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The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31P NMR
Chemistry and Physics of Lipids 163 (2010) 157–164
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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31 P NMR Dirk Dannenberger a , Rosmarie Süß b , Kristin Teuber b , Beate Fuchs b , Karin Nuernberg a , Jürgen Schiller b,∗ a b
Research Institute for Biology of Farm Animals, Research Unit of Muscle Biology and Growth, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Härtelstr. 16-18, D-04107 Leipzig, Germany
a r t i c l e
i n f o
Article history: Received 4 July 2009 Received in revised form 23 October 2009 Accepted 29 October 2009 Available online 10 November 2009 Keywords: Beef Phospholipids Lipids MALDI-TOF MS Grass and maize silage 31 P NMR TLC
a b s t r a c t The analysis of beef lipids is normally based on chromatographic techniques and/or gas chromatography in combination with mass spectrometry (GC/MS). Modern techniques of soft-ionization MS were so far scarcely used to investigate the intact lipids in muscle tissues of beef. The objective of the study was to investigate whether matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef. For the MALDI-TOF MS and 31 P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. Beside the triacylglycerols (TAGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) species the MALDI-TOF mass spectra of total muscle lipids gave also intense signals of cardiolipin (CL) species. The application of different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), leads to completely different mass spectra: 9-AA is particularly useful for the detection of (polar) phospholipids, whereas apolar lipids, such as cholesterol and triacylglycerols, are exclusively detected if DHB is used. Finally, the quality of the negative ion mass spectra is much higher if 9-AA is used. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Fat in cattle is present as membrane lipids (phospholipids), intermuscular fat (between the muscles), intramuscular fat (IMF), abdominal and subcutaneous fat. The fatty acyl compositions of adipose and muscle tissues in beef are affected by factors such as diet, species, fatness, age/weight, depot site, gender, breed, season and hormones. The most effective means of manipulating the related lipids and their fatty acyl profiles in muscle and adipose tissues of beef can be achieved through nutrition by strategic use of forage and dietary lipids (Nuernberg et al., 2005; Scollan et al., 2006; Wood et al., 2008). Intramuscular fat of beef mainly consists of triacylglycerols (TAGs) and phospholipids (PLs). TAGs serve as energy stores and are deposited in adipocytes. PLs are amphiphilic molecules with lipophilic acyl chains and a hydrophilic head and (in addition to cholesterol) abundant components of biological membranes. In beef muscle the presence of nine different phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), cardiolipin (CL), sphingomyelin (SM),
∗ Corresponding author. Tel.: +49 341 9715733; fax: +49 341 9715709. E-mail address: [email protected] (J. Schiller). 0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2009.10.011
phosphatidylserine (PS), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), and phosphatidic acid (PA) has been described (Yeo and Horrocks, 1988; Larick and Turner, 1989; Dannenberger et al., 2007). PC and PE are the most abundant constituents and SM, PI and CL are minor species in intramuscular beef lipids, and the fatty acyl profiles of the individual PL classes can be affected by different feeding regimes (Dannenberger et al., 2007). Beside diacyl PL species, there are also significant amounts of alkenyl-acyl and alkyl-acyl species, i.e. ether-linked compounds. However, studies on alkenylacyl species (plasmalogens) and the fatty aldehyde composition of beef lipids have been only sparsely described so far (Dannenberger et al., 2006). Nevertheless, it is known that plasmalogen compounds are particularly abundant in the PE subclass of muscle phospholipids. The focus regarding beef lipid composition was so far on the analysis of the fatty acyl composition. The traditional compositional analysis of beef lipids is based on chromatographic techniques (Caboni et al., 1994) as well as gas chromatography in combination with mass spectrometry (GC/MS) (Kim and Salem, 1993). Although nowadays highly established, modern techniques of softionization MS were so far only scarcely used to investigate the intact lipids (particularly TAGs) in muscle tissues of beef (Picariello et al., 2007).
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Due to the simple performance, the high sensitivity and the robustness against impurities, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) seems particularly suitable for that purpose. Although the quantitative information achievable from MALDI mass spectra is often assumed to be limited, it has been recently convincingly shown by using human blood plasma extracts that MALDI is a quantitative technique and the data obtained by MALDI agree favourably with data obtained by electrospray ionization (ESI) MS (Hidaka et al., 2007). Unfortunately, the different detectabilities of the individual lipid classes represent a major problem if complex lipid mixtures are analyzed because sensitively detectable lipids may suppress the less sensitively detectable ones (Schiller et al., 2007a; Petkovic´ et al., 2001; Gellermann et al., 2006). The sensitivity to detect a certain compound is also significantly influenced by the composition of the “matrix”, i.e. the presence or absence of other compounds. This normally makes previous separation of the total extract into the individual lipid classes necessary and there are nowadays (in addition to liquid chromatography) (LC/MS) methods available that allow the direct combination of thin-layer chromatography (TLC) (Touchstone, 1995) with MALDI MS (Fuchs et al., 2007a). Nevertheless, the careful selection of the matrix may already help to overcome some problems, making MALDI suitable for the investigation of complex lipid mixtures. 2,5-Dihydroxybenzoic acid (DHB) is unequivocally one of the most established UV matrices in the lipid field because it gives (in contrast to many other matrices, such as cinnamic acid derivates) only a rather moderate background in the lower mass region (Schiller et al., 1999). Due to its acidic properties, DHB is particularly useful as matrix for positive ion detection. Under these conditions, however, PC is most sensitively detected (Schiller et al., 2007a), whereas other lipids such as PE or PI are nearly completely suppressed – even as negative ions (Petkovic´ et al., 2001), i.e. the positive and the negative ion detection mode are not independent. Additionally, negative ion spectra obtained with DHB are dominated by signals of the matrix itself that may interfere with the mass regions of PL (Schiller et al., 2007b). It is reasonable to use less acidic matrices for the detection of negative ions. Some years before it was shown that paranitroaniline (PNA) is an excellent matrix for the detection of e.g. PE as negative ion even in the presence of significant amounts of PC (Estrada and Yappert, 2004). Unfortunately, however, the vacuum stability of this matrix is limited and, thus, spectra show time-dependent changes. Very recently, 9-aminoacridine (9-AA) was suggested as a powerful alternative to PNA: 9-AA was shown to detect lipids more sensitively than other matrices and to provide only a minimum background (Sun et al., 2008). However, although 9-AA was already tested with artificial and cellular lipids, this matrix was to our best knowledge not yet applied to the analysis of beef. The objective of this study is to investigate whether (a) matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef and (b) if 9-AA is the matrix of choice to detect negatively charged lipids, such as PI or CL in beef muscle lipids.
2. Materials and methods 2.1. Cattle experiment For the MALDI-TOF MS and 31 P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. The details of the diet experiment were very recently described (Mahecha et al., 2009). Briefly, 25 male German Simmental calves (3–4 months) were included
in an indoor experiment. They were randomly assigned into three groups with different feeding regimes. The control group (n = 9) was fed daily maize silage/grass silage (70/30, ad libitum), 1 kg of molasses, 1 kg of hay and concentrate including soybean. Treatment group I, conformed by unrestricted animals (n = 7) was fed grass silage (ad libitum), 1 kg of molasses, 1 kg of hay and concentrate including rapeseed in the same proportion as the control group. Treatment group II, conformed by restricted animals (n = 9), fed as treatment group I under a restriction of 1 kg of concentrate (50%) per day during the first 112 days of the fattening period. All bulls were slaughtered approximately at 635 kg live weight in the abattoir of the Research Institute for the Biology of Farm Animals in Dummerstorf (Germany). Longissimus muscle samples were isolated immediately after slaughter. Samples were taken at the 6th–13th rib of the left carcass side and were kept at −80 ◦ C until their analysis. Longissimus muscle samples from the control group and the treatment group I were selected in the present study. 2.2. Extraction of the muscle lipids The total lipids of 2 g muscle were extracted with 15 mL CHCl3 /CH3 OH (2:1, v/v) according to Folch et al. (1957) by homogenization (Ultra Turrax, 3 × 15 s, 12,000 rotations per minute) at room temperature. The solvent was evaporated and the amount of the extracted material was determined by weighing. CHCl3 /CH3 OH (2:1, v/v) was used to re-dissolve the isolated lipids. The details were described recently by Nuernberg et al. (2002). Solutions with a total lipid content of 2 or 10 mg/ml were prepared and used for subsequent analysis. 2.3. Reagents All phospholipid standards were obtained from AVANTI Polar Lipids Inc. (Alabaster, AL, USA) and were used as supplied. All chemicals and salts for sample preparation, MALDI-TOF MS (2,5-dihydroxybenzoic acid (DHB)) as well as all solvents for analysis (chloroform, methanol, isopropanol and acetonitrile) were obtained in highest commercially available purity from Fluka Feinchemikalien GmbH (part of Sigma–Aldrich Chemie GmbH, Taufkirchen) and used as supplied. 9-Aminoacridine hemihydrate was purchased from Acros Organics (Morris Plains, NJ). 2.4. Thin-layer chromatography Silage extracts were subjected to TLC separation prior to MALDITOF MS. 1 l lipid samples per spot were applied on HPTLC silica gel 60 plates (20 cm × 20 cm in size) (Merck, Darmstadt, Germany) and developed in TLC chambers (CAMAG, Switzerland) using chloroform, ethanol, water and triethylamine (30:35:7:35, v/v/v/v) as elution solvent in order to separate phospholipids. Chloroform/acetone (96:4, v/v) was used to separate triacylglycerols from the plant dyes. Lipids were visualized by spraying with a solution of PRIMULINE (Direct Yellow 59) according to White et al. (1998). Upon irradiation by UV light (366 nm), individual PLs become detectable as yellow spots. These spots were quantitatively assessed by using a digital image system in combination with the program Argus X1 delivered by BioStep (Jahnsdorf, Germany). Further details of combining MALDI-TOF MS directly with TLC are available in Fuchs et al. (2008a). 2.5. MALDI-TOF mass spectrometry Total lipid extracts and individual lipid fractions were independently investigated by MALDI-TOF MS. A 0.5 mol/l 2,5dihydroxybenzoic acid (DHB) solution in methanol (Schiller et al.,
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1999) or 10 mg/ml 9-aminoacridine (in isopropanol/acetonitrile (60/40, v/v)) was used in all cases (Sun et al., 2008). As the quality of the spectra recorded in the presence of 9-AA depends significantly on the applied solvent system, all lipids were diluted with isopropanol/acetonitrile (60/40, v/v) (Sun et al., 2008). This solvent mixture gave among all tested solvent systems (methanol, acetonitrile, etc.) the by far best results and this is in agreement with previous data (Sun et al., 2008). All samples were pre-mixed with the matrix prior to deposition onto the MALDI target and PC 13:0/13:0 (60 g/ml, final concentration) and PG 16:0/18:1 (4 g/ml, final concentration) were both added as reference compounds for the positive and negative ion mode, respectively. All MALDI-TOF mass spectra were acquired on a Bruker Autoflex mass spectrometer (Bruker Daltonics, Bremen, Germany). The system utilizes a pulsed nitrogen laser, emitting at 337 nm. The extraction voltage was 20 kV and gated matrix suppression was applied to prevent the saturation of the detector by matrix ions (Petkovic´ et al., 2001). 128 single laser shots were averaged for each mass spectrum. The laser fluence was kept about 10% above threshold to obtain optimum signal-to-noise ratios. In order to enhance the spectral resolution all spectra were acquired in the reflector mode using delayed extraction conditions. Under these conditions, resolutions of about 6000–8000 can be obtained with good reproducibility. Although this is low in comparison with the resolution indicated by the supplier of the device (ca. 20,000), it is absolutely sufficient to obtain isotopically resolved mass spectra of lipid samples. A more detailed methodological description of MALDI-TOF MS is given in Fuchs et al. (2008b) and Schiller et al. (2004). In the PSD (post source decay) experiments, the precursor ions of interest were isolated using a timed ion selector. The laser intensities for PSD spectra were maintained the same as in the reflector mode. The fragment ions were refocused onto the detector by stepping the voltage applied to the reflectron in appropriate increments. This can be done automatically by using the “FAST” (fragment analysis and structural TOF) subroutine of the Flex Control Program delivered by Bruker Daltonics. Further details are available in Fuchs et al. (2007b). 2.6.
31 P
NMR spectroscopy
In order to avoid the considerable line-widths of lipid aggregates, the dried organic extracts were solubilized in 50 mM TRIS (pH 7.65) containing 200 mM sodium cholate and 5 mM EDTA. After intense vortexing, 31 P NMR spectra were recorded on 0.5 ml samples in 5 mm NMR tubes on a Bruker DRX-600 spectrometer operating at 242.88 MHz for 31 P. All measurements were performed using a selective 31 P/1 H NMR probe at 37 ◦ C with composite pulse decoupling (Waltz-16) to eliminate 31 P–1 H coupling. Pulse intervals of the order of T1 were used to allow quantitative analysis of phospholipid integral intensities (Schiller et al., 2007c). Other NMR parameters were as follows: acquisition time 1 s, data size 8–16k, 60◦ pulse, pulse delay 2 s and a line-broadening (LB) of 1 Hz. Chemical shifts were referenced to the resonance of di-lauroyl-phosphatidic acid that was added as concentration and frequency standard. Further details are available in (Schiller et al., 2007c). Spectra were processed using the software “1D WINNMR” version 6.2® (Bruker Analytische Messtechnik GmbH, Rheinstetten) including the deconvolution (II) routine for peak area determination. 3. Results and discussion MALDI-TOF MS is increasingly used for – intact – lipid analysis because this method exhibits – beside the possibility to record spa-
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Fig. 1. Structures of the MALDI matrices 9-aminoacridine (9-AA) and 2,5dihydroxybenzoic acid (DHB) that were both used in this study. Please note that 9-AA is (due to its basic properties) the matrix of choice to detect negatively charged lipids, whereas DHB is more suitable for positive ion detection and particularly for apolar lipids such as in TAGs that are not detectable in the presence of 9-AA.
tially resolved spectra (“MALDI imaging”) – several advantages over other MS techniques. MALDI-TOF MS is fast, sensitive and tolerates impurities of the sample (salts, etc.) to a higher extent than e.g. electrospray ionization (ESI) MS that may be regarded as an alternative method (Pulfer and Murphy, 2003). In contrast, however, MALDI MS necessitates a “matrix” that enables the absorption of the energy emitted by the laser and must, thus, absorb at the laser wavelength (often 337 nm). Although there are also inorganic matrices available (e.g. graphite) (Fuchs and Schiller, 2009a), such matrices are not widely applied so far, and small organic molecules are still primarily used as matrices. DHB (Fig. 1) is one of the most frequently used matrix compounds (Schiller et al., 2004). Although the mass of this molecule (154 g/mol) is much smaller than the masses of typical PLs (about 700–900 g/mol), photoreactions may occur under the influence of the laser irradiation leading to the generation of a multitude of matrix peaks that were recently discussed in more detail (Schiller et al., 2007b). These “background” signals may interfere with the signals of the analytes of interest particularly if diluted samples are investigated and, even more severely, may decrease the sensitivity of detection. Therefore, the search for matrices with superior properties is still going on. Recently, 9-aminoacridine (9-AA), alone or in combination with other matrices has been shown to provide only a very moderate background and is, therefore, the matrix of choice for the detection of smaller compounds (Guo and He, 2007). Additionally, it was shown very recently (Sun et al., 2008) that 9AA is an excellent matrix for the analysis of the PL composition of biological extracts and exhibits three remarkable characteristics: (a) due to the basicity of 9-AA (cf. Fig. 1), the detection of negatively charged lipids is significantly improved in comparison to acidic matrices such as DHB and (b) using suitable solvent mixtures (especially acetonitrile and isopropanol) and a sufficient excess of matrix, H+ adducts are much more preferentially generated than alkali metal adducts (Sun et al., 2008). This is a clear advantage in the case of lipid mixtures because the superposition of different adducts, on one hand, and different acyl compositions, on the other hand, would otherwise complicate the assignments of the individual peaks (Schiller et al., 2001). Finally (c) the achievable sensitivity with 9-AA is much higher in comparison to DHB (Sun et al., 2008). Therefore, 9-AA might represent the most appropriate matrix to investigate the intact muscle lipids of beef. However, to our best knowledge such experiments were not yet performed. In Fig. 2 some selected positive ion MALDI-TOF mass spectra are compared. Spectra shown in traces (a and b) and (c and d) represent the muscle lipids of beef from two different animals fed with grass silage (treatment group I) and maize silage/grass silage (70/30, control group), respectively, but were recorded with 9-AA (a and c) or DHB (b and d) as MALDI matrix. Spectra recorded in the presence of DHB (b and d) are characterized by a higher number of peaks and, accordingly, some lipid classes are obviously exclusively detected in the presence of DHB, but not in the presence of 9-AA. For instance, TAGs and cholesterol are exclusively detected in the presence of DHB. This concerns TAG 54:2
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Fig. 2. Positive ion MALDI-TOF mass spectra of selected muscle lipid extracts of German Simmental bulls fed with grass silage (a and b) and with maize silage/grass silage (70/30) (c and d). Spectra in (a) and (c) were recorded in the presence of 9-AA, whereas DHB served as matrix in (b) and (d). The total lipid concentration was 2 mg/ml in all cases. Please note that the signals at m/z = 650.5 and 672.5 (marked with the dotted line) correspond to PC 13:0/13:0 that was added as internal standard (60 g/ml, final concentration). All samples were mixed 1:1 (v/v) with either DHB or 9-AA. All peaks are labeled according to their m/z ratios. Please note the complete disappearance of peaks of apolar lipids, such as triacylglycerols, if 9-AA is used as matrix (a and c). For details see text. Peaks marked with asterisks are stemming from the used DHB matrix. The small peaks between m/z = 612 and 633 cannot be assigned so far.
(m/z = 909.8), TAG 52:2 (m/z = 881.8), TAG 52:1 (m/z = 883.8), TAG 50:1 (m/z = 855.8) and TAG 48:1 (m/z = 827.7) as well as cholesterol (that is only detectable subsequent to water elimination but not as the intact molecule) at m/z = 369.3 (Schiller et al., 2000). TAGderived fragment ions (between m/z = 540 and 610; cf. Table 1 for detailed assignments) are accordingly also exclusively detected in the presence of DHB (Fig. 2b and d). Therefore, apolar lipids cannot be detected by MALDI-TOF MS if 9-AA is used as matrix. This may be regarded, on one hand, as a disadvantage because some potentially valuable pieces of information are lost, whereas, on the other hand, spectra can be significantly simplified in the presence of 9-AA. In contrast to the TAGs, PLs are detectable equally which matrix is used. Different phosphatidylcholine (PC) species are obvious between about m/z = 700 and 800 and more detailed peak assignments are provided in Table 1. The peaks at m/z = 650.5 and 672.5 (marked by the vertical dotted line) correspond to the H+ and Na+ adduct, respectively, of PC 13:0/13:0 that was artificially added to all samples as concentration standard. Considering the intensities of the H+ and Na+ adducts of the standard in dependence on the applied matrix, it is evident that the H+ adduct (m/z = 650.5) is more intense in the presence of 9-AA and the Na+ adduct (m/z = 672.5) in the presence of DHB. Thus, the intensities of both peaks have to be combined for quantitative data analysis. According to the results of MALDI-TOF mass spectra it seems that muscle lipids of the bulls fed with maize silage/grass silage (70/30) are characterized by a higher relative PC content (cf. the intensity ratios of the peaks at m/z = 650.5 and 760.6, for instance) compared to muscle lipids of grass silage-fed bulls. This is surprising because the grass and maize silage do not contain significant amounts of PL but only TAG (see Fig. 5) and, thus, only changes of the TAG compositions of beef were expected. Although analogous effects could be observed for all investigated muscle samples of both diet groups, no quantitative data are given here because a higher number of animals are clearly needed to obtain sufficient
statistical significance. However, deviations between individual MALDI spectra are expected to be about ±5% and, thus, much lower than the intra-individual deviations. Additionally, further efforts are necessary to investigate the potential influence of the applied solvents to extract the lipids. It must be emphasized that already from these spectra (that can be recorded in a few minutes only) important information about the fatty acyl compositions of the different lipid classes is also available. For instance, the contribution of saturated palmitic acid (16:0) is much higher in the TAGs from the grass silage-fed animals (cf. the peak at m/z = 827.7 that is exclusively detectable in trace (2b)). This is further confirmed by the peak at m/z = 549.5 that corresponds to the loss of sodium palmitate from the peak at m/z = 827.7. The loss of one fatty acyl residue is very characteristic for the MALDI mass spectra of TAGs (Fuchs and Schiller, 2009a). However, changes of the fatty acyl compositions do not only occur in view of the TAGs but also regarding the PC species. It is obvious that the contribution of PC 16:0/18:2 (m/z = 758.6 and 780.6) massively increases in the case of the maize silage-fed animals (maize lipids are high in 18:2 n − 6), where this species exhibits nearly the same intensity as PC 16:0/18:1 (m/z = 760.6 and 782.6). In the same manner as the content of linoleic acid (18:2 n − 6), the content of plasmalogen species (e.g. m/z = 742.6/744.6 and 764.6/766.6) also increases. Plasmalogens were not only identified by their characteristic masses but also by their extreme sensitivity towards even traces of acids (data not shown) (Fuchs et al., 2007c). It is, however, not yet clear if these changes concern exclusively the PC moiety or the remaining PL classes as well. Unfortunately, due to the permanent positive charge of the PC molecule, the positive ion spectra are dominated by the PC signals whereas the other PL classes (that are additionally present in smaller amounts than the PC) are suppressed (Petkovic´ et al., 2001). This problem can be easily overcome if the negative ion spectra of the same samples are recorded. In Fig. 3 two selected negative ion MALDI-TOF mass
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Fig. 3. Negative ion MALDI-TOF mass spectra of muscle lipid extracts of German Simmental bulls fed with grass silage (a) and with maize silage/grass silage (70/30) (b). Both spectra were recorded with 9-AA as matrix. Trace (c) was recorded with the same sample as in (b), but with DHB as matrix and is shown to illustrate the low quality of negative ion spectra achievable by the use of DHB. The total lipid concentration was 2 mg/ml in all cases. Please note that the signal at m/z = 747.5 (cf. the dotted line) corresponds to PG 16:0/18:1 that was added as internal standard (4 g/ml, final concentration). All peaks are labeled according to their m/z ratios. Peaks marked with asterisks are stemming from the applied DHB matrix.
spectra of total muscle lipid beef extracts subsequent to grass (3a) and maize silage/grass silage (70/30) (3b) feeding are shown. Both spectra were recorded with 9-AA as matrix. Trace (3c) is the same sample as shown in (3b) but this spectrum was recorded with DHB as matrix. It is obvious that DHB is a poor negative ion matrix in comparison to 9-AA. The spectrum given in trace (3c) is obviously dominated by matrix signals (cf. the most intense signal at m/z = 681.0 marked by an asterisk) leading in combination
with other factors to reduced sensitivity (Schiller et al., 2007b; Gellermann et al., 2006): Not even the PG 16:0/18:1 (m/z = 747.5) that was added as internal standard of known concentration (4 g/ml, final concentration) is detectable in the presence of DHB. Due to these obvious drawbacks, negative ion spectra recorded in the presence of DHB will not be discussed anymore and only spectra recorded with 9-AA as matrix will be considered. There are obviously two different groups of peaks (cf. traces (3a) and (3b)). The peaks at lower masses correspond to differ-
Fig. 4. 31 P NMR spectra of four different muscle lipid extracts of German Simmental bulls fed with grass silage (a and b) and with maize silage/grass silage (70/30) (c and d). The total lipid concentration was 10 mg/ml in all cases. Please note that the signal at about 3.15 ppm is caused by PA 8:0/8:0 that was added as internal concentration standard (167 g/ml corresponding to 375 mol/l). The indicated numbers indicate the concentrations of PC and PE as the most abundant PL. Abbreviations used in peak assignments: CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol.
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Fig. 5. Positive ion MALDI-TOF mass spectra of lipid extracts of the used maize (a) or grass silage (b). DHB was exclusively used as matrix in order to detect TAG signals. Traces (c) and (d) represent the TAG and chlorophyll fractions, respectively, obtained by TLC. Please note that the TAG contribution is not detectable if the total extracts (without previous separation) are investigated. For detailed peak assignments see Table 1.
ent PE species, while the peaks at higher masses (m/z = 863.6 and 885.6) represent two different PI species namely PI 18:0/18:1 and PI 18:0/20:4, respectively. A detailed list of all detected peaks as well as their individual assignments is given in Table 1. The detectability of PE species as negative ions is an additional important advantage of 9-AA because PE is otherwise completely suppressed by the presence of PC (Schiller et al., 2007a). In accordance with reference data for longissimus muscle of bulls (Dannenberger et al., 2006), it is evident that beside diacyl species there are also abundant alkenyl-acyl species that can be easily identified by the mass difference of 16 atomar mass units (amu) in comparison to the related diacyl species. For instance, the signal at m/z = 766.5 corresponds to PE 18:0/20:4 while the signal at m/z = 750.5 represents the related plasmalogen species. Of course, plasmalogens can not only be identified by their characteristic masses but as well by their typical fragments (Fuchs et al., 2007b) and their extreme sensitivity towards traces of acids. A simple method to identify plasmalogen species has been recently described (Fuchs et al., 2007c). In analogy to the positive ion spectra, it seems that maize silage/grass silage (70/30) feeding (trace (3b)) results in a higher PL content than feeding with grass silage alone (3a). This is supported by the comparison of the intensity of the PG 16:0/18:1 standard (m/z = 747.5, marked with a dotted line) with the other signals. It is also obvious that the PI/PE ratio increases in the case of the maize silage feeding. The detailed reasons for this effect are so far unknown. Unfortunately, investigations of the compositions of the individual PL classes in beef tissues were so far only sparsely described. Dannenberger et al. (2007) investigated the diet effect on the distribution of different PL classes and their fatty acyl profiles by high-performance thin-layer chromatography and gas chromatography. These authors observed no diet effect for the proportion of the PL classes PE, PC, PI and CL in the muscle lipids of German Holstein and German Simmental bulls. However, the diet affected the proportions of the SM and LPC fraction in the muscle lipids of German Simmental bulls. Pasture diet significantly increased the
SM and LPC proportions in the muscle lipids of German Simmental bulls. In addition to PE and PI, the MALDI-TOF MS mass spectra also show intense signals of cardiolipin (CL) at higher masses (cf. insert in trace (3a)). Although cardiolipin is rather difficult to detect by using common MALDI matrices (Rohlfing et al., 2007), the detection of CL is not a problem at all if 9-AA is used as matrix. CL 4 × 18:2 (m/z = 1470.1) is by far the most abundant species. This agrees with reference data where about 67% of 18:2 n − 6 acyl residues were determined in the CL fraction of beef muscle lipids (Dannenberger et al., 2007). The presented data cover only relative data in comparison to an internal standard because the “matrix” containing the analytes of interest would more significantly influence absolute data. Although ionization efficiencies of the individual lipid classes are surely different, their determination in a complex “matrix” is very difficult and has – to our best knowledge – not yet been attempted at all. The so far available data were exclusively determined with isolated PLs (Gellermann et al., 2006). Thus, in order to further confirm the obtained MALDI data, all samples were additionally investigated by high-resolution 31 P NMR spectroscopy (Fig. 4). To obtain absolute data, phosphatidic acid (PA 2 × 8:0) was added in a defined concentration (0.375 mmol/l) as concentration standard. Unfortunately, 31 P NMR does of course not provide any information about metabolites that lack phosphorous. This means that no information about e.g. TAGs can be provided. It is obvious at the first glance that animals fed with a mixture of maize silage/grass silage (traces (4c) and (4d)) instead of grass silage only (traces (4a) and (4b)) contain higher amounts of PL species, even if the relative ratio between the individual PL did not change significantly. Although NMR is a quantitative method, only the concentrations of PC and PE are provided because they are the most abundant PL of beef muscles and can, thus, be most accurately determined. The numbers given in Fig. 4 correspond to the molar concentrations of PC and PE. The resolution of 31 P NMR spectra is, unfortunately, not sufficiently high to allow to differentiate
D. Dannenberger et al. / Chemistry and Physics of Lipids 163 (2010) 157–164 Table 1 Assignment of the m/z values detected in the positive and negative ion mass spectra of beef muscle lipid and silage extracts. “DHB” indicates the molecular weight of the applied matrix (2,5-dihydroxybenzoic acid). Please note that only major signals are assigned and minor peaks are neglected. m/z ratio
Polarity
Assignment
369.3 375.0 551.0 551.5 577.5 599.5 601.5 603.5 605.5 650.5 672.5 673.5 675.0 681.0 698.5 699.5 715.5 726.5 742.5 742.6 744.5 744.6 747.5 748.5 750.5 758.6 760.6 766.5 766.6 776.5 782.6 808.6 827.7 855.8 863.6 871.5 871.7 873.7 875.7 877.7 879.7 881.8 883.8 885.6 893.5 895.7 897.7 899.7 901.7 902.6 903.7 905.7 907.7 909.5 907.7 909.7 910.6 911.6 912.6 926.6 1448.0 1468.1 1470.1 1472.1 1474.1
+ + + + + + + + + + + − − − − − − − − + − + − − − + + − + − + + + + − + + + + + + + + − + + + + + − + + + + + + − − − − − − − − −
Cholesterol + H+ –H2 O 2DHB–2H+ + 3Na+ 3DHB–3H+ + 4Na+ 855.8-Na-oleate 881.8-Na-oleate; 883.8-Na-stearate 903.8-Na-oleate 903.8-Na-linoleate 909.8-Na-stearate 909.8-Na-oleate PC 13:0/13:0 + H+ PC 13:0/13:0 + Na+ Unknown 4DHB–3H+ + Na+ + K+ 4DHB–4H+ + 3Na+ Unknown Unknown Unknown PE 18:0/18:2 (plasm)–H+ PE 18:0/18:2–H+ PC 16:0/18:2 (plasm) + H+ PE 18:0/18:1–H+ PC 16:0/18:1 (plasm) + H+ PG 16:0/18:1–Na+ PE 18:1/20:4 (plasm)–H+ PE 18:0/20:4 (plasm)–H+ PC 16:0/18:2 + H+ PC 16:0/18:1 + H+ PE 18:0/20:4–H+ PC 16:0/18:1 (plasm) + Na+ Unknown PC 16:0/18:1 + Na+ PC 18:0/18:2 + Na+ TAG 48:1 + Na+ TAG 50:1 + Na+ PI 18:0/18:1–Na+ Chlorophyll a–Mg2+ + 3H+ TAG 52:7 + Na+ TAG 52:6 + Na+ TAG 52:5 + Na+ TAG 52:4 + Na+ TAG 52:3 + Na+ TAG 52:2 + Na+ TAG 52:1 + Na+ PI 18:0/20:4–Na+ Chlorophyll a–Mg2+ + 2H+ + Na+ TAG 54:9 + Na+ TAG 54:8 + Na+ TAG 54:7 + Na+ TAG 54:6 + Na+ Unknown TAG 54:5 + Na+ TAG 54:4 + Na+ TAG 54:3 + Na+ Chlorophyll a–Mg2+ + 2H+ + K+ TAG 54:3 + Na+ TAG 54:2 + Na+ PC 16:0/18:2 + DHB–H+ PI 18:0/22:5 PC 16:0/18:1 + DHB–H+ Unknown CL 2 × 18:2/1 × 18:1/1 × 16:0 CL 3 × 18:2/1 × 18:3 CL 4 × 18:2 CL 3 × 18:2/1 × 18:1 CL 2 × 18:2/2 × 18:1
details of the fatty acyl compositions as well as the linkage between the glycerol and the apolar residues. It is, however, evident from the shape of the PE and the PC resonance that the composition of PE is much more complex regarding the fatty acyl composition. This is in complete agreement with the MALDI spectra (cf. Fig. 3) and
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reflects the significant contribution of alkenyl-acyl in addition to diacyl compounds. In addition to the analysis of the beef lipid extracts, the lipid compositions of the silages were also investigated. It turned out by 31 P NMR analysis that neither the maize silage nor the grass silage contained detectable amounts of PL (data not shown). As NMR is a rather insensitive method, the MALDI-TOF mass spectra of the total maize (a) and the grass silage (b) were also recorded and are shown in Fig. 5. As it was impossible to record convincing negative ion spectra in the presence of 9-AA and the detection of TAG is not possible with 9-AA as matrix (as already stated above), positive ion spectra were exclusively recorded in the presence of DHB. It is evident that both spectra differ significantly. Whereas the spectrum of the maize silage lipid extract (5a) can be easily explained by the presence of different TAG species (cf. Table 1), the signals of the grass silage lipid extract (5b) at m/z = 871.5, 893.5 and 909.5 do not agree with the mass of any known TAG. Therefore, it is assumed that the TAG species of the grass silage are suppressed by the presence of plant dyes. Chlorophyll a is one of the most abundant pigments in grass and is characterized by a monoisotopic mass of 892.5. Under (MALDI) MS conditions, the magnesium ion is lost, leaving a molecule with two negative charges. Subsequent to the addition of three H+ the molecule is detected at m/z = 871.5 (or as the sodiated ion at m/z = 893.5) (Vieler et al., 2007). Subsequent to TLC separation, the dye and the TAG can be easily identified and are shown in traces (5c) and (5d), respectively. For a more detailed assignment of the individual TAG species see Table 1. Although a comprehensive investigation of the silage composition was beyond the scope of this paper, it is evident by the strongly different signal-to-noise ratios that the TAG content of the grass silage (5c) is much lower in comparison to the maize silage (5a). It is also obvious that the grass silage contains much higher amounts of highly unsaturated fatty acyl residues, particularly linolenic acid (18:3 n − 3). Although the positions of the double bonds cannot be determined by standard MALDI-TOF MS as the “soft-ionization” does not lead to C–C bond fragmentations, the overall relative fatty acyl composition of lipid mixtures can be obviously easily determined.
4. Conclusions The majority of investigations of the lipid compositions of different beef tissues are so far primarily based on GC/MS data and, thus, on the analysis of the fatty acyl compositions by GC/FID (gas chromatography/flame ionization detectors) measurements only. GC/MS is laborious and time-consuming and has the additional disadvantage that only information about the fatty acyl residues is available, whereas information on the intact lipid species is lost as saponification and subsequent derivatization is required. In contrast, soft-ionization MS techniques enable the direct detection of the intact PL species with a minimum of sample pre-treatment and within a very short time. It has been also shown here that the use of different matrix compounds enables the selective identification of certain lipid classes. Although 9-AA is obviously a more suitable matrix than DHB (in particularly regarding negative ion detection) we do not claim that 9-AA is really the optimum matrix. Due to the so far widely unknown ionization process of MALDI MS (Fuchs and Schiller, 2009b), the matrix selection is still an empirical process and the prediction which matrix is the most appropriate one, is difficult. Therefore, the search for even more useful matrices is continuously going on. For instance, it was recently shown that 2-(2-aminoethylamino)-5-nitropyridine (Lorkiewicz and Yappert, 2009a) is a promising matrix because this compound gives lipid spectra of high quality – particularly in the negative ion mode – but is characterized by a significant background in the lower m/z range. Another approach by the same authors (Lorkiewicz and
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Yappert, 2009b) overcomes this problem because the suggested titania micro- or nanoparticles do not give any signal. Unfortunately, however, the applied nanoparticles are not commercially available. Although the number of investigated samples is clearly not high enough to enable sound data analysis differences in dependence on the animal diet could be also monitored. Due to the obtained promising preliminary data, further research will be focused on detailed investigations of diet effects on intact lipids and particularly the PL compositions of different beef tissues. Additionally, the detailed characterization of intact lipid species by MALDI-TOF MS investigations of different adipose and muscle tissues or cells in beef cattle is expected to contribute to the understanding of mechanisms regulating fatty acid biosynthesis in beef cattle under different dietary regimes. Acknowledgements This work was supported by the German Research Council (DFG Schi 476/5-1 and FU 771/1-1) as well as the Saxonian Excellence Initiative “LIFE”. References Caboni, M.F., Menotta, S., Lercker, G., 1994. High-performance liquid chromatography separation and light-scattering detection of phospholipids from cooked beef. J. Chromatogr. A 683, 59–65. Dannenberger, D., Nuernberg, G., Scollan, N.D., Ender, K., Nuernberg, K., 2007. Diets alters the fatty acid composition of individual phospholipid classes in beef muscle. J. Agric. Food Chem. 55, 452–460. Dannenberger, D., Lorenz, S., Nuernberg, G., Scollan, N.D., Ender, K., Nuernberg, K., 2006. Analysis of fatty aldehyde composition, including 12-methyltridecanal, in plasmalogens from longissimus muscle of concentrate- and pasture-fed bulls. J. Agric. Food Chem. 54, 182–188. Estrada, R., Yappert, M.C., 2004. Alternative approaches for the detection of various phospholipid classes by matrix-assisted laser desorption/ionization time-offlight mass spectrometry. J. Mass Spectrom. 39, 412–422. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Fuchs, B., Schiller, J., Süß, R., Schürenberg, M., Suckau, D., 2007a. A direct and simple method of coupling matrix-assisted laser desorption and ionization time-offlight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk. Anal. Bioanal. Chem. 389, 827–834. Fuchs, B., Schober, C., Richter, G., Süß, R., Schiller, J., 2007b. MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra. J. Biochem. Biophys. Methods 70, 689–692. Fuchs, B., Müller, K., Göritz, F., Blottner, S., Schiller, J., 2007c. Characteristic oxidation products of choline plasmalogens are detectable in cattle and roe deer spermatozoa by MALDI-TOF mass spectrometry. Lipids 42, 991–998. Fuchs, B., Schiller, J., Süß, R., Zscharnack, M., Bader, A., Müller, P., Schürenberg, M., Becker, M., Suckau, D., 2008a. Analysis of stem cell lipids by offline HPTLCMALDI-TOF MS. Anal. Bioanal. Chem. 392, 849–860. Fuchs, B., Arnold, K., Schiller, J., 2008b. Mass spectrometry of biological molecules. In: Meyers, R.A. (Ed.), Encyclopedia of Analytical Chemistry. John Wiley & Sons Ltd., Chichester, pp. 1–39. Fuchs, B., Schiller, J., 2009a. Application of MALDI-TOF mass spectrometry in lipidomics. Eur. J. Lipid Sci. Technol. 111, 83–98. Fuchs, B., Schiller, J., 2009b. Recent developments of useful MALDI matrices for the mass spectrometric characterization of apolar compounds. Curr. Org. Chem. 13, 1664–1681. Gellermann, G.P., Appel, T.R., Davies, P., Diekmann, S., 2006. Paired helical filaments contain small amounts of cholesterol, phosphatidylcholine and sphingolipids. Biol. Chem. 387, 1267–1274. Guo, Z., He, L., 2007. A binary matrix for background suppression in MALDI-MS of small molecules. Anal. Bioanal. Chem. 387, 1939–1944. Hidaka, H., Hanyu, N., Sugano, M., Kawasaki, K., Yamauchi, K., Katsuyama, T., 2007. Analysis of human serum lipoprotein lipid composition using MALDI-TOF mass spectrometry. Ann. Clin. Lab. Sci. 37, 213–221. Kim, H.Y., Salem Jr., N., 1993. Liquid chromatography–mass spectrometry of lipids. Prog. Lipid Res. 32, 221–245.
Larick, D.K., Turner, B.E., 1989. Influence of finishing diet on the phospholipid composition and fatty acid profile of individual phospholipids in lean muscle of beef cattle. J. Anim. Sci. 67, 2282–2293. Lorkiewicz, P., Yappert, M.C., 2009a. 2-(2-Aminoethylamino)-5-nitropyridine as a basic matrix for negative-mode matrix-assisted laser desorption/ionization analysis of phospholipids. J. Mass Spectrom. 44, 137–143. Lorkiewicz, P., Yappert, M.C., 2009b. Titania microparticles and nanoparticles as matrices for in vitro and in situ analysis of small molecules by MALDI-MS. Anal. Chem. 81, 6596–6603. Mahecha, L., Nuernberg, K., Nuernberg, G., Ender, K., Hagemann, E., Dannenberger, D., 2009. Effects of diet and storage on fatty acid profile, micronutrients and quality of muscle from German Simmental bulls. Meat Sci. 82, 365–371. Nuernberg, K., Nuernberg, G., Ender, K., Lorenz, S., Winkler, K., Rickert, R., Steinhart, H., 2002. 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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31 P NMR Dirk Dannenberger a , Rosmarie Süß b , Kristin Teuber b , Beate Fuchs b , Karin Nuernberg a , Jürgen Schiller b,∗ a b
Research Institute for Biology of Farm Animals, Research Unit of Muscle Biology and Growth, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Härtelstr. 16-18, D-04107 Leipzig, Germany
a r t i c l e
i n f o
Article history: Received 4 July 2009 Received in revised form 23 October 2009 Accepted 29 October 2009 Available online 10 November 2009 Keywords: Beef Phospholipids Lipids MALDI-TOF MS Grass and maize silage 31 P NMR TLC
a b s t r a c t The analysis of beef lipids is normally based on chromatographic techniques and/or gas chromatography in combination with mass spectrometry (GC/MS). Modern techniques of soft-ionization MS were so far scarcely used to investigate the intact lipids in muscle tissues of beef. The objective of the study was to investigate whether matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef. For the MALDI-TOF MS and 31 P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. Beside the triacylglycerols (TAGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) species the MALDI-TOF mass spectra of total muscle lipids gave also intense signals of cardiolipin (CL) species. The application of different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), leads to completely different mass spectra: 9-AA is particularly useful for the detection of (polar) phospholipids, whereas apolar lipids, such as cholesterol and triacylglycerols, are exclusively detected if DHB is used. Finally, the quality of the negative ion mass spectra is much higher if 9-AA is used. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Fat in cattle is present as membrane lipids (phospholipids), intermuscular fat (between the muscles), intramuscular fat (IMF), abdominal and subcutaneous fat. The fatty acyl compositions of adipose and muscle tissues in beef are affected by factors such as diet, species, fatness, age/weight, depot site, gender, breed, season and hormones. The most effective means of manipulating the related lipids and their fatty acyl profiles in muscle and adipose tissues of beef can be achieved through nutrition by strategic use of forage and dietary lipids (Nuernberg et al., 2005; Scollan et al., 2006; Wood et al., 2008). Intramuscular fat of beef mainly consists of triacylglycerols (TAGs) and phospholipids (PLs). TAGs serve as energy stores and are deposited in adipocytes. PLs are amphiphilic molecules with lipophilic acyl chains and a hydrophilic head and (in addition to cholesterol) abundant components of biological membranes. In beef muscle the presence of nine different phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), cardiolipin (CL), sphingomyelin (SM),
∗ Corresponding author. Tel.: +49 341 9715733; fax: +49 341 9715709. E-mail address: [email protected] (J. Schiller). 0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2009.10.011
phosphatidylserine (PS), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), and phosphatidic acid (PA) has been described (Yeo and Horrocks, 1988; Larick and Turner, 1989; Dannenberger et al., 2007). PC and PE are the most abundant constituents and SM, PI and CL are minor species in intramuscular beef lipids, and the fatty acyl profiles of the individual PL classes can be affected by different feeding regimes (Dannenberger et al., 2007). Beside diacyl PL species, there are also significant amounts of alkenyl-acyl and alkyl-acyl species, i.e. ether-linked compounds. However, studies on alkenylacyl species (plasmalogens) and the fatty aldehyde composition of beef lipids have been only sparsely described so far (Dannenberger et al., 2006). Nevertheless, it is known that plasmalogen compounds are particularly abundant in the PE subclass of muscle phospholipids. The focus regarding beef lipid composition was so far on the analysis of the fatty acyl composition. The traditional compositional analysis of beef lipids is based on chromatographic techniques (Caboni et al., 1994) as well as gas chromatography in combination with mass spectrometry (GC/MS) (Kim and Salem, 1993). Although nowadays highly established, modern techniques of softionization MS were so far only scarcely used to investigate the intact lipids (particularly TAGs) in muscle tissues of beef (Picariello et al., 2007).
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Due to the simple performance, the high sensitivity and the robustness against impurities, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) seems particularly suitable for that purpose. Although the quantitative information achievable from MALDI mass spectra is often assumed to be limited, it has been recently convincingly shown by using human blood plasma extracts that MALDI is a quantitative technique and the data obtained by MALDI agree favourably with data obtained by electrospray ionization (ESI) MS (Hidaka et al., 2007). Unfortunately, the different detectabilities of the individual lipid classes represent a major problem if complex lipid mixtures are analyzed because sensitively detectable lipids may suppress the less sensitively detectable ones (Schiller et al., 2007a; Petkovic´ et al., 2001; Gellermann et al., 2006). The sensitivity to detect a certain compound is also significantly influenced by the composition of the “matrix”, i.e. the presence or absence of other compounds. This normally makes previous separation of the total extract into the individual lipid classes necessary and there are nowadays (in addition to liquid chromatography) (LC/MS) methods available that allow the direct combination of thin-layer chromatography (TLC) (Touchstone, 1995) with MALDI MS (Fuchs et al., 2007a). Nevertheless, the careful selection of the matrix may already help to overcome some problems, making MALDI suitable for the investigation of complex lipid mixtures. 2,5-Dihydroxybenzoic acid (DHB) is unequivocally one of the most established UV matrices in the lipid field because it gives (in contrast to many other matrices, such as cinnamic acid derivates) only a rather moderate background in the lower mass region (Schiller et al., 1999). Due to its acidic properties, DHB is particularly useful as matrix for positive ion detection. Under these conditions, however, PC is most sensitively detected (Schiller et al., 2007a), whereas other lipids such as PE or PI are nearly completely suppressed – even as negative ions (Petkovic´ et al., 2001), i.e. the positive and the negative ion detection mode are not independent. Additionally, negative ion spectra obtained with DHB are dominated by signals of the matrix itself that may interfere with the mass regions of PL (Schiller et al., 2007b). It is reasonable to use less acidic matrices for the detection of negative ions. Some years before it was shown that paranitroaniline (PNA) is an excellent matrix for the detection of e.g. PE as negative ion even in the presence of significant amounts of PC (Estrada and Yappert, 2004). Unfortunately, however, the vacuum stability of this matrix is limited and, thus, spectra show time-dependent changes. Very recently, 9-aminoacridine (9-AA) was suggested as a powerful alternative to PNA: 9-AA was shown to detect lipids more sensitively than other matrices and to provide only a minimum background (Sun et al., 2008). However, although 9-AA was already tested with artificial and cellular lipids, this matrix was to our best knowledge not yet applied to the analysis of beef. The objective of this study is to investigate whether (a) matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef and (b) if 9-AA is the matrix of choice to detect negatively charged lipids, such as PI or CL in beef muscle lipids.
2. Materials and methods 2.1. Cattle experiment For the MALDI-TOF MS and 31 P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. The details of the diet experiment were very recently described (Mahecha et al., 2009). Briefly, 25 male German Simmental calves (3–4 months) were included
in an indoor experiment. They were randomly assigned into three groups with different feeding regimes. The control group (n = 9) was fed daily maize silage/grass silage (70/30, ad libitum), 1 kg of molasses, 1 kg of hay and concentrate including soybean. Treatment group I, conformed by unrestricted animals (n = 7) was fed grass silage (ad libitum), 1 kg of molasses, 1 kg of hay and concentrate including rapeseed in the same proportion as the control group. Treatment group II, conformed by restricted animals (n = 9), fed as treatment group I under a restriction of 1 kg of concentrate (50%) per day during the first 112 days of the fattening period. All bulls were slaughtered approximately at 635 kg live weight in the abattoir of the Research Institute for the Biology of Farm Animals in Dummerstorf (Germany). Longissimus muscle samples were isolated immediately after slaughter. Samples were taken at the 6th–13th rib of the left carcass side and were kept at −80 ◦ C until their analysis. Longissimus muscle samples from the control group and the treatment group I were selected in the present study. 2.2. Extraction of the muscle lipids The total lipids of 2 g muscle were extracted with 15 mL CHCl3 /CH3 OH (2:1, v/v) according to Folch et al. (1957) by homogenization (Ultra Turrax, 3 × 15 s, 12,000 rotations per minute) at room temperature. The solvent was evaporated and the amount of the extracted material was determined by weighing. CHCl3 /CH3 OH (2:1, v/v) was used to re-dissolve the isolated lipids. The details were described recently by Nuernberg et al. (2002). Solutions with a total lipid content of 2 or 10 mg/ml were prepared and used for subsequent analysis. 2.3. Reagents All phospholipid standards were obtained from AVANTI Polar Lipids Inc. (Alabaster, AL, USA) and were used as supplied. All chemicals and salts for sample preparation, MALDI-TOF MS (2,5-dihydroxybenzoic acid (DHB)) as well as all solvents for analysis (chloroform, methanol, isopropanol and acetonitrile) were obtained in highest commercially available purity from Fluka Feinchemikalien GmbH (part of Sigma–Aldrich Chemie GmbH, Taufkirchen) and used as supplied. 9-Aminoacridine hemihydrate was purchased from Acros Organics (Morris Plains, NJ). 2.4. Thin-layer chromatography Silage extracts were subjected to TLC separation prior to MALDITOF MS. 1 l lipid samples per spot were applied on HPTLC silica gel 60 plates (20 cm × 20 cm in size) (Merck, Darmstadt, Germany) and developed in TLC chambers (CAMAG, Switzerland) using chloroform, ethanol, water and triethylamine (30:35:7:35, v/v/v/v) as elution solvent in order to separate phospholipids. Chloroform/acetone (96:4, v/v) was used to separate triacylglycerols from the plant dyes. Lipids were visualized by spraying with a solution of PRIMULINE (Direct Yellow 59) according to White et al. (1998). Upon irradiation by UV light (366 nm), individual PLs become detectable as yellow spots. These spots were quantitatively assessed by using a digital image system in combination with the program Argus X1 delivered by BioStep (Jahnsdorf, Germany). Further details of combining MALDI-TOF MS directly with TLC are available in Fuchs et al. (2008a). 2.5. MALDI-TOF mass spectrometry Total lipid extracts and individual lipid fractions were independently investigated by MALDI-TOF MS. A 0.5 mol/l 2,5dihydroxybenzoic acid (DHB) solution in methanol (Schiller et al.,
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1999) or 10 mg/ml 9-aminoacridine (in isopropanol/acetonitrile (60/40, v/v)) was used in all cases (Sun et al., 2008). As the quality of the spectra recorded in the presence of 9-AA depends significantly on the applied solvent system, all lipids were diluted with isopropanol/acetonitrile (60/40, v/v) (Sun et al., 2008). This solvent mixture gave among all tested solvent systems (methanol, acetonitrile, etc.) the by far best results and this is in agreement with previous data (Sun et al., 2008). All samples were pre-mixed with the matrix prior to deposition onto the MALDI target and PC 13:0/13:0 (60 g/ml, final concentration) and PG 16:0/18:1 (4 g/ml, final concentration) were both added as reference compounds for the positive and negative ion mode, respectively. All MALDI-TOF mass spectra were acquired on a Bruker Autoflex mass spectrometer (Bruker Daltonics, Bremen, Germany). The system utilizes a pulsed nitrogen laser, emitting at 337 nm. The extraction voltage was 20 kV and gated matrix suppression was applied to prevent the saturation of the detector by matrix ions (Petkovic´ et al., 2001). 128 single laser shots were averaged for each mass spectrum. The laser fluence was kept about 10% above threshold to obtain optimum signal-to-noise ratios. In order to enhance the spectral resolution all spectra were acquired in the reflector mode using delayed extraction conditions. Under these conditions, resolutions of about 6000–8000 can be obtained with good reproducibility. Although this is low in comparison with the resolution indicated by the supplier of the device (ca. 20,000), it is absolutely sufficient to obtain isotopically resolved mass spectra of lipid samples. A more detailed methodological description of MALDI-TOF MS is given in Fuchs et al. (2008b) and Schiller et al. (2004). In the PSD (post source decay) experiments, the precursor ions of interest were isolated using a timed ion selector. The laser intensities for PSD spectra were maintained the same as in the reflector mode. The fragment ions were refocused onto the detector by stepping the voltage applied to the reflectron in appropriate increments. This can be done automatically by using the “FAST” (fragment analysis and structural TOF) subroutine of the Flex Control Program delivered by Bruker Daltonics. Further details are available in Fuchs et al. (2007b). 2.6.
31 P
NMR spectroscopy
In order to avoid the considerable line-widths of lipid aggregates, the dried organic extracts were solubilized in 50 mM TRIS (pH 7.65) containing 200 mM sodium cholate and 5 mM EDTA. After intense vortexing, 31 P NMR spectra were recorded on 0.5 ml samples in 5 mm NMR tubes on a Bruker DRX-600 spectrometer operating at 242.88 MHz for 31 P. All measurements were performed using a selective 31 P/1 H NMR probe at 37 ◦ C with composite pulse decoupling (Waltz-16) to eliminate 31 P–1 H coupling. Pulse intervals of the order of T1 were used to allow quantitative analysis of phospholipid integral intensities (Schiller et al., 2007c). Other NMR parameters were as follows: acquisition time 1 s, data size 8–16k, 60◦ pulse, pulse delay 2 s and a line-broadening (LB) of 1 Hz. Chemical shifts were referenced to the resonance of di-lauroyl-phosphatidic acid that was added as concentration and frequency standard. Further details are available in (Schiller et al., 2007c). Spectra were processed using the software “1D WINNMR” version 6.2® (Bruker Analytische Messtechnik GmbH, Rheinstetten) including the deconvolution (II) routine for peak area determination. 3. Results and discussion MALDI-TOF MS is increasingly used for – intact – lipid analysis because this method exhibits – beside the possibility to record spa-
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Fig. 1. Structures of the MALDI matrices 9-aminoacridine (9-AA) and 2,5dihydroxybenzoic acid (DHB) that were both used in this study. Please note that 9-AA is (due to its basic properties) the matrix of choice to detect negatively charged lipids, whereas DHB is more suitable for positive ion detection and particularly for apolar lipids such as in TAGs that are not detectable in the presence of 9-AA.
tially resolved spectra (“MALDI imaging”) – several advantages over other MS techniques. MALDI-TOF MS is fast, sensitive and tolerates impurities of the sample (salts, etc.) to a higher extent than e.g. electrospray ionization (ESI) MS that may be regarded as an alternative method (Pulfer and Murphy, 2003). In contrast, however, MALDI MS necessitates a “matrix” that enables the absorption of the energy emitted by the laser and must, thus, absorb at the laser wavelength (often 337 nm). Although there are also inorganic matrices available (e.g. graphite) (Fuchs and Schiller, 2009a), such matrices are not widely applied so far, and small organic molecules are still primarily used as matrices. DHB (Fig. 1) is one of the most frequently used matrix compounds (Schiller et al., 2004). Although the mass of this molecule (154 g/mol) is much smaller than the masses of typical PLs (about 700–900 g/mol), photoreactions may occur under the influence of the laser irradiation leading to the generation of a multitude of matrix peaks that were recently discussed in more detail (Schiller et al., 2007b). These “background” signals may interfere with the signals of the analytes of interest particularly if diluted samples are investigated and, even more severely, may decrease the sensitivity of detection. Therefore, the search for matrices with superior properties is still going on. Recently, 9-aminoacridine (9-AA), alone or in combination with other matrices has been shown to provide only a very moderate background and is, therefore, the matrix of choice for the detection of smaller compounds (Guo and He, 2007). Additionally, it was shown very recently (Sun et al., 2008) that 9AA is an excellent matrix for the analysis of the PL composition of biological extracts and exhibits three remarkable characteristics: (a) due to the basicity of 9-AA (cf. Fig. 1), the detection of negatively charged lipids is significantly improved in comparison to acidic matrices such as DHB and (b) using suitable solvent mixtures (especially acetonitrile and isopropanol) and a sufficient excess of matrix, H+ adducts are much more preferentially generated than alkali metal adducts (Sun et al., 2008). This is a clear advantage in the case of lipid mixtures because the superposition of different adducts, on one hand, and different acyl compositions, on the other hand, would otherwise complicate the assignments of the individual peaks (Schiller et al., 2001). Finally (c) the achievable sensitivity with 9-AA is much higher in comparison to DHB (Sun et al., 2008). Therefore, 9-AA might represent the most appropriate matrix to investigate the intact muscle lipids of beef. However, to our best knowledge such experiments were not yet performed. In Fig. 2 some selected positive ion MALDI-TOF mass spectra are compared. Spectra shown in traces (a and b) and (c and d) represent the muscle lipids of beef from two different animals fed with grass silage (treatment group I) and maize silage/grass silage (70/30, control group), respectively, but were recorded with 9-AA (a and c) or DHB (b and d) as MALDI matrix. Spectra recorded in the presence of DHB (b and d) are characterized by a higher number of peaks and, accordingly, some lipid classes are obviously exclusively detected in the presence of DHB, but not in the presence of 9-AA. For instance, TAGs and cholesterol are exclusively detected in the presence of DHB. This concerns TAG 54:2
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Fig. 2. Positive ion MALDI-TOF mass spectra of selected muscle lipid extracts of German Simmental bulls fed with grass silage (a and b) and with maize silage/grass silage (70/30) (c and d). Spectra in (a) and (c) were recorded in the presence of 9-AA, whereas DHB served as matrix in (b) and (d). The total lipid concentration was 2 mg/ml in all cases. Please note that the signals at m/z = 650.5 and 672.5 (marked with the dotted line) correspond to PC 13:0/13:0 that was added as internal standard (60 g/ml, final concentration). All samples were mixed 1:1 (v/v) with either DHB or 9-AA. All peaks are labeled according to their m/z ratios. Please note the complete disappearance of peaks of apolar lipids, such as triacylglycerols, if 9-AA is used as matrix (a and c). For details see text. Peaks marked with asterisks are stemming from the used DHB matrix. The small peaks between m/z = 612 and 633 cannot be assigned so far.
(m/z = 909.8), TAG 52:2 (m/z = 881.8), TAG 52:1 (m/z = 883.8), TAG 50:1 (m/z = 855.8) and TAG 48:1 (m/z = 827.7) as well as cholesterol (that is only detectable subsequent to water elimination but not as the intact molecule) at m/z = 369.3 (Schiller et al., 2000). TAGderived fragment ions (between m/z = 540 and 610; cf. Table 1 for detailed assignments) are accordingly also exclusively detected in the presence of DHB (Fig. 2b and d). Therefore, apolar lipids cannot be detected by MALDI-TOF MS if 9-AA is used as matrix. This may be regarded, on one hand, as a disadvantage because some potentially valuable pieces of information are lost, whereas, on the other hand, spectra can be significantly simplified in the presence of 9-AA. In contrast to the TAGs, PLs are detectable equally which matrix is used. Different phosphatidylcholine (PC) species are obvious between about m/z = 700 and 800 and more detailed peak assignments are provided in Table 1. The peaks at m/z = 650.5 and 672.5 (marked by the vertical dotted line) correspond to the H+ and Na+ adduct, respectively, of PC 13:0/13:0 that was artificially added to all samples as concentration standard. Considering the intensities of the H+ and Na+ adducts of the standard in dependence on the applied matrix, it is evident that the H+ adduct (m/z = 650.5) is more intense in the presence of 9-AA and the Na+ adduct (m/z = 672.5) in the presence of DHB. Thus, the intensities of both peaks have to be combined for quantitative data analysis. According to the results of MALDI-TOF mass spectra it seems that muscle lipids of the bulls fed with maize silage/grass silage (70/30) are characterized by a higher relative PC content (cf. the intensity ratios of the peaks at m/z = 650.5 and 760.6, for instance) compared to muscle lipids of grass silage-fed bulls. This is surprising because the grass and maize silage do not contain significant amounts of PL but only TAG (see Fig. 5) and, thus, only changes of the TAG compositions of beef were expected. Although analogous effects could be observed for all investigated muscle samples of both diet groups, no quantitative data are given here because a higher number of animals are clearly needed to obtain sufficient
statistical significance. However, deviations between individual MALDI spectra are expected to be about ±5% and, thus, much lower than the intra-individual deviations. Additionally, further efforts are necessary to investigate the potential influence of the applied solvents to extract the lipids. It must be emphasized that already from these spectra (that can be recorded in a few minutes only) important information about the fatty acyl compositions of the different lipid classes is also available. For instance, the contribution of saturated palmitic acid (16:0) is much higher in the TAGs from the grass silage-fed animals (cf. the peak at m/z = 827.7 that is exclusively detectable in trace (2b)). This is further confirmed by the peak at m/z = 549.5 that corresponds to the loss of sodium palmitate from the peak at m/z = 827.7. The loss of one fatty acyl residue is very characteristic for the MALDI mass spectra of TAGs (Fuchs and Schiller, 2009a). However, changes of the fatty acyl compositions do not only occur in view of the TAGs but also regarding the PC species. It is obvious that the contribution of PC 16:0/18:2 (m/z = 758.6 and 780.6) massively increases in the case of the maize silage-fed animals (maize lipids are high in 18:2 n − 6), where this species exhibits nearly the same intensity as PC 16:0/18:1 (m/z = 760.6 and 782.6). In the same manner as the content of linoleic acid (18:2 n − 6), the content of plasmalogen species (e.g. m/z = 742.6/744.6 and 764.6/766.6) also increases. Plasmalogens were not only identified by their characteristic masses but also by their extreme sensitivity towards even traces of acids (data not shown) (Fuchs et al., 2007c). It is, however, not yet clear if these changes concern exclusively the PC moiety or the remaining PL classes as well. Unfortunately, due to the permanent positive charge of the PC molecule, the positive ion spectra are dominated by the PC signals whereas the other PL classes (that are additionally present in smaller amounts than the PC) are suppressed (Petkovic´ et al., 2001). This problem can be easily overcome if the negative ion spectra of the same samples are recorded. In Fig. 3 two selected negative ion MALDI-TOF mass
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Fig. 3. Negative ion MALDI-TOF mass spectra of muscle lipid extracts of German Simmental bulls fed with grass silage (a) and with maize silage/grass silage (70/30) (b). Both spectra were recorded with 9-AA as matrix. Trace (c) was recorded with the same sample as in (b), but with DHB as matrix and is shown to illustrate the low quality of negative ion spectra achievable by the use of DHB. The total lipid concentration was 2 mg/ml in all cases. Please note that the signal at m/z = 747.5 (cf. the dotted line) corresponds to PG 16:0/18:1 that was added as internal standard (4 g/ml, final concentration). All peaks are labeled according to their m/z ratios. Peaks marked with asterisks are stemming from the applied DHB matrix.
spectra of total muscle lipid beef extracts subsequent to grass (3a) and maize silage/grass silage (70/30) (3b) feeding are shown. Both spectra were recorded with 9-AA as matrix. Trace (3c) is the same sample as shown in (3b) but this spectrum was recorded with DHB as matrix. It is obvious that DHB is a poor negative ion matrix in comparison to 9-AA. The spectrum given in trace (3c) is obviously dominated by matrix signals (cf. the most intense signal at m/z = 681.0 marked by an asterisk) leading in combination
with other factors to reduced sensitivity (Schiller et al., 2007b; Gellermann et al., 2006): Not even the PG 16:0/18:1 (m/z = 747.5) that was added as internal standard of known concentration (4 g/ml, final concentration) is detectable in the presence of DHB. Due to these obvious drawbacks, negative ion spectra recorded in the presence of DHB will not be discussed anymore and only spectra recorded with 9-AA as matrix will be considered. There are obviously two different groups of peaks (cf. traces (3a) and (3b)). The peaks at lower masses correspond to differ-
Fig. 4. 31 P NMR spectra of four different muscle lipid extracts of German Simmental bulls fed with grass silage (a and b) and with maize silage/grass silage (70/30) (c and d). The total lipid concentration was 10 mg/ml in all cases. Please note that the signal at about 3.15 ppm is caused by PA 8:0/8:0 that was added as internal concentration standard (167 g/ml corresponding to 375 mol/l). The indicated numbers indicate the concentrations of PC and PE as the most abundant PL. Abbreviations used in peak assignments: CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol.
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Fig. 5. Positive ion MALDI-TOF mass spectra of lipid extracts of the used maize (a) or grass silage (b). DHB was exclusively used as matrix in order to detect TAG signals. Traces (c) and (d) represent the TAG and chlorophyll fractions, respectively, obtained by TLC. Please note that the TAG contribution is not detectable if the total extracts (without previous separation) are investigated. For detailed peak assignments see Table 1.
ent PE species, while the peaks at higher masses (m/z = 863.6 and 885.6) represent two different PI species namely PI 18:0/18:1 and PI 18:0/20:4, respectively. A detailed list of all detected peaks as well as their individual assignments is given in Table 1. The detectability of PE species as negative ions is an additional important advantage of 9-AA because PE is otherwise completely suppressed by the presence of PC (Schiller et al., 2007a). In accordance with reference data for longissimus muscle of bulls (Dannenberger et al., 2006), it is evident that beside diacyl species there are also abundant alkenyl-acyl species that can be easily identified by the mass difference of 16 atomar mass units (amu) in comparison to the related diacyl species. For instance, the signal at m/z = 766.5 corresponds to PE 18:0/20:4 while the signal at m/z = 750.5 represents the related plasmalogen species. Of course, plasmalogens can not only be identified by their characteristic masses but as well by their typical fragments (Fuchs et al., 2007b) and their extreme sensitivity towards traces of acids. A simple method to identify plasmalogen species has been recently described (Fuchs et al., 2007c). In analogy to the positive ion spectra, it seems that maize silage/grass silage (70/30) feeding (trace (3b)) results in a higher PL content than feeding with grass silage alone (3a). This is supported by the comparison of the intensity of the PG 16:0/18:1 standard (m/z = 747.5, marked with a dotted line) with the other signals. It is also obvious that the PI/PE ratio increases in the case of the maize silage feeding. The detailed reasons for this effect are so far unknown. Unfortunately, investigations of the compositions of the individual PL classes in beef tissues were so far only sparsely described. Dannenberger et al. (2007) investigated the diet effect on the distribution of different PL classes and their fatty acyl profiles by high-performance thin-layer chromatography and gas chromatography. These authors observed no diet effect for the proportion of the PL classes PE, PC, PI and CL in the muscle lipids of German Holstein and German Simmental bulls. However, the diet affected the proportions of the SM and LPC fraction in the muscle lipids of German Simmental bulls. Pasture diet significantly increased the
SM and LPC proportions in the muscle lipids of German Simmental bulls. In addition to PE and PI, the MALDI-TOF MS mass spectra also show intense signals of cardiolipin (CL) at higher masses (cf. insert in trace (3a)). Although cardiolipin is rather difficult to detect by using common MALDI matrices (Rohlfing et al., 2007), the detection of CL is not a problem at all if 9-AA is used as matrix. CL 4 × 18:2 (m/z = 1470.1) is by far the most abundant species. This agrees with reference data where about 67% of 18:2 n − 6 acyl residues were determined in the CL fraction of beef muscle lipids (Dannenberger et al., 2007). The presented data cover only relative data in comparison to an internal standard because the “matrix” containing the analytes of interest would more significantly influence absolute data. Although ionization efficiencies of the individual lipid classes are surely different, their determination in a complex “matrix” is very difficult and has – to our best knowledge – not yet been attempted at all. The so far available data were exclusively determined with isolated PLs (Gellermann et al., 2006). Thus, in order to further confirm the obtained MALDI data, all samples were additionally investigated by high-resolution 31 P NMR spectroscopy (Fig. 4). To obtain absolute data, phosphatidic acid (PA 2 × 8:0) was added in a defined concentration (0.375 mmol/l) as concentration standard. Unfortunately, 31 P NMR does of course not provide any information about metabolites that lack phosphorous. This means that no information about e.g. TAGs can be provided. It is obvious at the first glance that animals fed with a mixture of maize silage/grass silage (traces (4c) and (4d)) instead of grass silage only (traces (4a) and (4b)) contain higher amounts of PL species, even if the relative ratio between the individual PL did not change significantly. Although NMR is a quantitative method, only the concentrations of PC and PE are provided because they are the most abundant PL of beef muscles and can, thus, be most accurately determined. The numbers given in Fig. 4 correspond to the molar concentrations of PC and PE. The resolution of 31 P NMR spectra is, unfortunately, not sufficiently high to allow to differentiate
D. Dannenberger et al. / Chemistry and Physics of Lipids 163 (2010) 157–164 Table 1 Assignment of the m/z values detected in the positive and negative ion mass spectra of beef muscle lipid and silage extracts. “DHB” indicates the molecular weight of the applied matrix (2,5-dihydroxybenzoic acid). Please note that only major signals are assigned and minor peaks are neglected. m/z ratio
Polarity
Assignment
369.3 375.0 551.0 551.5 577.5 599.5 601.5 603.5 605.5 650.5 672.5 673.5 675.0 681.0 698.5 699.5 715.5 726.5 742.5 742.6 744.5 744.6 747.5 748.5 750.5 758.6 760.6 766.5 766.6 776.5 782.6 808.6 827.7 855.8 863.6 871.5 871.7 873.7 875.7 877.7 879.7 881.8 883.8 885.6 893.5 895.7 897.7 899.7 901.7 902.6 903.7 905.7 907.7 909.5 907.7 909.7 910.6 911.6 912.6 926.6 1448.0 1468.1 1470.1 1472.1 1474.1
+ + + + + + + + + + + − − − − − − − − + − + − − − + + − + − + + + + − + + + + + + + + − + + + + + − + + + + + + − − − − − − − − −
Cholesterol + H+ –H2 O 2DHB–2H+ + 3Na+ 3DHB–3H+ + 4Na+ 855.8-Na-oleate 881.8-Na-oleate; 883.8-Na-stearate 903.8-Na-oleate 903.8-Na-linoleate 909.8-Na-stearate 909.8-Na-oleate PC 13:0/13:0 + H+ PC 13:0/13:0 + Na+ Unknown 4DHB–3H+ + Na+ + K+ 4DHB–4H+ + 3Na+ Unknown Unknown Unknown PE 18:0/18:2 (plasm)–H+ PE 18:0/18:2–H+ PC 16:0/18:2 (plasm) + H+ PE 18:0/18:1–H+ PC 16:0/18:1 (plasm) + H+ PG 16:0/18:1–Na+ PE 18:1/20:4 (plasm)–H+ PE 18:0/20:4 (plasm)–H+ PC 16:0/18:2 + H+ PC 16:0/18:1 + H+ PE 18:0/20:4–H+ PC 16:0/18:1 (plasm) + Na+ Unknown PC 16:0/18:1 + Na+ PC 18:0/18:2 + Na+ TAG 48:1 + Na+ TAG 50:1 + Na+ PI 18:0/18:1–Na+ Chlorophyll a–Mg2+ + 3H+ TAG 52:7 + Na+ TAG 52:6 + Na+ TAG 52:5 + Na+ TAG 52:4 + Na+ TAG 52:3 + Na+ TAG 52:2 + Na+ TAG 52:1 + Na+ PI 18:0/20:4–Na+ Chlorophyll a–Mg2+ + 2H+ + Na+ TAG 54:9 + Na+ TAG 54:8 + Na+ TAG 54:7 + Na+ TAG 54:6 + Na+ Unknown TAG 54:5 + Na+ TAG 54:4 + Na+ TAG 54:3 + Na+ Chlorophyll a–Mg2+ + 2H+ + K+ TAG 54:3 + Na+ TAG 54:2 + Na+ PC 16:0/18:2 + DHB–H+ PI 18:0/22:5 PC 16:0/18:1 + DHB–H+ Unknown CL 2 × 18:2/1 × 18:1/1 × 16:0 CL 3 × 18:2/1 × 18:3 CL 4 × 18:2 CL 3 × 18:2/1 × 18:1 CL 2 × 18:2/2 × 18:1
details of the fatty acyl compositions as well as the linkage between the glycerol and the apolar residues. It is, however, evident from the shape of the PE and the PC resonance that the composition of PE is much more complex regarding the fatty acyl composition. This is in complete agreement with the MALDI spectra (cf. Fig. 3) and
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reflects the significant contribution of alkenyl-acyl in addition to diacyl compounds. In addition to the analysis of the beef lipid extracts, the lipid compositions of the silages were also investigated. It turned out by 31 P NMR analysis that neither the maize silage nor the grass silage contained detectable amounts of PL (data not shown). As NMR is a rather insensitive method, the MALDI-TOF mass spectra of the total maize (a) and the grass silage (b) were also recorded and are shown in Fig. 5. As it was impossible to record convincing negative ion spectra in the presence of 9-AA and the detection of TAG is not possible with 9-AA as matrix (as already stated above), positive ion spectra were exclusively recorded in the presence of DHB. It is evident that both spectra differ significantly. Whereas the spectrum of the maize silage lipid extract (5a) can be easily explained by the presence of different TAG species (cf. Table 1), the signals of the grass silage lipid extract (5b) at m/z = 871.5, 893.5 and 909.5 do not agree with the mass of any known TAG. Therefore, it is assumed that the TAG species of the grass silage are suppressed by the presence of plant dyes. Chlorophyll a is one of the most abundant pigments in grass and is characterized by a monoisotopic mass of 892.5. Under (MALDI) MS conditions, the magnesium ion is lost, leaving a molecule with two negative charges. Subsequent to the addition of three H+ the molecule is detected at m/z = 871.5 (or as the sodiated ion at m/z = 893.5) (Vieler et al., 2007). Subsequent to TLC separation, the dye and the TAG can be easily identified and are shown in traces (5c) and (5d), respectively. For a more detailed assignment of the individual TAG species see Table 1. Although a comprehensive investigation of the silage composition was beyond the scope of this paper, it is evident by the strongly different signal-to-noise ratios that the TAG content of the grass silage (5c) is much lower in comparison to the maize silage (5a). It is also obvious that the grass silage contains much higher amounts of highly unsaturated fatty acyl residues, particularly linolenic acid (18:3 n − 3). Although the positions of the double bonds cannot be determined by standard MALDI-TOF MS as the “soft-ionization” does not lead to C–C bond fragmentations, the overall relative fatty acyl composition of lipid mixtures can be obviously easily determined.
4. Conclusions The majority of investigations of the lipid compositions of different beef tissues are so far primarily based on GC/MS data and, thus, on the analysis of the fatty acyl compositions by GC/FID (gas chromatography/flame ionization detectors) measurements only. GC/MS is laborious and time-consuming and has the additional disadvantage that only information about the fatty acyl residues is available, whereas information on the intact lipid species is lost as saponification and subsequent derivatization is required. In contrast, soft-ionization MS techniques enable the direct detection of the intact PL species with a minimum of sample pre-treatment and within a very short time. It has been also shown here that the use of different matrix compounds enables the selective identification of certain lipid classes. Although 9-AA is obviously a more suitable matrix than DHB (in particularly regarding negative ion detection) we do not claim that 9-AA is really the optimum matrix. Due to the so far widely unknown ionization process of MALDI MS (Fuchs and Schiller, 2009b), the matrix selection is still an empirical process and the prediction which matrix is the most appropriate one, is difficult. Therefore, the search for even more useful matrices is continuously going on. For instance, it was recently shown that 2-(2-aminoethylamino)-5-nitropyridine (Lorkiewicz and Yappert, 2009a) is a promising matrix because this compound gives lipid spectra of high quality – particularly in the negative ion mode – but is characterized by a significant background in the lower m/z range. Another approach by the same authors (Lorkiewicz and
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