Direct qualitative analysis of triacylglycerols by electrospray mass spectrometry using a linear ion trap

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

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

“…In spite of this level of both chromatographic and mass spectral separation, each [M+NH 4 ] + was composed of numerous isobaric, yet distinct components. For example, the ion at m/z 876.8, corresponding to TAGs 52:2 (calculated fatty acyl carbon atoms: total number of double bonds), was previously reported to contain over 15 distinct species having unique fatty acyl groups and not counting positional isomers [20]. In part, this can be observed by MS/MS of m/z 876.8 ( Figure 3B…”

“…For the example, the [M+NH 4 ] + present in this mixture of TAGs observed at m/z 876.8, corresponded to a family of 52:2 TAG species. The MS 2 product ions corresponding to the eight different fatty acyl groups described above were subjected to MS 3 analysis and observed product ions compared to expected product ions for fatty acyl groups 2:0 to 25:2 ( (20). The neutral losses from suspected RC≡O + and/or R′ + 74 ions in the MS 3 mass spectra, the third fatty acyl group was determined (Table 3).…”

“…In recent years there has been renewed interest in the development of mass spectrometric methods using electrospray ionization and atmospheric pressure chemical ionization to analyze TAGs at the molecular species level [16][17][18][19]. We recently reported a strategy using MS 3 to analyze TAGs from a complex mixture as the NH 4 + adduct ions generated by electrospray ionization [20]. This method was used to identify positional isomers of unique molecular species at the level of individual esterified fatty acyl groups present within the macrophage derived cell line, called RAW 264.7 cells, with the estimated total of unique molecular species being well over 500.…”

A comparative study of the composition of triacylglycerol molecular species in equine and human milks

“…TAGs ammonium adducts were prepared as described by McAnoy et al (2005). Each TAG fraction collected by preparative NARP-HPLC was diluted in 0.5 mL of chloroform/methanol (1:1, v/v) and 25 μL aqueous NH 4 OAc (10 mM) was then added.…”

Application of Probe Electrospray Ionization Mass Spectrometry (PESI-MS) to Clinical Diagnosis: Solvent Effect on Lipid Analysis

“…Although it is widely known that TAGs are hallmarks of RCC, our findings indicated that TAG with different fatty acyl substituents that are not observed in noncancerous tissue could be useful for the application of PESI-MS to cancer diagnosis, as well as to metabolomics underlying the disease [22]. Additionally, sequential and exhaustive ionization of analytes by PESI-MS would guarantee significant potential for the application of mass spectrometry to rapid and real-time clinical diagnosis to guide surgery.…”