Simultaneous Quantification of Galactolipids

Galactolipids such as monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) are crucial lipids involved in photosynthesis and metabolic regulation. They are affected by various stress conditions such as nutrient limitation. Often when galactolipids are quantified, it is only performed as a sum parameter using methods that do not differentiate between the individual MGDG and DGDG. However, to obtain an impression of the distribution and to evaluate whether the formation of individual MGDG and DGDG changes depending on sulfur nutrition, it was necessary to develop a method that simultaneously determines individual MGDG and DGDG. The development of the new LC-ESI-MS/MS method, as well as the method validation process, are based on procedures described by Fischer et al. [35 (link)] with slight modifications. The galactolipids MGDG and DGDG were analyzed on an Agilent 1260 Infinity Quarternary LC System (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) coupled to a triple quadrupole API 4000 QTrap mass spectrometer (Sciex Germany GmbH, Darmstadt, Germany) equipped with a turbo spray source, which was operating in positive ion mode, with the following mass spectrometer settings: ion spray voltage: 4500 V; ion source heater  =  650 °C source gas 1: 50 psi; source gas 2: 0 psi; and curtain gas: 10 psi. The injection volume for all samples was 5 μL, the column oven temperature was set to 20 °C, and the autosampler was maintained at 4 °C. The separation of analytes was achieved using a Kinetex^® C18 column (2.6 μm, 150 mm × 2.1 mm), equipped with a Kinetex^® C18 security guard column (Phenomenex Inc., Torrance, CA, USA), using a constant flow rate of 300 μL min⁻¹. Eluent A was water with 2 mM ammonium acetate and eluent B was acetonitrile. The elution started with 55% eluent B for 2 min and linearly increased to 87.5% eluent B within 2 min, which was kept constant for 41 min. Then, the composition was readjusted to 55% eluent B within 1 min, followed by 4 min of re-equilibration. MRM transitions of the different galactolipids were obtained by direct flow injection of the MGDG and DGDG mix (in methanol with 2 mM ammonium acetate) into the ESI source in positive mode. Each glycolipid (MGDG, DGDG) precursor ion was determined based on three specific fragment ions (product ions). Table 1 and Table 2 summarize the precursor and fragment ions of all compounds tested in the present study. Mass analyzer settings were optimized for all analytes to maximize the transmission and sensitivity of each characteristic mass transition. These optimizations were acquired automatically by using autotune mode provided by the Analyst^® software 1.6.1 (AB Sciex Germany GmbH, Darmstadt, Germany).