Abstract
Lipidomics approaches provide quantitative characterization of hundreds of lipid species from biological samples. Recent studies highlight the value of these methods in studying circadian biology, and their potential goes far beyond studying lipid metabolism per se. For example, lipidomics analyses of subcellular compartments can be used to determine daily rhythmicity of different organelles and their intracellular dynamics. In this chapter we describe in detail the procedure for around the clock shotgun lipidomics, from sample preparation to bioinformatics analyses. Sample preparation includes biochemical fractionation of nuclei and mitochondria from mouse liver harvested throughout the day. Lipid content is determined and quantified, in unbiased manner and with wide coverage, using multidimensional mass spectrometry shotgun lipidomics (MDMS-SL). Circadian parameters are then determined with nonparametric statistical tests. Overall, the approach described herein is applicable for various animal models, tissues, and organelles, and is expected to yield new insight on various aspects of circadian biology and lipid metabolism.
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Notes
- 1.
Melt sucrose on a slightly warm plate, prior to addition of other ingredients. Prepare buffer in advance, at least 1 day prior to fractionation. This ensures hardening which will sustain the biological sample. This buffer can be stored in 4 °C for up to 1 month. Titer to pH with either KOH or NaOH.
- 2.
See footnote 1.
- 3.
See footnote 1.
- 4.
Polycarbonate is important for reuse of tubes, from our experience other materials might damage when reusing.
- 5.
Titer with KOH to improve extraction.
- 6.
Its purpose is to remove fat tissue from the sample.
- 7.
Else, could be performed on 106 cells, 100 μL body fluids
- 8.
We purchase all of the lipid internal standards from Avanti Polar Lipids, Inc., Alabaster, AL, USA except if otherwise noted.
- 9.
In case of liver analysis without fractionation rapidly freeze the tissue in liquid nitrogen and keep frozen until lipidomics.
- 10.
Nuclei isolation can be done with a liver which has been frozen in −80 °C.
- 11.
This is a crucial step: materials must be very cold to avoid turbulence. Transfer the sample in slope, gently on the wall of the tube. The two phases must not mix, this would destroy the separation. If mixture occurs cease immediately and resume with the rest of the sample on top of a new Cushion. The mixed samples cannot be used.
- 12.
This contains crude cytosolic fraction, which, if desired, can be used for western blot. In that case, collect with a pipette and store at −80 °C until use.
- 13.
Vacuum far from the pellet, tilt tube to distance the liquid from the pellet.
- 14.
Avoid scratching the tube for further use
- 15.
For maximal yield first resuspend in half the volume, transfer it, and thoroughly collect the remaining pellet with the rest.
- 16.
Only fresh liver is to be used, mitochondria do not fractionate well from frozen liver.
- 17.
Manual homogenization; Avoid creating vacuum force and/or air bubbles!
- 18.
For maximal yield first resuspend in half the volume, transfer it, and thoroughly collect the remaining pellet with the rest.
- 19.
Avoid the fat layer on top and the lower layer at the bottom. Aim for ∼10–13 mL to be on the safe side.
- 20.
Steps 8 and 9 are an optional wash, we perform it.
- 21.
Equivalent to 10–20 mg wet weight of tissue or ∼1 × 106 cells.
- 22.
The stock solution of each single internal standard is prepared in chloroform/methanol (1:1, v/v) with a concentration approximately 1Â mg/mL. The amount of each single lipid species in the premixture is based on the abundance of the corresponding lipid class in the samples. The molecular species of internal standards are selected because they represent <0.1% of the endogenous cellular lipid mass levels as predetermined by ESI-MS lipid analysis.
- 23.
See footnote 22.
- 24.
In order to eliminate the contamination from the top layer (aqueous phase) to the bottom phase, insert the glass Pasteur pipet into the upper layer with slowly air bubbling until the pipet inserts into the bottom layer, which could avoid the upper layer liquid going into the pipet. When taking the glass pipet out from the upper layer, touch the outside of the pipet on the edge of the glass tube, and quickly transfer the bottom layer to a new glass tube.
- 25.
The total lipid concentration of a lipid extract can be estimated through the protein content or on the basis of the lipid analysis results from previous experiments [12]. This knowledge is useful for estimation of the concentrations of total lipids from different lipid extracts to prevent lipid aggregation during analysis through mass spectrometry. The lithium hydroxide is made of 80-time dilution of a saturated methanol solution.
- 26.
Since sample ionization (ChipSoft) and spectral collection (Xcalibur) are operated with two separate software programs, the ionization polarity and time controlled by the ChipSoft should be matched to those of the mass spectrometer. The mass spectrometer is triggered to start collecting spectra with the start of the nanospray.
- 27.
For the triple-quadrupole mass spectrometer, the first and third quadrupoles are used as independent mass analyzer with a mass resolution of 0.7 Th, and the second quadrupole serves as a collision cell for tandem mass spectrometry [13]. For the analysis of cardiolipin, a high-resolution mass spectrometer (at least Δm 10,000 full width at half maximum, FWHM) is applied to detect and differentiate the doubly charged ions.
- 28.
The isotope effects from other atoms (such as hydrogen, nitrogen, or phosphorus) are usually neglected due to extremely low abundance of its isotope or very small differences between the analyzed species and the selected internal standard.
- 29.
The differences in the number of total carbon atoms and the number of total double bonds present in fatty acyl chains of each individual species from those of the selected standards can be considered as two important variables with multivariate least-square regression to determine the correction factors for each individual molecular species for the second-step quantification. The linear dynamic range of quantification is extended dramatically by using this second step to quantify the overlapping and/or low-abundance species with one or more MS/MS scans through reduce background noise, increase S/N ratios of low-abundance species, and filter the overlapping molecules with class-specific PIS or NLS.
References
van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9(2):112–124. https://doi.org/10.1038/nrm2330
Adamovich Y, Aviram R, Asher G (2015) The emerging roles of lipids in circadian control. Biochim Biophys Acta 1851(8):1017–1025. https://doi.org/10.1016/j.bbalip.2014.11.013
Asher G, Sassone-Corsi P (2015) Time for food: the intimate interplay between nutrition, metabolism, and the circadian clock. Cell 161(1):84–92. https://doi.org/10.1016/j.cell.2015.03.015
Gooley JJ, Chua EC (2014) Diurnal regulation of lipid metabolism and applications of circadian lipidomics. J Genet Genomics 41(5):231–250. https://doi.org/10.1016/j.jgg.2014.04.001
Adamovich Y, Rousso-Noori L, Zwighaft Z, Neufeld-Cohen A, Golik M, Kraut-Cohen J, Wang M, Han X, Asher G (2014) Circadian clocks and feeding time regulate the oscillations and levels of hepatic triglycerides. Cell Metab 19(2):319–330. https://doi.org/10.1016/j.cmet.2013.12.016
Kasukawa T, Sugimoto M, Hida A, Minami Y, Mori M, Honma S, Honma K, Mishima K, Soga T, Ueda HR (2012) Human blood metabolite timetable indicates internal body time. Proc Natl Acad Sci U S A 109(37):15036–15041. https://doi.org/10.1073/pnas.1207768109
Aviram R, Manella G, Kopelman N, Neufeld-Cohen A, Zwighaft Z, Elimelech M, Adamovich Y, Golik M, Wang C, Han X, Asher G (2016) Lipidomics analyses reveal temporal and spatial lipid organization and uncover daily oscillations in intracellular organelles. Mol Cell 62(4):636–648. https://doi.org/10.1016/j.molcel.2016.04.002
Han X, Gross RW (2005) Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom Rev 24(3):367–412
Han X, Yang K, Gross RW (2012) Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses. Mass Spectrom Rev 31(1):134–178. https://doi.org/10.1002/mas.20342
Yang K, Cheng H, Gross RW, Han X (2009) Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics. Anal Chem 81(11):4356–4368. https://doi.org/10.1021/ac900241u
Hughes ME, Hogenesch JB, Kornacker K (2010) JTK_CYCLE: an efficient nonparametric algorithm for detecting rhythmic components in genome-scale data sets. J Biol Rhythm 25(5):372–380. https://doi.org/10.1177/0748730410379711
Han X (2016) Lipidomics: comprehensive mass spectrometry of lipids. Wukey series on mass spectrometry. Wiley, Hoboken, New Jersey
Han X, Yang K, Yang J, Cheng H, Gross RW (2006) Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples. J Lipid Res 47(4):864–879. https://doi.org/10.1194/jlr.D500044-JLR200
Yang K, Cheng H, Gross RW, Han X (2009) Automated lipid identification and quantification by multi-dimensional mass spectrometry-based shotgun lipidomics. Anal Chem 81:4356–4368
Yang K, Fang X, Gross RW, Han X (2011) A practical approach for determination of mass spectral baselines. J Am Soc Mass Spectrom 22:2090–2099
Han X, Gross RW (2001) Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal Biochem 295(1):88–100
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Aviram, R., Wang, C., Han, X., Asher, G. (2021). A Lipidomics View of Circadian Biology. In: Brown, S.A. (eds) Circadian Clocks. Methods in Molecular Biology, vol 2130. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0381-9_12
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DOI: https://doi.org/10.1007/978-1-0716-0381-9_12
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