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Annals of Clinical & Laboratory Science 37:213-221 (2007)
© 2007 Association of Clinical Scientists

Analysis of Human Serum Lipoprotein Lipid Composition Using MALDI-TOF Mass Spectrometry

Hiroya Hidaka1, Noboru Hanyu2, Mitsutoshi Sugano3, Kenji Kawasaki3, Kazuyoshi Yamauchi3 and Tsutomu Katsuyama4
1 Department of Biomedical Laboratory Science, School of Health Sciences, Shinshu University, Matsumoto; 2 Clinical Laboratories, Shinonoi General Hospital, Shinonoi; 3 Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto; and 4 Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Nagano Prefecture, Japan

Address correspondence to Hiroya Hidaka, Ph.D., Department of Biomedical Laboratory Science, School of Health Sciences, Shinshu University, Asahi 3-1-1, Matsumoto, Nagano, 390-8621, Japan; tel 81 263 37 2800; fax 81 263 34 5316; e-mail hiroyan{at}hsp.md.shinshu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
This study used matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify all lipid classes in human serum lipoproteins. After the major lipoproteins classes were isolated from serum by ultracentrifugation, the lipids were extracted and mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) dissolved in Folch’s solution (chloroform/methanol 2:1, v/v). MALDI-TOF MS analysis of the samples identified phospholipids (PLs), lysophospholipids (lysoPLs), sphingolipids (SLs), triglycerides (TGs), cholesteryl esters (CEs), and free cholesterol; it also showed the characteristics of individual fatty acid chains in serum lipids. MALDI-TOF MS allowed analysis of strongly hydrophobic and non-polar molecules such as CEs and TGs as well as hydrophilic molecules such as phospholipids. Direct analysis of fatty acids was not possible. The concentrations of lipids were not consistent with the ion peak intensities, since the extent of polarity affected the ionization characteristics of the molecules. However, lipid molecules with similar molecular structures but various fatty acid chains, such as phosphatidylcholine (PCs), were analyzed quantitatively by MALDI-TOF MS. Quantitative measurement of cholesterol was possible with the use of an internal standard. This study shows that MALDI-TOF MS can be used for direct investigation and quantitative analysis of the phospholipid composition of serum lipoproteins.

Keywords: MALDI-TOF mass spectrometry, lipoproteins, lipids, phospholipids, lysophospholipids


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Blood lipids are dynamic in terms of their interactions, efflux, and transfer between serum lipoproteins and peripheral cell membranes [1–8]. Phospholipids (PLs) are the main constituents of cell membranes and play an important role as second messengers in signal transduction [9,10]. PLs in lipoproteins are structurally classed as either glycerophosphatides or sphingolipids. Glycerophosphatides include phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), and lysophospholipids. Sphingolipids include mainly sphingomyelins (SMs), which are important membrane structure molecules. PLs contain a variety of hydrophilic bases and hydrophobic fatty acid residues.

Complexes containing cholesterol, glycerophospholipid, and sphingomyelin, such as rafts or caveoli, have been suggested as binding or metabolic sites of cell membranes [11,12]. The physical chemical property of a lipid molecule is affected by its fatty acid groups, which influence its physiological function [13,14]. The transfer and degradation of fatty acid groups of cell surface lipids can affect the function and metabolism of plasma lipoproteins, since lipid molecules are constantly exchanged between cell membranes and plasma lipoproteins.

Lipid analysis is traditionally based on chromatographic techniques such as thin layer and column chromatography [15–17]. High performance liquid chromatography (HPLC) is a useful method for analysis of phospholipid classes [18,19], while analysis of cholesterol, CEs, and TGs is often accomplished using gas chromatography [20]. Analyzing the fatty acid composition of lipids usually involves removing any esters, and the free fatty acid is analyzed with derivatization or methylation [21–23].

Lipidomics is an expanding research field involving the analysis of lipids, particularly those of human serum lipoproteins and cell membranes [24–30]. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) analysis of lipids is sensitive, relatively unaffected by impurities, and offers convenient sample preparation, making it an excellent analytical approach for rapid screening of lipid components in biological matrices [31]. While PL analysis by MALDI-TOF MS has been reported, there are few reports of such analysis of cholesterol, CEs, and TGs. In the present study, we analyzed the lipid composition of human serum lipoproteins using MALDI-TOF MS and we applied this technique for quantitative analyses of phospholipid fatty acyl chain composition and cholesterol.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Chemicals.  Reagents for centrifugal separation of lipoproteins (NaCl, NaBr, and EDTA-2Na) and lipid extraction (trifluoro-acetic acid, methanol, and chloroform) were purchased from Wako Pure Chemical Co., Osaka, Japan; 2,5-dihydroxybenzoic acid (2,5-DHB), cholesterol, and 4-cholesten-3-one were from Sigma Chemical Co., St. Louis, MO, USA.

Serum samples.  Fifty healthy male and female volunteers between 23 and 63 years of age were recruited from the University Hospital staff. Informed consent was obtained from each volunteer and the study protocol was approved by the Ethical Review Board of Shinshu University School of Medicine. All procedures were performed according to the Helsinki Declaration of 1975 as revised in 1996. Fresh blood samples, drawn after overnight fasting for ≥12 hr, were allowed to clot at room temperature. After centrifugation (3500 rpm, 10 min), serum samples were collected in plastic tubes. Pooled serum was prepared by mixing randomly selected serum samples.

Preparation of human lipoproteins.  Ultracentrifugation was performed using a Beckman TLA 100.3 rotor in an Optima TLX ultracentrifuge (Beckman/Coulter Corp., Fullerton, CA, USA). Human chylomicrons and their remnants (d <1.000) were isolated from fresh human serum. Briefly, distilled water (0.5 ml) was carefully laid over 0.5 ml of serum in a 1.5 ml tube and centrifuged at 20,000 rpm (15 min, 5°C). After ultracentrifugation, the top 0.4 ml and the bottom 0.4 ml were collected separately. The lipoproteins recovered in the top (water) fraction (d <1.000) were chylomicrons and their remnants. Very low density lipoproteins (VLDL: d <1.006), low density lipoproteins (LDL: d 1.006–1.063), and high density lipoproteins (HDL: d 1.063–1.21) were prepared from the bottom fraction (d >1.000 g/ml) using ultracentrifugation as described by Hatch [32]. After separation by ultracentrifugation, the lipoprotein fractions were dialyzed overnight in phosphate buffered saline (PBS) solution.

Extraction of lipids.  Lipid extraction from lipoproteins was performed according to Folch et al [33]. After the lower phase (chloroform) containing lipids was separated from the aqueous upper phase (water and methanol), the chloroform fraction was stored under N2 in the dark at 4°C.

Mass spectrometry.  Mass spectrometric measurements were performed using a MALDI-TOF MS instrument (Voyager Elite XL, PerSeptive Biosystems, Framingham, MA, USA). The system utilizes a pulsed nitrogen laser (emission, 337 nm; delay, 100 ns; accelerating voltage, 25 kV). The resolution of the ion peak is M/ M determined by the resolution calculator using the GRAMS/386 software supplied with the instrument. In order to enhance spectral resolution, the device was used in reflector mode, so that the total field-free time-of-flight distance was 3.0 m. In the positive ion mode with 2,5-DHB as the matrix, angiotensin I ([M+H]+: 1296.7) and des-Arg-bradykinin ([M+H]+: 904.5) were used for calibration of the instrument. To 1.0 µl of lipid solution in a 0.4 ml micro-glass tube, 1.0 µl of matrix solution (10 mg of 2,5-DHB in 1 ml of chloroform/methanol, 2/1 v/v) was added, and then applied to a metal sample plate for MS analysis. After the sample had dried, the metal plate was inserted into the MALDI-TOF MS analyzer. The lipid mass was measured at approximately 2000 absorption laser intensity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Mass spectra of pure 2,5-DHB matrix.  The matrix used for analysis of lipoprotein lipid extracts by MALDI-TOF MS was 2,5-DHB. The positive ion laser desorption mass spectra resulting from analysis of 1 µl of the solution of 10 mg/ml 2,5-DHB matrix in Folch’s solution (chloroform/methanol, 2:1, v/v) are shown in Fig. 1Go. The major mass spectrum peaks were at m/z 154.1 (M: neutral material of 2,5-DHB); 155.2 (M+H)+; 177.1 (M+Na)+; 184.2, 273.2 (2M-2H2O+H)+; 375.1 (2M-2H++3Na)+; 413.1; and 551.2 (3M-3H++4Na)+. The peak at m/z 551.2 was detected as the origin peak of the matrix even in the lipid analysis pattern described below.


Figure 1
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  		Fig. 1. Positive ion laser desorption mass spectra of 2,5-DHB. Insert table is an assignment of detectable molecular ions derived from the matrix. One µl of the solution of 2,5-DHB in C/M (2:1 v/v) was loaded on a sample plate with 100 sample positions. The plate was loaded into the mass spectrometer and the mass spectrum of 2,5-DHB was acquired by the instrumental software with a N2 laser (337 nm) in the reflector mode.

 
Using control serum lipids, we also examined the major mass peaks of lipoprotein lipids. Lipids were mixed with 2,5-DHB in C/M (2:1 v/v) and subjected to MALDI-TOF MS. Peaks from free cholesterol at m/z 369.6 (M-H2O+H)+, lysophosphatidyl choline at m/z 496.6 and 524.7, phosphatidyl choline at m/z 725.9, 758.9, 782.9, 809.0, 833.0, and 2,5-DHB were detected (Fig. 2Go).


Figure 2
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  		Fig. 2. Positive ion laser desorption mass spectra of the control serum lipids with 2,5-DHB in C/M (2:1 v/v) solution. The peaks with asterisks are derived from molecules of 2,5-DHB.

 
Optimization of MALDI-TOF MS conditions.  Since lipoprotein lipids interact differently with different organic solvents, we explored the optimal conditions for lipoprotein lipid analysis by MALDI-TOF MS. Lipid extracts from the lipid control serum were prepared using the modified Folch’s method, and then dissolved in chloroform and methanol. Three different matrices were tested; 10 mg/ml 2,5-DHB in either Folch’s solution (C/M, 2:1 v/v), chloroform/methanol/water (C/M/W, 4:2:0.3 v/v), or 0.1% TFA in methanol (0.1% TFA/ M). Extracts were mixed with an equal volume of matrix solution, and 1 µl of the mixture was applied to the sample plate. We found that the major PL mass spectral peaks were similar in both C/M and C/M/W at m/z 759, 781, 809, and 833. In contrast, the cholesterol mass intensity peak ([M-H2O+H]+, at m/z 369) in C/M was approximately twice that observed in C/M/W. In 0.1% TFA/M, the mass intensity of cholesterol and PLs was low in comparison to C/M or C/M/W (Fig. 3AGo). Based on these data, in subsequent experiments we used C/M (2:1 v/v) as the matrix solvent. The matrix was mixed with the sample at a 1:1 (v/v) ratio, and 1 µl of this mixture was applied to the mass spectrometer sample plate. Further studies on MALDI-TOF MS optimization showed that altering the laser intensity between 2000 and 2200 had little effect on PL mass intensities, whereas it dramatically increased the cholesterol mass intensity (Fig. 3BGo). Altering the laser shot frequency on the sample plate had little effect on the mass intensity of any lipid species (Fig. 3CGo).


Figure 3
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  		Fig. 3. The effect of sample solvent, laser shot frequency, and laser intensity on absorption intensity. Panel A: Lipoprotein lipid extracts were prepared in C/M (2:1 v/v) solution, and mixed at a ratio of 1:1 with 2,5-DHB in chloroform/methanol/water (C/M/W, 4:2:0.3 v/v)(open bars), chloroform/methanol (C/M, 2:1 v/v)(closed bars), or 0.1%TFA in methanol (0.1%TFA/M)(shadow bars). The mixtures then underwent MALDI TOF MS analysis. Panel B: Samples were prepared using a 2,5-DHB/C/M matrix, and then subjected to various laser intensities. Panel C: Samples were prepared using a 2,5-DHB/C/M matrix, and then subjected to various frequencies of laser shots. Significance of the peak intensity difference from the baseline was determined by t-test: * p <0.05: ** p <0.01.

 
Quantitative determination of cholesterol.  While the ionization peak intensity ratio of PLs remained virtually constant, the cholesterol peaks varied with the strength of laser radiation. For quantitative determination of cholesterol, 4-cholesten-3-one was chosen as the internal standard. Cholesterol (1 mg/ml) and 4-cholesten-3-one (1 mg/ml) dissolved in C/M (2:1 v/v) solution were mixed at various ratios, and then 1 µl of mixture was applied to the mass spectrometer sample plate. The relationship of the peak intensity vs the concentration ratio of cholesterol to 4-cholesten-3-one was curvilinear (Fig. 4Go). By this approach it was possible to measure cholesterol levels quantitatively using MALDI-TOF MS.


Figure 4
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  		Fig. 4. Calibration curve of peak intensity ratio vs cholesterol/4-cholesten-3-one concentration ratio. Cholesterol (1 mg/ml) and 4-cholesten-3-one (1 mg/ml) as an internal standard dissolved in C/M (2:1 v/v) solution were mixed at the various ratios (x-axis), and then 1 µl of each mixture was applied to the mass spectrometer sample plate. The mixtures underwent MALDI TOF MS analysis. The y-axis shows the ratio of peak intensity of mass in cholesterol and 4-cholesten-3-one.

 
Mass spectra of plasma lipoproteins.  Lipoprotein lipids extracted in C/M (2:1 v/v) solution were analyzed using MALDI-TOF MS with 2.5-DHB in C/M (2:1 v/v) solution as the matrix. MALDI-TOF MS showed that CM-remnant lipids were mainly TGs at m/z 854.1, 882.1, and 906.3 and free cholesterol at m/z 369.6 (M-H2O+H)+, with a small amount of PL at m/z 782.9 corresponding to either PC 16:0/20:4 [+H]+ or PC16:0/18:1 [+Na]+ (Fig. 5AGo). VLDL lipids were mostly cholesterol at m/z 369.5, PLs at m/z 669.7, 725.9, 759.0, 781.0, 809.0, and 829.0, and TGs at m/z 854.2, 882.2, and 906.3. The peaks at m/z 669.7 and 725.9 corresponded to sphingomyelin (Fig. 5BGo). LDL lipids were mainly PLs and cholesterol, with low levels of TGs and lysoPLs. Also detected were glycerophospholipids at m/z 759.0, 781.0, 809.1, and 829.0, SM at m/z 704.0 and 726.0, cholesteryl ester at m/z 672.0 (CE C18:2), and triglyceride at m/z 880.3 (Fig. 5CGo). Other than differences in the phospholipid mass peak intensities, the HDL mass spectrum was similar to that for LDL (Fig. 5DGo). The molar mass range of lipoprotein lipids is approximately between m/z 360 – 910.


Figure 5
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  		Fig. 5. Positive ion laser desorption mass spectra of lipids extract from human serum lipoproteins with 2,5-DHB in C/M (2:1 v/v) solution. Lipoprotein lipid extracts were prepared in C/M (2:1 v/v) solution, mixed at a ratio of 1:1 with 2,5-DHB in C/M (2:1 v/v) solution, and analyzed using MALDI TOF MS. Panel A: chylomicrons; panel B: VLDL; panel C: LDL; and panel D: HDL. The complete assignment of all detected molecular ions is listed in Table 1Go.

 
Assignments of the matrix and the lipids.  The assignment of all peaks detectable in the positive mass spectra of lipids from human serum lipoproteins is listed in Table 1Go. The lipid molecular mass spectrum ranged from m/z 369 to m/z 906 (m/z), while the matrix ranged from m/z 154 to m/z 551. The peaks of lipoprotein lipid, the lysophospholipid mass spectrum ranging from m/z 496.6 to m/z 542.3, did not overlap with the peaks of the matrix. Protonated, Na+-associated, and dehydrated forms of the molecules were detected by MALDI-TOF MS.


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  		Table 1. Assignment of the lipids molecular ions detectable in lipid extract from human serum lipoprotein
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
This study investigated the usefulness of MALDI-TOF MS for analyzing the lipid composition of human serum lipoproteins. Using lipid extracts of lipoproteins with 2,5-DHB as the matrix, MALDI-TOF MS analysis was able to identify different lipid classes with various fatty acid chains, such as PLs, lysoPLs, sphingolipids (SLs), TGs, CEs, and free cholesterol. The ratio of molecular species showing differences in the fatty acid chain of PL was quantitatively analyzed by MALDI-TOF MS. Furthermore, it was possible to measure cholesterol quantitatively with an internal standard.

Lipid analysis by MALDI-TOF MS requires only a small amount of sample, involves simple and rapid sample preparation, and is able to analyze various lipid molecules simultaneously. Molecular species of phospholipids and fatty acid chains in phospholipid molecules are analyzed directly with the extraction of lipids. Previously, the investigation of fatty acid chains in lipid molecules generally involved separating lipid molecules using thin layer chromatography (TLC) [15–17] or column chromatography. These are established methods, but the analytical processes are not only complicated and time-consuming, but require comparatively large amounts of sample and organic solvents. Furthermore, the analysis of fatty acid chain in the lipid molecule involves the acidic methylation of lipids for gas chromatography [20] or fluorescence labeling of lipids for high performance liquid chromatography [21–25]. Mass spectrometry has advantages over such methods since isolation of molecular species of PLs and identification of fatty acid chains are performed directly from extracted lipids. Furthermore, the MALDI-TOF MS analysis allows rapid measurement, high mass resolution, and sensitivity for lipid molecular species [31].

Previously, some ionization methods, including electron impact [34], plasma desorption [35], chemical ionization [36], and fast atom bombardment [26] have been applied for mass spectrometric analysis of lipids. These methods have respective advantages and disadvantages. The coupling of mass spectrometry with gas chromatography (GC/ MS) is an important and essential analytical tool in lipid research, especially for non-polar lipids such as cholesterol ester or triglyceride, but it is difficult to analyze the fatty acid chains in CEs and TGs by GC-MS directly [37]. The lipid sample has to be hydrolyzed prior to analysis and the resulting free fatty acids have to be converted into corresponding trimethyl silyl or methyl esters to enhance their volatility. MALDI-TOF MS gives high sensitivity and resolution of molecular mass as with the above methods, and the fatty acid chains in lipid molecules can be analyzed directly from lipid extracts in Folch’s solution. The operation of the MALDI-TOF MS instrument is also quick and convenient.

Peaks of ionized PLs, lyso-PLs, and SMs were detected by MALDI-TOF MS as a result of the additions of hydrogen [H]+ or sodium [Na]+ ions, as described previously [31,38]. While cholesterol was not detected in either of these ionized forms, it was detected in a dehydrated form with [H]+ addition ([-H2O+H]+) [39]. CEs were detected with [Na]+ addition. The mass ion peak intensities for cholesterol and CEs did not reflect their proportions in serum. Therefore, the ionization of cholesterol was increased by the stimulated emission of laser beam energy of MALDI-TOF MS. The peak intensity of molecule classes with different polarities in the same mass spectrogram was not comparable to their concentrations in the lipid extract. Polar lipids such as PLs were analyzed with high sensitivity by MALDI-TOF MS, but CE and TG levels of ionization showed lower sensitivity than PLs due to their strong hydrophobicity and non-polarity. Further study of optimal conditions for quantitative analysis of the fatty acid composition of CE and TG may be required.

We anticipated that reproducibility and quantitative determinations might be unsatisfactory using MALDI-TOF MS. However, uniform sample preparation and laser beam irradiation for 50–100 shots in a random manner resulted in reproducible and quantifiable peaks. The concentrations of molecules with different polarities were inconsistent with the intensity of the peaks in the same mass spectrogram since molecule polarity affected laser absorption by TOF-MS. However, we found that use of an internal standard allowed quantitative determination of lipid molecular masses by MALDI-TOF MS. Molecular species of similar polarity such as cholesterol and 4-cholesten-3-one (internal standard) could be measured quantitatively in the same mass spectrogram. It is difficult to compare different molecular species quantitatively, but MALDI-TOF MS is an effective method of analyzing the variation of fatty acid compositions in molecular structures such as glycerophospholipids.

Fatty acid chains of phospholipids were similar in VLDL, LDL, and HDL. This similarity can be attributed to the production of LDL by hydrolysis of VLDL with LPL, and phospholipid distribution in the lipoprotein membrane by intermolecular transfer from HDL to VLDL/LDL with phosphatide transfer protein. It is thought that PL in chylomicrons and their remnants is affected by the alimentary PL composition, because the chylomicrons and their remnants are metabolized by a course unlike that of VLDL/LDL. Thus, the analysis of variations in fatty acid composition in PL may be useful to account for the actions of the hydrolysis enzyme and the transfer protein in lipoproteins.

Phospholipids play important roles as lipids constituting the neurilemma cell membrane and plasma lipoproteins, and additionally in the mobilization of cholesterol from the cell membrane and the control of genetic functions and protein metabolism [9,10]. Metabolites of PLs function as lipid mediators, controlling apoptosis, differentiation, growth, and cellular motility. Saturated and unsaturated fatty acids that are able to dissociate from PLs have important functions as precursors of bioactive molecules such as prostaglandins, in the reconstitution of lipids and energy materials, and in the activity of molecules like adipocytocaine. Analysis of fatty acid chains in plasma PLs may offer important information regarding fatty acid metabolism or the absorption mechanism of PLs from the intestine to the intravascular compartment. The analysis may be applicable to nutritional management and to elucidating pathophysiologic mechanisms and diagnosis of conditions such as arteriosclerosis and metabolic syndromes.

The current study shows that MALDI-TOF MS allows analysis of lipid molecules with strong hydrophobicity and non-polarity, such as cholesterol, CEs, and TGs. In addition, MALDI-TOF MS analysis allows simultaneous detection of multiple hydrophilic molecule types, such as phospholipids. The direct analysis of fatty acids has not yet been achieved. MALDI-TOF MS appears to be useful for screening and quantitative determination of lipid classes from lipoproteins, liposomes, and lipid extracts of cells and tissues. In addition, MALDI-TOF MS analysis appears capable of providing detailed information regarding lipid composition through the use of internal standards.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Emeritus Professor Tamotsu Taketomi of Shinshu University for valuable advice and comments. This study was supported in part by a grant-in-aid for exploratory research (#18659159) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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