Lipidomic Analysis of Insect Fat Body

Lipids were extracted from wandering 3^rd instar larval fat body as previously described [60 (link)]. The lipidomic analyses were carried out on an analytical system comprising an Agilent HPLC 1260 coupled with a SCIEX 5500 QTRAP. Separation of individual classes of polar lipids by normal phase HPLC was carried out using a Phenomenex Luna 3u silica column (i.d. 150x2.0 mm). Multiple reaction monitoring (MRM) transitions were set up for quantitative analysis of various polar lipids. Individual lipid species were quantified by referencing to spiked internal standards. PC-14:0/14:0, LPC-C20, PE-14:0/14:0, PS-14:0/14:0, PA-17:0/17:0, PG-14:0/14:0 were obtained from Avanti Polar Lipids and dioctanoyl phosphatidylinositol (PI, 16:0-PI) was obtained from Echelon Biosciences, Inc. Separation of glycerol lipids (DAG and TAG) by reverse phase HPLC/ESI/MS/MS was carried out on a Phenomenex Kinetex 2.6μ-C18 column (i.d. 4.6x100mm). Using neutral loss-based MS/MS techniques, the levels of TAG were calculated as relative contents to the spiked d5-TAG 48:0 internal standard (CDN Isotopes), while DAG species were quantified using 4ME 16:0 Diether DG as an internal standard (Avanti Polar Lipids). Free cholesterols and ergosterols were analyzed using HPLC/APCI/MS/MS with the corresponding d6-Cho (CDN Isotopes) as the internal standard.

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Fan W., Lam S.M., Xin J., Yang X., Liu Z., Liu Y., Wang Y., Shui G, & Huang X. (2017). Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition. PLoS Genetics, 13(3), e1006664.

Publication 2017

5500 QTRAP Luna 3u silica column PC-14:0/14:0 PE-14:0/14:0 PS-14:0/14:0 PA-17:0/17:0 PG-14:0/14:0 Kinetex 2.6μ-C18 column d5-TAG 48:0 4ME 16:0 Diether DG

Cholesterols Fat body Glycerol Hplc Isotopes Larval Lipids Ms ms Phosphatidylinositol Silica

Corresponding Organization : Academy of Mathematics and Systems Science

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Variable analysis

independent variables

Wandering 3rd instar larval fat body

dependent variables

Levels of various polar lipids (PC, LPC, PE, PS, PA, PG, PI)
Levels of glycerol lipids (DAG and TAG)
Levels of free cholesterols and ergosterols

control variables

Lipid extraction protocol as previously described [60]
Analytical system comprising Agilent HPLC 1260 and SCIEX 5500 QTRAP
Normal phase HPLC separation using Phenomenex Luna 3u silica column
Reverse phase HPLC/ESI/MS/MS separation of glycerol lipids using Phenomenex Kinetex 2.6μ-C18 column
HPLC/APCI/MS/MS analysis of free cholesterols and ergosterols
Spiked internal standards: PC-14:0/14:0, LPC-C20, PE-14:0/14:0, PS-14:0/14:0, PA-17:0/17:0, PG-14:0/14:0, d5-TAG 48:0, 4ME 16:0 Diether DG, d6-Cho

positive controls

Spiked internal standards for quantification of lipid species

negative controls

Not explicitly mentioned

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