 min. The flow rate of the nano-flow pump was set to 200 nL/min and the 10-port switching valve was switched every 1 min. The ESI spray potential was -4000 V in negative-ion mode, curtain gas flow was 0.8 L/min, Ex lens potential was 120 V, and AP1 temperature was 140 °C. The scan mass range was m/z 200 to 2000, an accumulation time was 20 ms, and a microsequence was 52 times. In the MS/MS experiment, the data-dependent MS/MS scan mode was used. Isolation time was 10 ms, collision-induced dissociation (CID) time was 10 ms, and CID gain energy was determined automatically for the target m/z.
Quantitation of PtdGlc MS analysis is expected to facilitate the estimation of the amount of the very minor lipid, PtdGlc. For the quantification of PtdGlc, we first examined the internal standard. Isotopically labeled compounds have been used extensively to improve the quantitation performance of LC-MS analysis [26,27]. Indeed, such compounds are useful to correct for variation because they possess essentially the same chemical properties and retention times as their nonlabeled counterparts. At present, because it is difficult to synthesize isotopically labeled PtdGlc, we decided to use commercially available C17:0/C17:0 PtdGly as an internal standard. C17:0/C17:0 PtdGly was useful because its retention behavior is similar to that of PtdGlc in normal-phase liquid chromatography and it was sensitively detected in negative ion mode electrospray mass spectrometry. To determine the detection limits (s/n = 3) of our system, we extracted the ion chromatographic peak of the deprotonated molecule of PtdGlc by injecting 100 nL of the standard solution. MS/ MS chromatogram data were not used for quantitation because
Results and discussion Detection and identification of PtdGlc We first tested detection sensitivity of PtdGlc in mass spectrometry in both positive- and negative-ion modes and found that detection of PtdGlc by negative-ion mode gave better sensitivity than positive-ion mode. Therefore, in this communication, all mass spectra were obtained in negative-ion mode. Parameters such as curtain gas flow, spray potential, and AP1 temperature were optimized by monitoring the base peak ion of PtdGlc at m/z 893.6 as a standard sample. Next, we applied a mixture of PtdGlc and other standard phospholipids to a Si column and found that the major phospholipids were easily separable from one another as shown in Fig. 2. We previously reported that the retention of PtdGlc by Si column HPLC was almost the same as that of PtdGly [8]. In the present study,
a TIC
100 0 100
b
XIC; m/z 749.5
c
XIC; m/z 893.6
C17:0/C17:0 PtdGly
Relative Intensity
0 100
C18:0/C20:0 PtdGlc
0 100
d XIC; m/z 746.5
C18:0/C18:0 PtdEth
0 100
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XIC; m/z 809.5
f
XIC; m/z 772.6
C16:0/C16:0 PtdIns
0 100 0
0
12
C18:0/C18:1 PtdCho
24
36
48
60
Retention Time (min) Fig. 2. Total ion chromatogram and extracted ion chromatograms of the glycerophospholipids standard mixture (each 1 lM). A 100-nL sample was injected into a 150 0.075-mm I.D. Si column and eluted at a flow rate of 200 nL/min.
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C18:0/C20:0 PtdGlc
1.0
b
[M-H]893.6
Relative Intensity
Relative Intensity
a
0.8 0.6 0.4 0.2 0
200
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749.6
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m/z [R1COO]283.3
1.0 0.8
d [M-H-Glc-R2COOH]419.3
[R2COO] -
0.6
[M-H-Glc-R1COOH]-
311.3
[M-H-R2COOH]-
0.4 447.3
0.2
599.3 0
200
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520
680
840
1000
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m/z
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Relative Intensity
c
[M-H]-
C17:0/C17:0 PtdGly
1.0
[M-H-Glc]731.6 680
840
1000
m/z
[RCOO]269.3
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[M-H-Gly-RCOOH]-
0.6 [M-H-RCOOH]-
0.4 405.3
0.2 0
200
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497.3 520
680
m/z
Fig. 3. Mass spectra of C18:0/C20:0 PtdGlc (a) and C17:0/C17:0 PtdGly (c) and their respective product ion spectra at m/z 893.6 (b) and m/z 749.6 (d) obtained by collisioninduced dissociation, injecting 100 nL of the standard solution (each 1 lM).
insufficient chromatogram data were obtained in the data-dependent MS/MS scan mode. The detection limit of PtdGlc was 10 nmol/ L; therefore, the detection limit of the injection amount was 1.0 fmol. The PtdGlc calibration curve obtained through LC-MS analysis is shown in Fig. 4. Linearity for solutions containing 6.3–800 fmol of PtdGlc was found to be good for this range (correlation coefficient, R2>0.995). The intercept of the calibration curve was 0.025 relative response units. However, the linearity was obtained from highly purified sample. The crude or partially purified sample is considered to contain causative compounds for signal suppression or interference effects by other ions. To check the applicability to real samples, the control
5.0 y = 0.0053x + 0.0131
4.5
2
Peak Area (PtdGlc/PtdGly)
R = 0.9956
4.0 3.5 3.0
2.5 2.0
5
1.5 1.0 0.5 0.0 0
100
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400
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PtdGlc amount (fmol) Fig. 4. PtdGlc calibration curve. A given amount of PtdGlc was applied to a Si column (150 0.075-mm I.D.) and eluted at a flow rate of 200 nL/min. Peak intensities obtained at m/z 893.6 were plotted against the amounts of PtdGlc applied to the column. The amount of the internal standard (PtdGly) applied to the column was 25 fmol.
C6 glioma cell extract was spiked with a given amount of PtdGlc and PtdGly (as an internal standard) solutions and the calibration was checked again. When PtdGlc amounts spiked into the control cell extracts were 20 and 200 fmol/injection, respectively, quantitative amounts of PtdGlc by this LC-MS method were 19.0 ± 3.0 and 191 ± 15 fmol/injection (mean ± SD, n=3), respectively. Consequently, signal suppressions and interferences effects caused by the C6 glioma cell solutions were negligibly small in the extract samples. Quantitative analysis of PtdGlc in C6 rat glioma cells We identified PtdGlc to be a novel cell-surface marker of radial astroglial cells in developing brains [5]. C6 glioma cells have been used as a model cell line to study the molecular mechanisms underlying the c-AMP/theophylline-induced differentiation of neural cells into GFAP-positive glia (astroglia) [22]. Thus, we examined whether PtdGlc in C6 glioma cells was involved in glial differentiation. We found that treatment of C6 glioma cells with DIM21 caused increased expression of GFAP protein (data not shown). To study further the role of PtdGlc in glial cells, we examined whether PtdGlc levels change during C6 glioma cell differentiation. First, we used the TLC-immunostaining technique to examine the presence of PtdGlc in C6 glioma cells. Although PtdGlc in control cells was undetectable, we detected a significant increase of DIM21 antigen when C6 glioma cells were treated with DIM21 in the presence of cAMP (data not shown). Next, we used our LC-MS method to analyze the PtdGlc content in C6 glioma cells. As shown in Fig. 5 and Table 1, mass spectrometric analysis did not detect PtdGlc in control cells. However, PtdGlc was found in cells cultured in serum-free conditions at 0.51 nmol/ 106 cells. Similar levels of PtdGlc were also detected in cells treated with DIM21. In cAMP/theophylline-plus-DIM21-treated cells, PtdGlc levels increased significantly to 4.61 nmol/106 cells. These findings supported the results obtained by the TLC-immunostaining method.
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100
a
10% FBS (control) m/z 893.6
0
Relative Intensity
100
b
Serum-free medium m/z 893.6
0 100
c
DIM21 m/z 893.6
d
DIM21 + cAMP m/z 893.6
0 100
0 0
12
24
36
48
60
Retention Time (min) Fig. 5. Representative extracted ion chromatograms of PtdGlc in C6 rat glioma cells. y axes were normalized by the chromatogram of (d). LC conditions were same as those in Fig. 2.
Table 1 Quantitative estimate of PtdGlc in C6 glioma cells using an internal standard for calibration Culture conditions
Amount of PtdGlca (nmol/106 cells)
10% FBS (control) Serum-free medium cAMP DIM21 DIM21 + cAMP
N.D.b 0.51 ± 0.16 N.D. 0.51 ± 0.14 4.61 ± 0.11
Lipids extracted from one million cells were analyzed. a Mean ± SD; n=3. b N.D., not detected.
Previously, we demonstrated that in HL60 cells, stimulation of the cell-surface PtdGlc-rich microdomain with anti-PtdGlc antibody causes granulocytic differentiation accompanied by an increase of PtdGlc content [5]. The present results from C6 glioma cells indicated that PtdGlc also forms lipid signaling domains or lipid rafts involved in astroglial differentiation. We have succeeded in developing a method for rapid identification and quantification of minor glucosylated phospholipids, such as PtdGlc, using a LC-MS system. Recently the multiple-reaction monitoring (MRM) approach has been widely used for quantitative analysis. However, the instrument used in the present experiment does not equip the detector for MRM mode. In this communication we showed that PtdGlc could be quantified using the present system even when the mixture of lipid extract was analyzed. The amounts of PtdGlc obtained with differentiated C6 glioma cells is very similar to the TLC-immunostaining results. Our novel method does not require the time-consuming step of purifying phospholipids prior to LC-MS analysis. In cells and tissues thus far examined, PtdGlc possesses C18:0 and C20:0 as its sole fatty acids, indicating that PtdGlc has very important biological functions and that its biosynthesis is critically controlled in cells and tissues. The present analytical method should elucidate the biological importance of PtdGlc in biological membranes.
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