A direct infusion ESI-MS/MS approach was used to detect and quantify SGs and ASGs containing sitosterol, campesterol and stigmasterol (). A major advantage of this approach is that it is not necessary to separate SGs and ASGs from other compounds prior to analysis. Both SGs and ASGs can be analyzed as [M + NH4
ions, and detected in positive mode by a series of neutral loss scans specific for the hexose or acyl hexose moieties. SGs and ASGs with greater than one sugar are very rare [7
] and we did not target these compounds in our analysis. shows the mass spectral scan values that were used to detect SG and ASG molecules. A previous report on analysis of cholesteryl esters using ESI-MS/MS indicated that mass spectral response varied considerably among steryl esters with different acyl groups [20
]. To determine whether various SG and ASG molecular species have variable mass spectral responses, isolated soybean-derived SG and ASG mixtures from a commercial source were separately analyzed by GC-FID and the results were compared with those obtained by ESI-MS/MS. The data indicate that, although there was a slight tendency of the mass spectrometer to be more sensitive to SGs and ASGs containing campesterol relative to those containing the sitosterol and stigmasterol, the mole fractions of each sterol in the SG and ASG mixtures detected by the two methods were very similar (). Sitosterol was the most prominent sterol found in both commercially-obtained soybean SGs and ASGs. Campesterol and stigmasterol were the other major sterols, while minor sterols comprised ~2% or less.
Mass spectral scan values for steryl glucosides and acyl steryl glucosides.
Figure 2 Sterols from purified soybean SGs and ASGs as mole fraction of total detected sterol compounds. After hydrolysis of the SGs or ASGs, sterols were determined by GC-FID, or the SGs and ASGs were analyzed intact by ESI-MS/MS. In the ESI-MS/MS data, the signals (more ...)
Analysis of the purified ASG mixture by GC-FID and ESI-MS/MS showed a tendency of the mass spectrometer to be less sensitive to 18:2 (linoleic acid)-containing ASGs than to other fatty-acyl SGs, but the fatty acid compositions of the purified ASGs determined by the two methods were similar overall (). The major fatty acid found in purified commercially-obtained soybean ASGs was 16:0 (palmitic acid), comprising greater than 50% of the total, followed by 18:2 (linoleic), 18:0 (stearic), 18:1 (oleic), 18:3 (linolenic) and 16:1 (palmitoleic) acids. Taken together, the data indicate that the composition of SG and ASG determined by ESI-MS/MS of the isolated compounds is very close to that determined by GC-FID, indicating that various SG and ASG molecular species have similar molar mass spectral responses. In addition, our data are comparable to the percentile compositions provided by Matreya LLC, the commercial source of the SG and ASG mixtures (see Experimental Procedure).
Figure 3 Fatty acids of purified soybean ASG as a mole fraction of total detected fatty acids. Fatty acids were determined by GC-FID after formation of methyl esters, or the ASGs were analyzed intact by ESI-MS/MS. In the ESI-MS/MS data, the signals for various (more ...)
We next applied ESI-MS/MS to seed samples from wild-type Arabidopsis and the ugt80A2,B1 mutant using a spike-in experiment to verify linearity of response of the normalized mass spectral signals. SGs and ASGs were analyzed by direct infusion of unseparated seed extracts, and known amounts of soybean SGs and ASGs were spiked-in to wild-type and mutant samples to determine the linearity of their ESI-MS/MS response in the presence of other seed lipids. The linearity of SG and ASG response was good over the range in which the mutant and wild-type are compared, and the mutant and wild-type spike-in data form parallel lines (). The data show that wild-type values fall within the linear range of the spike-in to the mutant samples, indicating that the amounts of SGs and ASGs in the wild-type samples are linearly related to the amounts in the mutant samples. These data indicate that the ESI-MS/MS method is useful for comparison of biological samples.
Figure 4 Spike-in experiments indicate linear responses in mass spectral signals from ESI-MS/MS analysis of SGs and ASGs. Normalized mass spectral signals are shown for compounds from Arabidopsis seeds of wild-type (squares) and the ugt80A2,B1 mutant (circles) (more ...)
Levels of SGs and ASGs in Arabidopsis
seeds of ugt80A2,B1
mutants and wild-type are shown in (Table S1
) with individual molecular species of SGs and ASGs indicated. The masses detected were consistent with identification of sitosterol, campesterol, and stigmasterol as the three major sterols in Arabidopsis
seed SGs and ASGs. Minor sterols, with masses consistent with identifications of cholesterol and brassicasterol, were also detected by ESI-MS/MS as ~2% or less of the total SGs (data not shown). The data show that sitosteryl, campesteryl, and stigmasteryl glucosides were significantly reduced by 85%, 92%, and 82% (7-, 13- and 6-fold), respectively, in ugt80A2,B1
in comparison to wild-type seeds, and total SGs were reduced by 86% (7-fold) (; Table S1
). Although SG levels were reduced in ugt80A2,B1
, the relative proportions of SGs that contained sitosterol, campesterol and stigmasterol were similar to those in wild-type. In addition, we observed that the levels of phospholipids PtdCho and PtdEtn were very similar in the mutant and wild-type (Table S2
), indicating that the ugt80A2,B1
mutant is specifically deficient in SGs and ASGs.
Figure 5 SG and ASG levels are reduced in seeds from ugt80A2,B1 mutants of Arabidopsis. ESI-MS/MS was applied to analyze SGs and ASGs from seeds of wild-type (WT) (solid bars) and ugt80A2,B1 (stippled bars). Normalized mass spectral signals per mg seed weight (more ...)
The major ASG molecular species, 16:0 (palmitic), 18:1 (oleic), 18:2 (linoleic), and 18:3 (linolenic) acids were reduced to a slightly greater extent than SGs in ugt80A2,B1
compared to wild-type seeds. Total ASG levels were reduced 96% (23-fold) in the double mutant (; Table S1
). Among the ASGs detected in seeds, 16:0 (palmitic acid) was the predominant acyl group accounting for 57% and 66% of the total ASG signal in wild-type and mutant, respectively. Strikingly, campesterol-containing ASGs were reduced 98% (61-fold), while decreases in sitosterol- and stigmasterol-containing ASGs were reduced to a lesser extent. Campesterol-containing ASGs accounted for only 10% of the ASGs in ugt80A2,B1
in comparison to 25% in wild-type. In wild-type, the signal from SGs accounted for 88% of the total glucosylated sterol signal (SGs + ASGs), and in the ugt80A2,B1
mutant the signal from SGs accounted for 96% percent of the total glucosylated sterols. Overall, there was a reduction of 87% (8-fold) in SG + ASG content in the mutant in comparison to wild-type.