No abstract

Triacylglycerols (TAGs) isolated from a biological sample provide a challenge for mass spectrometric analysis because of the complexity of naturally occurring TAGs, which may contain different fatty acyl substituents resulting in a large number of molecular species having the identical elemental composition. We have investigated the use of mass spectrometry to obtain unambiguous information as to the individual TAG molecular species present in a complex mixture of triacylglycerols using a linear ion trap mass spectrometer. Ammonium adducts of TAGs, [M ϩ NH 4 ] ϩ , were generated by electrospray ionization, which permitted the molecular weight of each TAG molecular species to be determined. The mechanisms involved in the decomposition of the [M ϩ NH 4 ] ϩ and subsequent fragment ions were investigated using deuterium labeling, MS/MS, and MS 3 experiments. Collision induced decomposition of [M ϩ NH 4 ] ϩ ions resulted in the neutral loss of NH 3 and an acyl side-chain (as a carboxylic acid) to generate a diacyl product ion. MS/MS data were used to identify each acyl group present for a given [M ϩ NH 4 ] ϩ ion, and this information could be combined with molecular weight data to identify possible TAG molecular species present in a biological extract. Subsequent MS 3 experiments on the resultant diacyl product ions, which gave rise to acylium (RCO ϩ ) and related ions, enabled unambiguous TAG molecular assignments. These strategies of MS, MS/MS, and MS 3 experiments were applied to identify components within a complex mixture of neutral lipids extracted from RAW 264.7 cells. (J Am Soc Mass Spectrom 2005, 16, 1498 -1509

Triacylglycerols are neutral lipids present in all mammalian cells as energy reserves and diacylglycerols as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of triacylglycerols and diacylglycerols present in mammalian cells are quite complex and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600-900 daltons, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed using tandem mass spectrometry 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 triacylglycerols 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 hr time course after Kdo 2 -lipid A exposure whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.

The gas phase degradation reactions of the chemical warfare agent (CWA) simulant, dimethyl methylphosphonate (DMMP), with the hydroperoxide anion (HOO(-)) were investigated using a modified quadrupole ion trap mass spectrometer. The HOO(-) anion reacts readily with neutral DMMP forming two significant product ions at m/z 109 and m/z 123. The major reaction pathways correspond to (i) the nucleophilic substitution at carbon to form [CH(3)P(O)(OCH(3))O](-) (m/z 109) in a highly exothermic process and (ii) exothermic proton transfer. The branching ratios of the two reaction pathways, 89% and 11% respectively, indicate that the former reaction is significantly faster than the latter. This is in contrast to the trend for the methoxide anion with DMMP, where proton transfer dominates. The difference in the observed reactivities of the HOO(-) and CH(3)O(-) anions can be considered as evidence for an alpha-effect in the gas phase and is supported by electronic structure calculations at the B3LYP/aug-cc-pVTZ//B3LYP/6-31+G(d) level of theory that indicate the S(N)2(carbon) process has an activation energy 7.8 kJ mol(-1) lower for HOO(-) as compared to CH(3)O(-). A similar alpha-effect was calculated for nucleophilic addition-elimination at phosphorus, but this process--an important step in the perhydrolysis degradation of CWAs in solution--was not observed to occur with DMMP in the gas phase. A theoretical investigation revealed that all processes are energetically accessible with negative activation energies. However, comparison of the relative Arrhenius pre-exponential factors indicate that substitution at phosphorus is not kinetically competitive with respect to the S(N)2(carbon) and deprotonation processes.

The collision-induced spectra of [M À H] À ions of a variety of natural and synthetic amphibian peptides containing Asp and/or Glu exhibit characteristic g backbone cleavage ions that identify the positions of these residues in the peptide. A theoretical study suggests that the Glu cleavage involves an S N i reaction of the carboxylate anion from the Glu a side chain to form a deprotonated cyclic lactone. The presence of either Asp or Glu or other residues that effect pronounced side-chain cleavages (e.g. Ser or Thr) results in the normal a and b backbone cleavages being reduced in comparison to those cleavages which originate from side chains.