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

Abstract

Background: Wax 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.

Results: We 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.

Conclusions: We 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.

Figure 1

Schematic workflow of wax ester profiling by nano-ESI-MS/MS.

Figure 2

Thin layer chromatographic separation of seed lipid extracts. Separation of seed lipid extracts of wild type plants (*marked with asterisk) or wax ester accumulating transgenic plants. The silica TLC plate was developed with hexane:diethyl ether:acetic acid (80:20:0.1, v/v/v). Bands for the triacylglycerols (TAG), wax esters (WE) and steryl esters (SE) were identified by analysis of respective lipid class standards. Lipids were visualized by charring after immersion in cupric sulfate solution.

Figure 3

Collision-induced dissociation mass spectra of wax ester standards. Spectra were obtained from positive nano-ESI-MS/MS of the ammonium adduct [M+NH₄]⁺ precursor ion representing the fragmentation pattern of seven wax ester prototype groups. Wax ester dissociate into characteristic analytical product ions (A) presenting the protonated acid ion a: [RCO₂H₂]⁺, the acylium ion b: [RCO]⁺, the dehydrated acylium ion c: [RCO – H₂O]⁺ and the alcohol-specific loss of fatty acid fragment [R´ ]⁺ with R and R´ representing the fatty acid or the alcohol moiety, respectively. The mass spectra (B-H) show fragmentation patterns of wax esters as labeled. Analytical fragment ions a-d are labeled in accordance to (A).

Figure 4

Wax ester profiles of (A) Arabidopsis transgenic lines expressing MaFAR and JoWS and (B) jojoba seed oil. Shown are the means (+SD) of the relative accumulation of the ten most abundant wax ester species in mol % of total wax ester accumulation of ten individual transgenic lines for (A) and four replicate measurements for (B).

Figure 5

Acyl chain profile calculated from wax ester composition. The relative incorporation of specific acyl groups at the (A) alcohol moiety and the (B) acyl moiety that was calculated based on the wax ester profiles of transgenic MaFAR/ScWS-expressing Arabidopsis thaliana (MaFAR+ScWS) and jojoba seed oil.