Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Summary

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

Abstract

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (<1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Introduction

When analyzing lipid data, stable isotope labeling (SIL) is an effective method to assess metabolic pathways in living organisms. In this method, atoms within an analyte are replaced by stable isotopes containing ¹³C or ²H. These isotopes are further incorporated into precursors, which are later embedded within the fatty acids, labeling the areas in which they reside. With these isotopes, one is able to identify the distribution and metabolism of lipids, as they will contrast against the unlabeled background¹. Common mass spectrometry platforms are not able to distinguish the signal coming from endogenous molecules

Protocol

1. Sample preparation

1. Insect dissection
   1. Dissect Aedes aegypti mosquito ovaries when they are 7-days old in a phosphate buffered saline solution using microneedles.
   2. Collect eight ovaries from phosphate buffered saline solution using microneedles and store in 1.5 mL microcentrifuge tubes at -80 ˚C.
   3. Collect ovaries in biological replicates.
2. Lipid extraction
   1. Add 10 µL of internal standard (ISTD) into the eigh.......

Discussion

When assessing the use of this protocol, it is essential to ensure the DIA-PASEF and MS/MS parameters are applicable to the triglycerides in question. Specifically, mobility windows and window width should follow the pattern of the triglycerides. This can be determined with a previous run using the DDA method. When tracking the triglycerides in the internal standard, the windows for the DIA set up should cover all the pre-identified molecules of interest. The windows should all be of equal sizes, and the ranges fall with.......

Materials

Name	Company	Catalog Number	Comments
Accucore C30 colum	Thermo Fisher Scientific	27826-252130
Bruker Compass Data Analysis	Bruker Daltonics Inc	2363525870	Version 5.2
d-Glucose-13C6	Sigma-Aldrich	389374
eppendorf tubes	Fisher Scientific	14-282-300
EquiSplash Lipidomix	Avanti Polar Lipids	330731-1EA
glass vials	Thermo Fisher Scientific	11-417-236
heavy water (2H2O)	Sigma-Aldrich	1.13366
polypropylene pestles	Fisher Scientific	BAF199230001
Prominence LC-20 CE ultrafast liquid chromatograph	Shimadzu	L20234651907
silanized glass inserts	Thermo Fisher Scientific	03-251-826
Sucrose-13C12	Sigma-Aldrich	605417
timsTOF	Bruker Daltonics Inc	1.84443E+11
Tuning Mix Calibration Standard	Agilent Technologies	36