Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry

Abstract

BackgroundWax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.ResultsWe present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.ConclusionsWe developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.

Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions

“…The authors attributed it to an ammoniated carboxylic acid group of an odd electron species[17]; however, their assignment is unlikely as radical ions are not typically formed during ESI-MS analysis of wax esters. A peak corresponding to [RCOOH 2 + H 2 O] + was also apparent in Figure 3B of reference [23]. …”

“…The typical fragments that contain the fatty alcohol moiety derived from ESI-CID of wax esters were [R′ ] + ions [15, 17, 23], which most likely resulted from charge-induced dissociation as proposed for short chain esters by Munson et al [33] Interestingly, for dissociation of unsaturated fatty alcohol moiety-containing wax esters, the intensities of the [R′ ] + peaks were much higher (Figure 5, Supplementary Figure S35 and Supplementary Table S2), which is reasonably explained by the mechanism we propose that involves the formation of secondary carbocation (in contrast, primary carbocation is formed from dissociation of saturated wax esters) and further delocalization of the charge with hydride migration (Supplementary Scheme S1b). Again, the steps include ammonium adduct formation with the double bond via cation-π interaction [44, 45] and charge-remote dissociation involving a six-membered ring transition state [46, 47].…”

“…The latter format was adopted in this work. Please note that some studies present a mixed format of wax ester shorthand (e.g., 18:0/16:1 for stearyl palmitoleate) [23] and one needs to keep this in mind when comparing the results from different reports.…”

“…The CID spectra of wax esters using ESI [17, 20, 23–25], CI [13] and APCI [15] have been reported and the major product ions have been applied for identification and quantitation of wax esters. Some of these studies utilized a method known as multiple-reaction monitoring (MRM) [26] in which only one or very few product/precursor ion pairs (or transition) were used for detection and quantification of specific wax esters in complex biological samples such as seed oils and meibomian gland secretions [23–25].…”

“…Some of these studies utilized a method known as multiple-reaction monitoring (MRM) [26] in which only one or very few product/precursor ion pairs (or transition) were used for detection and quantification of specific wax esters in complex biological samples such as seed oils and meibomian gland secretions [23–25]. This MRM approach negates the need for scanning resulting in increased detection sensitivity and greater analysis throughput.…”