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. 2007 Jul 1;366(1):59-70.
doi: 10.1016/j.ab.2007.03.012. Epub 2007 Mar 18.

Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry

Robert C Murphy  1 , Patrick F James, Andrew M McAnoy, Jessica Krank, Eva Duchoslav, Robert M Barkley
Affiliations

Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry

Robert C Murphy et al. Anal Biochem. .

Abstract

Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH(3). Deuterium-labeled internal standards were used to normalize the signal for each nominal [M+NH(4)](+) ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo(2)-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo(2)-lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.

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Figures

Figure 1
Figure 1
(A) Positive ion electrospray ionization of synthetic 1-hexadecanoyl-2-oleoyl-3-linoleoyl glycerol (16:0/18:1/18:2-TAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 874.8. Abundant product ions correspond to neutral loss of each fatty acyl group as a free carboxylic acid and ammonia (NH3). (B) Positive ion electrospray ionization of synthetic [1,1,2,3,3]-D5-1-arachidonoyl-2-linoleoyl-3- arachidonoyl glycerol (D5-20:4/18:2/20:4-TAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 949.8. Abundant product ions correspond to neutral loss of each fatty acyl group as a free carboxylic acid and ammonia (NH3).
Figure 1
Figure 1
(A) Positive ion electrospray ionization of synthetic 1-hexadecanoyl-2-oleoyl-3-linoleoyl glycerol (16:0/18:1/18:2-TAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 874.8. Abundant product ions correspond to neutral loss of each fatty acyl group as a free carboxylic acid and ammonia (NH3). (B) Positive ion electrospray ionization of synthetic [1,1,2,3,3]-D5-1-arachidonoyl-2-linoleoyl-3- arachidonoyl glycerol (D5-20:4/18:2/20:4-TAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 949.8. Abundant product ions correspond to neutral loss of each fatty acyl group as a free carboxylic acid and ammonia (NH3).
Figure 2
Figure 2
(A) Positive ion electrospray ionization of synthetic 1,2-stearoyl-2-glycerol (18:0/18:0-DAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 642.5. Abundant product ion corresponds to neural loss of water plus ammonia and each fatty acyl group as the identical carboxylic acid and ammonia (NH3). (B) Positive ion electrospray ionization of synthetic [1,1,2,3,3]-D5-1,2-palmitoyl-glycerol (D5-16:0/16:0-DAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 591.4 Abundant product ions correspond to neural loss of water plus ammonia and each fatty acyl group as the identical carboxylic acid and ammonia (NH3).
Figure 2
Figure 2
(A) Positive ion electrospray ionization of synthetic 1,2-stearoyl-2-glycerol (18:0/18:0-DAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 642.5. Abundant product ion corresponds to neural loss of water plus ammonia and each fatty acyl group as the identical carboxylic acid and ammonia (NH3). (B) Positive ion electrospray ionization of synthetic [1,1,2,3,3]-D5-1,2-palmitoyl-glycerol (D5-16:0/16:0-DAG) followed by collision induced dissociation of the [M+NH4]+ at m/z 591.4 Abundant product ions correspond to neural loss of water plus ammonia and each fatty acyl group as the identical carboxylic acid and ammonia (NH3).
Figure 3
Figure 3
(A) Positive ion electrospray ionization of RAW 264.7 cell glyceryl lipids extracted from 2 × 108 cells followed by TLC purification. No D5-internal standards were present in this sample. The electrospray mobile phase contained 10 mM NH4OAc so that the ammonium adduct ion for each molecular species of TAGs and DAGs predominated. (B) Collision induced dissociation of m/z 876.8 corresponding to the [M+NH4]+ for 52:2-TAG molecular species present in the RAW cell glyceryl lipid extract. Each product ion corresponds to neural loss each fatty acyl group plus ammonia (NH3) present in these isobaric molecular species of TAGs. (C) Collision induced dissociation of m/z 878.8 corresponding to the [M+NH4]+ for 52:1-TAG molecular species present in the RAW cell glyceryl lipid extract. Each product ion corresponds to the neutral loss of each fatty acyl group plus ammonia (NH3) present in these isobaric molecular species of TAGs.
Figure 3
Figure 3
(A) Positive ion electrospray ionization of RAW 264.7 cell glyceryl lipids extracted from 2 × 108 cells followed by TLC purification. No D5-internal standards were present in this sample. The electrospray mobile phase contained 10 mM NH4OAc so that the ammonium adduct ion for each molecular species of TAGs and DAGs predominated. (B) Collision induced dissociation of m/z 876.8 corresponding to the [M+NH4]+ for 52:2-TAG molecular species present in the RAW cell glyceryl lipid extract. Each product ion corresponds to neural loss each fatty acyl group plus ammonia (NH3) present in these isobaric molecular species of TAGs. (C) Collision induced dissociation of m/z 878.8 corresponding to the [M+NH4]+ for 52:1-TAG molecular species present in the RAW cell glyceryl lipid extract. Each product ion corresponds to the neutral loss of each fatty acyl group plus ammonia (NH3) present in these isobaric molecular species of TAGs.
Figure 3
Figure 3
(A) Positive ion electrospray ionization of RAW 264.7 cell glyceryl lipids extracted from 2 × 108 cells followed by TLC purification. No D5-internal standards were present in this sample. The electrospray mobile phase contained 10 mM NH4OAc so that the ammonium adduct ion for each molecular species of TAGs and DAGs predominated. (B) Collision induced dissociation of m/z 876.8 corresponding to the [M+NH4]+ for 52:2-TAG molecular species present in the RAW cell glyceryl lipid extract. Each product ion corresponds to neural loss each fatty acyl group plus ammonia (NH3) present in these isobaric molecular species of TAGs. (C) Collision induced dissociation of m/z 878.8 corresponding to the [M+NH4]+ for 52:1-TAG molecular species present in the RAW cell glyceryl lipid extract. Each product ion corresponds to the neutral loss of each fatty acyl group plus ammonia (NH3) present in these isobaric molecular species of TAGs.
Figure 4
Figure 4
Glyceryl lipids containing palmitic acid (16:0) as revealed by the neutral loss scan for 273.3u during electrospray ionization of neutral lipids from control RAW 264.7. The identity of abundant [M+NH4]+ ions are indicated by the abbreviations for each fatty acyl group present in the diacylglycerol ions and the total number of fatty acyl carbon atoms and total number of fatty acyl double bonds present in triacylglycerol molecular species. The glyceryl lipids were isolated from RAW 264.7 cells (approximately 5 × 106 cells) after treatment for 1 hr with Kdo2-lipid A to which a stock solution of D5-TAG and D5-DAG internal standards (600 pmol each internal standard) was added prior to extraction.
Figure 5
Figure 5
A quantity of synthetic 16:0/18:1/18:2-TAG (spike) was added to an aliquot of the RAW glyceryl lipid extract along with a constant amount of the internal standard mixture of D5-TAG molecular species. The abundance of the [M+NH4]+ at m/z 874.4 obtained in the neutral loss scan for (A) palmitate, neutral loss of 273u (NL 273), (B) oleate, neutral loss of 299u (NL 299), and (C) linoleate, neutral loss of 297u (NL 297) was divided by the abundance of the [M+NH4]+ from each internal standard and plotted as to the amount of synthetic spike added (n=3, SEM).
Figure 6
Figure 6
Abundance of [M+NH4]+ ions measured in sequential neutral loss scans of the glyceryl lipid extract from approximately 8 × 106 RAW 264.7 cells to which 600 pmol of each D5-TAG and D5-DAG internal standard was added prior to extraction. Positive ion nanoelectrospay ionization was employed using the Nanomate interface on an aliquot of the sample. The setting for each neutral loss is indicated above each segment. The abundance (cps, counts per second) of the summed ion current for each neutral loss appears on the ordinate while the lapsed time of nanospray is on the abscissa.
Figure 7
Figure 7
Time course of changes in absolute abundance of RAW 264.7 cell triacylglycerol and diacylglycerol molecular species following stimulation with either Kdo2-lipid A (100 nM) or not (Control) as measured by neutral loss experiments such as that in Figure 6. (A) Changes in abundance of those 50:1-TAG molecular species containing oleate (18:1). (B) Changes abundance of those 50:1-TAG molecular species containing palmitoleate (16:1). (C) Changes in abundance of those 36:4-DAG molecular species containing arachidonate (20:4). The other fatty acyl group for this DAG species would correspond to 16:0.
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