De novo synthesis of ovarian lipid reserves.
The use of stable isotope labelling combined with ultrahigh resolution mass spectrometry has shown significant advantages for the analysis of biological pathways.6,26,28,41,42 Different from other labelling techniques, the incorporation of stable isotopes does not change the chemical properties of the lipid and allows the detection of the labeled molecule by their isotopic profile. This approach enables direct analysis of nutrient distribution, mobilization and metabolism.4,23,37,43-45 While the concept is simple, for the analysis of complex biological samples it requires: i) the use of complementary pre-separation techniques to diminish matrix effects and increase the sensitivity of the analysis 27,37,46-53 and ii) the use of high magnetic fields to achieve ultrahigh mass resolution with short transient duration for compatibility with LC separation (e.g., resolving power greater than m/Dm50% > 1000000 and mass accuracy better than 1 ppm).27,36,41-43,46-48 More recently, in addition to the use of higher magnetic fields, alternative detection strategies (e.g., hexapolar detection, 3W+) has allowed better sensitivity and shorter analysis time, making more efficient the coupling of LC-FT-ICR MS.41,46,47,54 Adult female mosquitoes were offered either 20% sucrose/water or 20% sucrose/2H2O water for a period of 2, 3, 4, 6 and 7 days after emergence. Inclusion of deuterated water (2H2O) in the mosquito diet during the PVG stage, provided unequivocal identification of de novo TG dynamics. When offered a sucrose-2H2O diet, adult female mosquitoes synthesized de novo FA using isotopically labeled substrates, such as acetyl CoA, NADPH and water, which became metabolically enriched with 2H (Figure 1).
Identification and quantification of TGs from mosquito ovaries
Typical LC and MS profiles for unlabeled TGs extracted from ovaries generated using the LC-URMS workflow are shown in Figure 2. TG signals were in the 800-900 m/z range, eluting at 35-45 minutes after injection of the sample. The molecular ammonium adduct ion forms ([M+NH4]+) of TG species showed higher abundance than the sodium adduct ions ([M+Na]+), and were used for quantitation analysis (Figure 2). In most cases, a single LC band was observed per TG; however, in the case of TG 50:4 and TG 50:5, the double bands corresponded to the presence of positional isomers. Table 1 displays a summary of the TGs detected using LC-FT-ICR MS with a mass accuracy lower than 300 ppb. TGs extracted from ovaries of females fed 2H2O labeled sugar diets revealed similar EIC profiles; although, the addition of 2H creates distinct isotopic patterns (Figure S1). The use of ultrahigh resolution FT-ICR MS allowed the observance of the isotopic fine structure. This permits direct discrimination of the signals containing 2H from that of naturally occurring isotopes (e.g., 13C).
The de novo synthesized TGs have the characteristic presence of 2H in their elemental composition (Figure 3). For example, the mass profile of de novo synthesized TG 48:2 showed the substitution of 30 hydrogens by deuterium atoms (Figure 3A). These substitutions were absent in ovarian samples from females that received a non-labeled sucrose diet (Figure 3B). A closer inspection to the 820 – 834 m/z range highlights the importance of ultrahigh resolution MS for this type of analysis (Figure 3C). For example, at the nominal mass level, the 826.70 – 826.80 range contains signals from TG 48:2 [M+Na+] 1x13C isotope, TG 48:2 [M+NH4+] 5x2H and 1x13C isotopes, and TG 48:2 [M+NH4+] with 6x2H. These signals measured at lower resolving powers would result in convolution of the two peaks, contributing ambiguous peak assignment and spurious quantification values.
The trends of changes and quantities of TG were similar in ovaries from females raised on non-labeled diet and labeled diet (Figure 4). In good agreement with previous reports, TGs containing 48, 50, 52, and 54 carbons were the most abundant species.27 As a general tendency, the amount of TGs increased with the days after eclosion, reaching a steady state towards the last days. Most significantly, deuterium incorporation was observed in all TGs species, but with unique dynamics for each of them. In terms of total lipid amounts, three-quarters of TG lipids were labeled during the six-day experiment, with the percentage of 2H incorporation increasing as a function of time (Figures 4 and S2).
Dynamics of de novo TG incorporation into the ovary
The investigation of the dynamic changes of ovarian lipids during the PVG period was assisted by the incorporation of stable isotopes, providing an additional dimension of absolute quantitative values. The analysis of the number of deuterium atoms incorporated into eighteen different TG species at different days after adult eclosion, indicated a dynamic constant adjustment of ovarian TG stores during the experiment. In Figure 5, we display the distribution of the number of deuterium incorporated into each TG as a function of time. Results are expressed as amount of ovarian TG deuterated species relative to the TG 48:1 (d7) internal standard using a color scale. The replacement of hydrogen by deuterium in TGs was already detected at day 2 after eclosion. At day 3 we observed a significant increase of 2H-TGs. The day when we recorded the highest number of incorporated 2H was different depending on the carbon number and unsaturation degree of the TG. At day 4, TGs 54:2, 54:5 and 54:6 have at least 20x 2H incorporated into their lipid chains; while TG 50:4, TG 52:1 and TG 52:2 have less than 10x 2H incorporated. We observed a positive correlation between the number of 2H incorporated into a TG and the carbon number, molecular size and number of unsaturated bonds. TG 54 and TG 52 were more abundant than TG 48, validating a relation between chain length and ability to incorporate deuterium. Interestingly, at day 7 the median number of 2H incorporations and total amount of TGs species in the ovary decreased; except for TGs 52:3, 52:4 and 52:5, suggesting that some of the follicles might have been resorbed. In terms of kinetics, the saturated species, such as TG 50:1; TG 52:1; TG 54:2, showed high deuterium incorporation at day 3, suggesting the deuteriums incorporated in single bonds are more stable than those incorporated in species rich in double bonds, such as TG 50:5; TG 52:5 and TG 54:6.