Identification of Fatty Acids, Phospholipids, and Their Oxidation

In the Laboratory

Identification of Fatty Acids, Phospholipids, and Their Oxidation Products Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry and Electrospray Ionization Mass Spectrometry Christopher W. Harmon, Stephen A. Mang,† John Greaves, and Barbara J. Finlayson-Pitts* Department of Chemistry, University of California, Irvine, California 92697 *[email protected] † Current address: Department of Chemistry & Biochemistry, University of Maryland, Baltimore, MD 21250.

Ozone is ubiquitous in the atmosphere, existing in the lower atmosphere (troposphere) at a mixing ratio of approximately 30 ppb by volume in remote regions but at levels in excess of 100 ppb in polluted urban areas. Whereas ozone in the upper atmosphere (stratosphere) provides a shield for UV radiation received at the Earth's surface, tropospheric ozone is harmful. Exposure to tropospheric ozone has well-documented negative health effects on humans and animals, as well as on plants and materials (1), and as a result, it has been designated as a criteria pollutant by the U.S. Environmental Protection Agency. The National Ambient Air Quality Standard for ozone in the United States, which is aimed at protecting public health, is 75 ppb ozone for 8 h. The interaction of ozone with biological systems consists, in many cases, of reactions between ozone and endogenous unsaturated lipids. One molecule in the lungs that is a target for ozonolysis is POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), a common component of lipid bilayers, as well as the pulmonary surfactant that is found in the alveolar region of the lung where gas exchange occurs (2). A deficit of phospholipids in the lung is associated with hyaline membrane disease in newborns and acute respiratory distress syndrome in adults (e.g., resulting from exposure to hanta virus) (2-6). Unsaturated phospholipids such as POPC react with ozone via the Criegee mechanism (Scheme 1) (7, 8), resulting in scission of the hydrocarbon chain and the production of stable carboxylic acids and aldehydes or ketones. In the condensed phase, secondary ozonides (SOZ, see E1 and E2 in Scheme 1) are also typically formed. All three types of stable products may be sensitively and selectively detected using mass spectrometric techniques. Two analytical techniques to detect the ozone-reaction products are matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). These techniques are ideally suited for analyzing biomolecules because of their ability to handle high relative molecular masses (Mr), and undergraduate experiments have been designed that illustrate this capability (9-19). ESI-MS is particularly useful because of its ability to sensitively and quantitatively detect small amounts of analyte and also because of the ease with which it is coupled with liquid chromatography. 186

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Students also learn that ESI-MS can be readily used to analyze smaller molecular masses, whereas MALDI is mainly useful for molecular masses greater than 500 Da (although it can be used for small biomolecules when less common matrix materials such as porphyrins are used) (20). For undergraduate students interested in a career in the modern chemical and pharmaceutical industries, familiarity with these two instruments is rapidly becoming indispensable. We present an experiment designed to introduce mass spectrometry, in particular MALDI-MS and ESI-MS, to upper-division chemistry undergraduates and to illustrate the application of the techniques to the analysis of compounds commonly found in biological samples. In addition, the use of internal standards is demonstrated. This experiment is used for a 7 h laboratory period, although our experience is that it can be completed in 4-5 h. Details of the experimental procedures are found in the online supporting material. Hazards Hexane, methanol, and ethanol are flammable liquids and are irritating to the skin, eyes, and respiratory tract. Methanol may be fatal if swallowed. Oleic, linoleic, and stearic acids may be irritating to the skin, eyes, and respiratory tract. The toxicological properties of DPPC and POPC have not been fully investigated; they may cause eye, skin, respiratory, and digestive irritation. Gloves, goggles, and protective clothing should be worn at all times during the sample preparation step. Care should be taken to ensure that the gloves are not coated with a surfactant, as this type of contamination can significantly complicate a MALDIMS mass spectrum. Ozone is a known air pollutant capable of inducing adverse health effects in people even at relatively low exposure levels. If students smell ozone during the experiment they should turn off all ozone generators, notify the instructor immediately, and leave the laboratory until notified it is safe to return. Results Synthetic mixtures of the phospholipids or fatty acids are used for most of this experiment. An extract from soybeans

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Vol. 87 No. 2 February 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed800041v Published on Web 01/12/2010

In the Laboratory

prepared by the students is also used in the last part of the experiment and compared to the synthetic fatty acid mixture. Extraction of crushed soybeans is carried out using a 9:1 (v/v) hexane/ethanol mixture with sonication as described in detail in the supporting material. MALDI-MS A MALDI mass spectrum for a mixture of POPC (Mr = 760) and the saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC (Mr = 734), at a molar ratio (POPC/DPPC) of 1.2:1 is shown in Figure 1A. DPPC serves as an internal standard because it is not oxidized by ozone. DPPC is detected as the [M þ H]þ peak at m/z = 735 (A1) and also as the sodium adduct [M þ Na]þ at m/z = 757 (A2). The corresponding peaks for POPC are the [M þ H]þ peak at m/z = 761 (B1, Scheme 1) and Scheme 1. Ozonolysis of POPC and Resulting Products of Mr = 650, 666, and 808a

the [M þ Na]þ peak m/z = 783 (B2, Scheme 1), respectively. Small peaks at lower masses are due to trace contaminants resulting from oxidation during storage and handling. Ozone adds to the double bond of the oleoyl chain to form an unstable primary ozonide, which decomposes into an aldehyde and a Criegee intermediate (CI) (7, 8). As seen in Scheme 1, the CI rearranges to form a carboxylic acid. The -CHO and -COOH groups can be attached to either the phospholipid backbone (R1) or to the oleic acid moiety (R2); however, only those attached to the phospholipids are seen in this experiment because of the focus on higher molecular mass species (C1, C2, D1, D2, E1, and E2). In addition, the aldehyde and CI can recombine to form the secondary ozonide, SOZ (Scheme 1, E1 and E2). The mass spectra after oxidation with 1% ozone for 15 min is shown in Figure 1B. The peaks due to POPC are smaller compared to the fully saturated, and hence unreactive, DPPC. All of the expected high molecular mass products of POPC ozonolysis are detected: the phospholipid aldehyde is seen as the [M þ H]þ at m/z = 651 (C1) and as the [M þ Na]þ at m/z = 673 (C2). The CI forms a carboxylic acid, which is detected as the [M þ H]þ at m/z = 667 (D1) and as the [M þ Na]þ at m/z = 689 (D2), and the smaller peaks of the secondary ozonide can be seen as the [M þ H]þ at m/z = 809 (E1) and as the [M þ Na]þ at m/z = 831 (E2), respectively (the small peak at m/z = 700 was not reproducible and may be due to oxidation of an impurity in the POPC). Quantitative measurements of the product yields from the MALDI mass spectra are not possible owing to varying signal intensities from the highly variable matrix-to-analyte ratios across the sample (21-24). ESI-MS

a B1, C1, D1, and E1 are the [M þ H]þ adducts of these products, and B2, C2, D2, and E2 are the corresponding [M þ Na]þ adducts.

The ESI mass spectra confirm the results obtained from MALDI-MS. The positive-ion ESI mass spectrum of a mixture of DPPC [M þ Na]þ at m/z = 757 (A2) and POPC [M þ Na]þ at m/z = 783 (B2) at a molar ratio (POPC/DPPC) of 1.2:1 is shown in Figure 2A. The smaller peaks at m/z = 758 and 784 are due to the 13C isotopes. The positive-ion mode was chosen because the analytes contain nitrogen, which are more stable as cations and give a strong signal. A mass spectrum of this mixture after oxidation with ozone (Figure 2B) shows that the peak attributed to POPC (B2) has decreased to approximately 50% relative to DPPC (A2). The ESI mass spectrum of the oxidized sample in the product region is shown in Figure 2C. All products that are detected in MALDI are detected in ESI. The phospholipid aldehyde peaks appear at [M þ H]þ at m/z = 651 (C1) and

Figure 1. MALDI mass spectra of a mixture of the phospholipids POPC and DPPC before (A) and after (B) exposure to ozone. Peak labels are defined in the text: the A-labeled peaks are from DPPC and other labeled peaks are related to POPC.

r 2010 American Chemical Society and Division of Chemical Education, Inc.

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In the Laboratory

[M þ Na]þ at m/z = 673 (C2). These are dwarfed, however, by its sodiated methanol adduct [M þ CH3OH þ Na]þ at m/z = 705 (C3). The carboxylic acid product is seen predominantly as the sodium adduct ion [M þ Na]þ at m/z = 689 (D2). (The [M þ H]þ peaks are relatively stronger if 0.1% formic acid

Figure 2. ESI mass spectra (positive-ion mode) of a mixture of the phospholipids POPC and DPPC before (A) and after (B) exposure to ozone. Parts (C) and (D) show expanded regions where expected reaction products are observed.

Figure 3. Calibration relating measured amounts and experimentally determined peak heights of a mixture of POPC and DPPC. Peak heights were obtained via ESI-MS in positive-ion mode (y = 1.03x; R2 = 0.993).

is added when methanol or acetonitrile is used as the solvent). The SOZ peaks at m/z = 809 and 831 (E1 and E2, respectively) are shown in Figure 2D. The peaks located in the vicinity of m/z = 770 in Figures 2A and B are irreproducible impurities not detected in MALDI and are likely from ESI-MS sample preparation. The mixture of POPC and DPPC can be quantitatively analyzed using ESI-MS. DPPC serves as an internal standard because it does not react with ozone. The results for a typical calibration mixture prepared by the students (described in the experimental procedures in the supporting material) are shown in Figure 3. The filled circles in Figure 3 are data from standard mixtures in which increasing concentrations of POPC are combined with a fixed amount of DPPC. The open square in Figure 3 shows the peak height of a mixture that had an initial POPC:DPPC ratio equal to that of the most concentrated standard, but which had been exposed to 1% ozone for 15 min. The amount of POPC that reacted was calculated with a least-squares analysis, using a fit forced through the origin. The decrease in POPC relative to DPPC shows that 41% of the initial POPC has been reacted. The ionization efficiency in ESI-MS is compound dependent and can vary substantially based on the functional groups present in a molecule. However, because of the structural similarities of the products and reactants, differences in ionization efficiencies are expected to be small. Soybean Oil The goals of the next part of the experiment were to determine the most prevalent fatty acids in soybean oil and to demonstrate the application of ESI-MS to the analysis of the products of ozonolysis. Identification of the fatty acids in the soybean extract is based on matching the mass spectrum to that

Figure 5. Negative-ion mode ESI mass spectra of ozonolysis products derived from a synthetic mixture of linoleic, oleic, and stearic fatty acids after ozonolysis (see Table 1 for product identification).

Figure 4. Negative-ion mode ESI mass spectra of (A) soybean oil extracts; (B) a synthetic mixture of linoleic (I), oleic (II), and stearic (III) acids; and (C) the same synthetic mixture of linoleic, oleic, and stearic acids after ozonolysis.

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In the Laboratory Table 1. Relative Molecular Masses, Ozonolysis Products and Their Relative Molecular Masses, and the Ions Observed in ESI for a Synthetic Mixture of Oleic, Linoleic, and Stearic Acids Parent Compound (Mr)a

Ions in ESIb,c

Oleic acid:

281 (II)

CH3(CH2)7CHdCH(CH2)7COOH (282) Linoleic acid:

279 (I)

CH3(CH2)4CHdCHCH2CHdCH(CH2)7COOH (280) Stearic acid:

Ozonolysis Productc

Mr of Ozonolysis Product

Ion in ESI

CH3(CH2)7COOH (IV)

158

157

HOOC(CH2)7COOH (VI)

188

187

OHC(CH2)7COOH (V)

172

171

HOOC(CH2)7COOH (VI)

188

187

283 (III)

CH3(CH2)16COOH (284) a

Mr is the relative molecular mass. b Negative-ion mode. c Roman numeral corresponds to designation of peaks in Figures 4 and 5.

of a synthetic mixture of stearic, oleic, and linoleic acids. ESI-MS was then used to identify the products of reaction of the synthetic fatty-acid mixture with O3. The negative-ion mode ESI-MS of the soybean extract is shown in Figure 4A, and that of the synthetic fatty-acid mixture is shown in Figure 4B. The negative-ion mode was used because it is particularly sensitive to carboxylic acids. The [M - H]- peaks of linoleic acid (Mr = 280, I), oleic (Mr = 282, II), and stearic (Mr = 284, III) acids are clearly seen in both samples. The corresponding ESI-MS spectra of the same synthetic mixture after reaction with 1% O3 for 15 min is shown in Figure 4C. A dramatic reduction in the unsaturated fatty-acid components relative to the saturated and unreactive stearic acid is seen. The ESI-MS (negative-ion mode) in the m/z = 150-190 region for the synthetic fatty-acid mixture after ozonolysis is shown in Figure 5. Product peaks are observed at m/z = 157, 171, and 187. These are the [M - H]- ions of products IV, V, and VI (shown in Table 1) that are expected from ozonolysis of the double bonds in oleic and linoleic acids. The secondary ozonides from linoleic and oleic acids are not seen in the ESI mass spectra. Since secondary ozonides have been reported using ESI (25), it seems likely that in the present experiments they undergo secondary reactions under the high concentrations of ozone used and hence are not present in detectable amounts. Educational Value Students learn techniques that are relevant to the analysis of biological samples by mass spectrometry, including MALDI-MS and ESI-MS. They learn how to use these techniques for qualitative identification of species as well as quantitative measurement using internal standards. The amount of data analysis and interpretation may be tailored to the capabilities and backgrounds of the students. Students are also introduced to concepts in air pollution chemistry through this experiment; however, it should be noted that much higher concentrations of O3 are used in this experiment than found in air (10,000 ppm vs 0.1 ppm). This is necessary because the samples form a thick film on the walls of the vials and diffusion of O3 into the film is slow at low concentrations. The higher concentrations apparently speed up the oxidation sufficiently to disrupt the surface so that reaction of more of the bulk of the film to produce measurable amounts of products occurs. Instructors may wish to add questions to the laboratory experiment that explore air pollution chemistry and its impacts on biological systems in more detail. Literature Cited 1. U.S. E.P.A. Air Quality Criteria For Ozone and Related Photochemical Oxidants; U.S. Environmental Protection Agency: Washington, DC, 2006; Vols. 1-3, EPA/600/R-05/004aF-cF, 2006.


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Supporting Information Available Instructor notes and a student handout including details of the experimental procedures, extraction of crushed soybeans, and preparation of the calibration mixture. This material is available via the Internet at http://pubs.acs.org.


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Identification of Fatty Acids, Phospholipids, and Their Oxidation

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