ABCD-E-FG-H123
• Characterisation of positional isomers of neutral lipids
• True high-energy CID (20 KeV) was performed on triacylglycerols
• High m/z region of CID spectrum is dominated by abundant charge-remote fragmentation of the fatty acid substituent
• Low mass fragment ions provide important structural information
• The presence of double bonds or hydroxyl groups within the fatty acid chain can be determined
• High mass accuracy measurements
Again, in all three of these cases the positions of the various fatty acid substituents could be unequivocally assigned using the distinctive E1/3-, F1/3-, G1/3- and J2-type ions. Closer inspection reveals additional information regarding the lipid. In particular in the case of 1,3-dioleoyl-2-palmitoyl-glycerol , the mass difference of 54 Da in the high mass region (m/z 727.7 and m/z 781.6) indicates the position of the double bond within the fatty acid. Furthermore the resolving power in MS2mode is demonstrated in the spectrum of 1,2-distearoyl-3-oleoyl-glycerol, where the observed E1/3-type ions are separated by 2 Da (m/z 417.4 and m/z 419.4). In this case, the product ions from other charge-remote fragmentation coincide with those defining the position of the double bond in the mono-unsaturated fatty acid substituent.
RELATIVE ABUNDANCE
Figure 5: MALDI-TOF mass spectrum of the triacylglycerols in cocoa butter. Abbreviations used: O = oleic acid, P = palmitic acid, S = stearic acid
Figure 6: Comparison of the low m/z region of the high-energy CID spectra of cocoa butter components m/z 855.8 and 883.8 (blue traces) and synthetic triacylglycerols (red traces): 1,3-dipalmitoyl-2-oleoyl-glycerol and
Cocoa butter precursor m/z 855.8 1,3-dipalmitoyl-2-oleoyl-glycerol
Cocoa butter precursor m/z 883.8 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol
High-energy CID of precursor ions m/z 855.8 and 883.8 produced identical spectra in the low mass diagnostic region to those shown in Figures 3 and 4b indicating they are 1,3-dipalmitoyl-2-oleoyl-glycerol and 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol respectively (Figures 6a and b). Results
Triacylglycerols could be readily desorbed and ionised by MALDI as sodiated adducts without significant in-source decay. Only B- and C-type ions were observed in the PSD spectrum of the [M+Na]+adduct ion of 1,2-dipalmitoyl-3-oleoyl-glycerol. These are formed through the loss of one free fatty acid and one sodium fatty acid carboxylate residue as shown in Figure 1. These ions do not allow the distinction between the two outer fatty acid-substituents (sn-1, sn-3) and the middle position (sn-2). Therefore only structural information regarding the fatty acid composition of this triacylglycerol could be obtained using PSD conditions.
In contrast to the PSD spectrum, the 20 keV CID spectrum of the same triacylglycerol yields a rich array of various structurally diagnostic product ions such as E-, F-, G- and J-type ions(1)in the low mass region in addition to the ions observed under PSD conditions (Figure 2).
x200
Figure 1: PSD analysis of 1,2-dipalmitoyl-3-oleoyl-glycerol
The strong simultaneous charge-remote fragmentation of the three fatty acids substituents observed in the high mass region of the high-energy CID spectra is thought to be the result of a formal 1,4-elimination of molecular hydrogen (H2) and loss of neutral alkenes.
Additionally, in the high-energy CID spectrum only, the low mass region displays D-type product ions and more significantly the structurally important ions E1/3-, F1/3- and G1/3-type ions only present in sn-1 and sn-3 substituents and the J2-type ion characteristic of the sn-2 substituent.
These low mass product ions can be used to accurately differentiate structural isomers of triacylglycerols.
In particular E1/3(m/z 417.2 and m/z 391.4), F1/3(m/z 403.2 and m/z 377.5), G1/3(m/z 345.4 and m/z 319.4) and J2(m/z 305.3) indicate that, in the case shown, the palmitic acid substituent is linked to the middle position (sn-2) of the glycerol backbone.
Figure 3 shows the high-energy CID spectrum of a structural isomer of the triacylglycerol shown in Figure 2, namely 1,3-dipalmitoyl-2-oleoyl-glycerol.
In this case the outer two fatty acids are identical in mass, yielding an E1/3ion at m/z 391.4, an F1/3ion at m/z 377.4 and G1/3ion at m/z 319.3. The J2ion now appears at m/z 331.2, indicating the oleoyl-substituent to be linked to the sn-2 position. Three additional synthetic triacylglycerols were considered, to further demonstrate the value of high-energy CID in isomer differentiation.
Figure 4 illustrates the high-energy CID spectra of the sodiated adducts of 1,3-dioleoyl-2-palmitoyl-glycerol, 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol and
300 400 500 600 700 800
Figure 3: High-energy CID analysis of 1,3-dipalmitoyl-2-oleoyl-glycerol. Inset: low mass region showing diagnostic E-, F-, G-and J-ions
Figure 2: High-energy CID analysis of 1,2-dipalmitoyl-3-oleoyl-glycerol. Inset: low mass region showing diagnostic E-, F-, G-and J-ions
300 400 500 600 700 800
Figure 4: High-energy CID spectra of the sodiated adducts of (a) 1,3-dioleoyl-2-palmitoyl-glycerol, (b) 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol and (c) 1,2-distearoyl-3-oleoyl-glycerol
RELATIVE ABUNDANCE
This technique was applied to the differentiation of triacylglycerols positional isomers in plant oils, in particular cocoa butter. The three principal triacylglycerol components of cocoa butter (m/z 855.8, m/z 883.8 and m/z 911.9) are displayed in Figure 5.
Again, in all three of these cases the positions of the various fatty acid substituents could be unequivocally assigned using the distinctive E1/3-, F1/3-, G1/3- and J2-type ions. Closer inspection reveals additional information regarding the lipid. In particular in the case of 1,3-dioleoyl-2-palmitoyl-glycerol , the mass difference of 54 Da in the high mass region (m/z 727.7 and m/z 781.6) indicates the position of the double bond within the fatty acid. Furthermore the resolving power in MS2mode is demonstrated in the spectrum of 1,2-distearoyl-3-oleoyl-glycerol, where the observed E1/3-type ions are separated by 2 Da (m/z 417.4 and m/z 419.4). In this case, the product ions from other charge-remote fragmentation coincide with those defining the position of the double bond in the mono-unsaturated fatty acid substituent.
RELATIVE ABUNDANCE
Figure 5: MALDI-TOF mass spectrum of the triacylglycerols in cocoa butter. Abbreviations used: O = oleic acid, P = palmitic acid, S = stearic acid
Figure 6: Comparison of the low m/z region of the high-energy CID spectra of cocoa butter components m/z 855.8 and 883.8 (blue traces) and synthetic triacylglycerols (red traces):
1,3-dipalmitoyl-2-oleoyl-glycerol and
Cocoa butter precursor m/z 855.8 1,3-dipalmitoyl-2-oleoyl-glycerol
Cocoa butter precursor m/z 883.8 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol
High-energy CID of precursor ions m/z 855.8 and 883.8 produced identical spectra in the low mass diagnostic region to those shown in Figures 3 and 4b indicating they are 1,3-dipalmitoyl-2-oleoyl-glycerol and 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol respectively (Figures 6a and b).
Results
Triacylglycerols could be readily desorbed and ionised by MALDI as sodiated adducts without significant in-source decay. Only B- and C-type ions were observed in the PSD spectrum of the [M+Na]+adduct ion of 1,2-dipalmitoyl-3-oleoyl-glycerol. These are formed through the loss of one free fatty acid and one sodium fatty acid carboxylate residue as shown in Figure 1. These ions do not allow the distinction between the two outer fatty acid-substituents (sn-1, sn-3) and the middle position (sn-2). Therefore only structural information regarding the fatty acid composition of this triacylglycerol could be obtained using PSD conditions.
In contrast to the PSD spectrum, the 20 keV CID spectrum of the same triacylglycerol yields a rich array of various structurally diagnostic product ions such as E-, F-, G- and J-type ions(1)in the low mass region in addition to the ions observed under PSD conditions (Figure 2).
x200
Figure 1: PSD analysis of 1,2-dipalmitoyl-3-oleoyl-glycerol
The strong simultaneous charge-remote fragmentation of the three fatty acids substituents observed in the high mass region of the high-energy CID spectra is thought to be the result of a formal 1,4-elimination of molecular hydrogen (H2) and loss of neutral alkenes.
Additionally, in the high-energy CID spectrum only, the low mass region displays D-type product ions and more significantly the structurally important ions E1/3-, F1/3- and G1/3-type ions only present in sn-1 and sn-3 substituents and the J2-type ion characteristic of the sn-2 substituent.
These low mass product ions can be used to accurately differentiate structural isomers of triacylglycerols.
In particular E1/3(m/z 417.2 and m/z 391.4), F1/3(m/z 403.2 and m/z 377.5), G1/3(m/z 345.4 and m/z 319.4) and J2(m/z 305.3) indicate that, in the case shown, the palmitic acid substituent is linked to the middle position (sn-2) of the glycerol backbone.
Figure 3 shows the high-energy CID spectrum of a structural isomer of the triacylglycerol shown in Figure 2, namely 1,3-dipalmitoyl-2-oleoyl-glycerol.
In this case the outer two fatty acids are identical in mass, yielding an E1/3ion at m/z 391.4, an F1/3ion at m/z 377.4 and G1/3ion at m/z 319.3. The J2 ion now appears at m/z 331.2, indicating the oleoyl-substituent to be linked to the sn-2 position. Three additional synthetic triacylglycerols were considered, to further demonstrate the value of high-energy CID in isomer differentiation.
Figure 4 illustrates the high-energy CID spectra of the sodiated adducts of 1,3-dioleoyl-2-palmitoyl-glycerol, 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol and
300 400 500 600 700 800
Figure 3: High-energy CID analysis of 1,3-dipalmitoyl-2-oleoyl-glycerol. Inset: low mass region showing diagnostic E-, F-, G-and J-ions
Figure 2: High-energy CID analysis of 1,2-dipalmitoyl-3-oleoyl-glycerol. Inset: low mass region showing diagnostic E-, F-, G-and J-ions
300 400 500 600 700 800
Figure 4: High-energy CID spectra of the sodiated adducts of (a) 1,3-dioleoyl-2-palmitoyl-glycerol, (b) 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol and (c) 1,2-distearoyl-3-oleoyl-glycerol
RELATIVE ABUNDANCE
This technique was applied to the differentiation of triacylglycerols positional isomers in plant oils, in particular cocoa butter. The three principal triacylglycerol components of cocoa butter (m/z 855.8, m/z 883.8 and m/z 911.9) are displayed in Figure 5.
First Edition: May, 2012
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More significant is the case of the final main triacylglycerol component of cocoa butter. The low mass region of the high-energy CID of m/z 911.9 is displayed in Figure 7 and overlaid with that of 1,2-distearoyl-3-oleoyl-glycerol (previously shown in Figure 4c).
References
(1) Cheng C, Gross ML, Pittenauer E. Complete structural elucidation of triacylglycerols by tandem mass spectrometry.
Anal. Chem. 1998; 70: 4417-4426.
(2) Al-Saad KA et al. MALDI TOF MS of lipids: ionization and prompt fragmentation patterns.
RCM 2003; 17: 87-96.
(3) Belgacem O, Bowdler A, Brookhouse I, Brancia FL, Raptakis E. Dissociation of biomolecules using ultra-violet matrix-assisted laser desporption/ionisation time-of-flight/curved field reflectron tandem mass spectrometer equipped with a differential-pumped collision cell.
Rapid Commun. Mass Spectrom. 2006; 20: 1653-1660.
(4) Pittenauer E, Allmaier G. The renaissance of high-energy CID for structural elucidations of complex lipids: MALDI-TOF/RTOF_MS of alkali cationized triacylglycerols.
JASMS 2009; 20: 1037-1047.
m/z 419.3
405.3 331.3
347.3 J2
G1/G3
F1/F3
E1/E3
333.4J2 345.3 347.4G3 G1
403.4F3 405.5F1 417.4 E3
419.4E3 Cocoa butter precursor m/z 911.9
1,2-distearoyl-3-oleoyl-glycerol CID [M+Na] +
350 400
RELATIVE ABUNDANCE
Figure 7: Comparison of the low m/z region of the high-energy CID spectra of cocoa butter component m/z 911.9 and synthetic triacylglycerol: 1,2-distearoyl-3-oleoyl-glycerol
It is evident from these spectra that there are significant differences between the two CID spectra. Of particular interest is the 2 Da shift in the mass of the diagnostic J2-type ion (m/z 333.4 for 1,2-distearoyl-3-oleoyl-glycerol and m/z 331.3 for the cocoa butter component). This confirms that the fatty acid-substituent linked to the hydroxy group of the sn-2 position of the glycerol backbone was oleic acid in case of the cocoa butter triacylglycerol. Therefore the two triacylglycerols (1,2-distearoyl-3-oleoyl-glycerol and 1,3-distearoyl-2-oleoyl-glycerol) are simply positional isomers, the natural one corresponding to 1,3-distearoyl-2-oleoyl-glycerol.
Conclusion
MALDI-MS of sodiated triacylglycerols using true high-energy CID TOF/TOF conditions (ELAB = 20 keV) has proven a valid alternative to sector-field analyser based experiments. More significantly, the AXIMA Performance has been shown to allow accurate differentiation of positional isomers within this class of lipids.
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