Characterization of Polyunsaturated Phospholipid Remodeling in

Downloaded by STANFORD UNIV GREEN LIBR on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch013

Chapter 13

Characterization of Polyunsaturated Phospholipid Remodeling in Mammalian Cells by High-Performance Liquid Chromatography—Electrospray Ionization Mass Spectrometry 1

2

Hee-Yong Kim , Tao-Chin Lin Wang , and Yee-Chung Ma

1

1

National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Building 10, Room 3C-103, Bethesda, MD 20892 National Institute of Mental Health, National Institutes of Health, Building 10, Room 3D-40, Bethesda, MD 20892 2

Characteristics of cell membranes are greatly affected by the composition of phospholipid bilayers. The cellular membrane phospholipid composition is maintained by complex biosynthetic and remodeling processes, including phospholipase, base exchange and acyltransferase reactions. In order to understand these complex biochemical mechanisms, analysis of the phospholipid profile in biomembranes is necessary. Using electrospray mass spectrometry coupled to reversed phase HPLC, phospholipid molecular species of different head groups and fatty acyl compositions could be separated and detected in a single run. Sensitivity at the picomole level was attained with a linear response range over 2 orders of magnitude. Application of this technique to remodeling of polyunsaturated phospholipids in C-6 glia cells and the rodent brain is demonstrated. Phospholipid bilayers constitute the major part of cell membranes. Since the characteristics of cell membranes are influenced by the phospholipid composition, the maintenance of proper phospholipid composition in membranes is of great importance. Distinctive lipid composition is maintained in specific cell membranes, probably through complex biosynthetic and remodeling processes including phospholipase, base exchange and acyltransferase reactions (1, 2). Neuronal membranes are highly enriched in polyunsaturated fatty acids (3). These polyunsaturates can modulate various neuronal functions as membrane components (4) or as the free fatty acid form after mobilization upon stimulation (5). Polyunsaturated fatty acids, released from distinctive lipid pools in response to stimuli, are mostly reincorporated into membrane lipids while a certain portion escapes to be metabolized to biologically active compounds through various oxygenative pathways. In order to understand these complex biochemical processes, analysis of the phospholipid profile in cell membranes is necessary. This chapter not subject to U.S. copyright Published 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


268

BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS

Analysis of phospholipidsfroma biological matrix is a difficult task since most of the biomembranes contain a mixture of a variety of molecular species with different phospho-head groups. Conventional analysis techniques including column and argentation thin-layer chromatography (6,7) are not acceptable due to limited resolving power as well as limited sensitivity. High performance chromatographic analysis after derivatization with a chromophore (8) improves the sensitivity and resolving power, however, the added analysis step is laborious and also time consuming. In addition, confirmation of each peak identity must be established using other techniques such as gas chromatography (GC), GC-mass spectrometry (MS) or fast atom bombardment MS (9) after collecting and derivatizing each fraction. Therefore, an alternative technique which can provide reasonable separation, sensitivity and structural information in an efficient manner is desirable. Such techniques have been developed in our laboratory using thermospray liquid chromatography / mass spectrometry (LC/MS) which allows the direct detection of samples eluting from an LC column without any derivatization (10,11). However, difficulties in quantification imposed by non-linear response curves (12) together with insufficient sensitivity to monitor the remodeling of labeled lipid molecules prompted us to develop another LC/MS technique using electrospray LC/MS for phospholipid analysis (13). Experimental A Hewlett-Packard 5989 mass spectrometer with HP electrospray source was used for collecting most of the data presented here. In this system, a liquid flow at typically 1-5 μΙ7πύη is introduced through a needle with the aid of a nebulizing gas, nitrogen, to a high electric field which generates the charged droplets. Typically the column effluent was split 1:100 using a commercial splitter. In some cases, a Finnigan TSQ-700 mass spectrometer with a Finnigan electrospray/atmospheric pressure chemical ionization (APCI) source was employed, and a flow rate of 0.1 mL/min was introduced into the ion source after the flow was split by 1:5 or 1:4. The heated nitrogen gas was supplied to aid desolvation and orientation of produced ion clusters through a capillary. Ions exiting from the capillary were introduced into the mass analyzer through focusing lenses. Spectra generated in this way usually contain molecular ion species with minimal fragmentation. However, changing the electric voltage at the end of the capillary can inducefragmentationfor certain compounds. Electrospray Mass Spectra of Phospholipids Various phospholipids were detected as their protonated or natriated molecules as shown in figure 1. The spectra were obtained using a capillary exit voltage set at 200 V. Sodium attachment was more noticeable for acidic phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). Under this condition only PI produced significant diacylglyceryl (DG) fragments. Diacylglyceryl fragment ions contain molecular species information and were common to all phospholipid classes with a given fatty acyl composition. Therefore, producing diacylglyceryl fragments was beneficial, since less ions needed to be monitored in comparison to the case with molecular ions. Formation of diacylglyceryl fragments was induced by raising the capillary exit voltage as shown for 18:0 22:6-PE infigure2. Generally, the molecular ion intensity was maximum at an exit voltage of 200 V and decreased at higher exit voltages. At around 300 V, the intensity of the diacylglyceryl fragment was at a maximum. Similarfragmentationcould be induced for phosphatide acid (PA), PI and PS, although the extent of fragmentation was molecular species dependent. Phosphatidylcholine (PQ was resistant tofragmentationto DG even with the highest capillary exit potential applicable under the present configuration.


13. KIM ET AL.

Polyunsaturated Phospholipid Remodeling

269

Abundance (M+Na)+


969

18:0, 20:4-PI (mw=886)

20000-

DG 627

(M-H+2Na)+

5000·

200

300 00

400

500

600

700

800

900

(M+H)+ 2

18:0, 22:6-PS (mw=835) 18:0, 20-.4-PS (mw=8!1)

1000

1 (M+Na)+

834

(M+Na)+^ 800

600

(M-H+2Na)+

400-

o J

H7

100

, 1 . .1 200

360 ' '

800

900

m/z Figure 1. Positive ion electrospray mass spectra of PI, PC, PS and sphingomyelin (from ref. 13). Approximately 40-80 pmoles (0.4 to 0.8 femtomole after split) of standards were directly introduced by the flow injection technique using methanol:hexane:water 96:3:1. Continued on next page


270


Abundance U (M+H)+


50000 ^ 45000

18:0, 22-.6-PC (mw=833)

40000 35000 30000

(M+Na)+ B56

25000·] 20000 15000 10000 5000 0

' I

300

800

500

3000

1

900

'

(M+H)+

18:0-SM (mw=730) 2000

(M+Na)

1500 H

1000

0 >•!••••, 150 200 250 300 350 400 4 5 0 500 550 600 650 700 750

m/z Figure 1. Continued


13. KIM ET AL.

Polyunsaturated Phospholipid Remodeling

271


Sensitivity of the technique The sensitivity of the technique was assessed using the protonated molecule of 18:0 20:4-PC (Figure 3). The ion trace was generated from the injection of 1.35 pmoles of 18:0 20:4-PC by direct flow injection at a flow rate of 5 μί/πύη after a 100:1 flow split. This split allows only 1 % of the sample to be introduced into the mass spectrometer. Under this condition the present detection limit assessed for various PC molecular species was in the 0.5-1 pmole range, which represents approximately a 20 fold greater sensitivity in comparison to the limit (20 pmoles) obtained by the thermospray technique (11), even after the split Response was reproducible with no significant peak broadening, which indicates that direct coupling of L C for chromatographic peak detection is possible with this technique. High Performance Liquid Chromatography (HPLC) / Electrospray MS of Standard Phospholipids Chromatographic integrity of the HPLC separation was well maintained during transit through the capillary line to the mass spectrometer. An example is shown infigure4 for 18:0 20:4 containing phospholipids. Each molecular species can be monitored by using either the DG fragment or the (M+H) . Due to the difficulty to induce DG fragments from PC, monitoring protonated molecules was required for PC. Since diacylglyceryl ion resulted from die loss of the head group from the intact molecule, all phospholipids with the same fatty acyl composition of 18:0 20:4 can be detected as DG fragments at the same mass to charge ratio of 627 Da regardless of their head group identity. Therefore, with a proper chromatographic system which can separate a given molecular species of PS, PI and PE, only one diacylglyceryl fragment ion needed to be monitored. We have developed such a rapid chromatographic system using reversed phase HPLC (2.1 mm χ 15 cm) with the mobile phase containing a mixture of 0.5% ammonium hydroxide in water : methanol : hexane changing from 12:88:0 to 0:88:12 after holding at the initial solvent composition for 3 min (12). As shown in this figure, PS eluted first, followed by PE and then PC. Under die same condition, PI and PA eluted between PS and PE (data not shown). With ESI the intensity of DG fragment ions as well as protonated molecular ions appeared to be affected by the nature of the phospho-head group and was less dependent on fatty acyl composition within a given phospholipid class. This phenomenon is opposite to the results obtained by the thermospray technique (12) where the response was more dependent on the fatty acyl composition than on the head group characteristics. Therefore, this technique might be more suited for studies involving comparison of molecular species within a phospholipid class while the thermospray technique may be more useful for phospholipid remodeling between phospholipid classes. An example is shown for brain cytosolic phospholipids containing 20:4n6 or 22:6n3 infigure5. The cytosolic fraction was obtained after centrifugation of brain homogenate at 100,000xg for 1 hour. All phospholipid classes except PC could be analyzed using common DG fragment ions as 18:0 22:6,16:0 22:6,18:0 20:4 and 16:0 20:4 species were monitored by the ion traces of 651, 624, 628 and 599 Da, respectively in this figure. Unexpectedly, after centrifugation the cytosolic fraction still contained considerable levels of phospholipids, indicating that phospholipids exist in soluble forms in the cytosol, possibly associated with protein. The cytosolic phospholipids, however, were enriched in phosphatidylinositol-containing 20:4n6 in comparison to synaptosomal or whole brain phospholipids (data not shown), suggesting that preferential processes may exist in association of phospholipids with proteins such as ones involved in phospholipid transport (14). +



272

DG(oVz651)

2JXXN-7 η

MH+

Characterization of Polyunsaturated Phospholipid Remodeling in

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