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US 10,705,100 B1Granted
Methods for analyzing lipids and membrane proteins in biological matter using stable isotopes and mass spectrometry
OwnerScott B. Hansen, Andrew S. Hanseninventors
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Abstract
Methods for accurately comparing the levels of ionizable lipids in two cell populations that differ in some respect from each other using mass spectroscopy and isotopic labeling are provided. The methods can be used to identify a change in a lipid of interest in response to a cellular, chemical, genetic, or environmental change to the cell population (i.e., a lipid response to a cell perturbation). The change in the lipid of interest can be a change in composition, rate of synthesis, and/or location of the lipid.
Claims13
A method for determining the relative abundance of a lipid of interest in a cell population, comprising:
- (i) culturing a first sample of the cell population in a first medium, the first medium including a lipid precursor having a first abundance of at least one isotope, the lipid precursor selected to label 2 to 6 atoms of a membrane lipid;
- (ii) culturing a second sample of the cell population in a second medium, the second medium including the lipid precursor with a second and different abundance of the at least one isotope,
- wherein the lipid precursor in each of the samples is incorporated into a biological lipid produced within the respective samples;
- (iii) modulating one of the samples of the cell population, wherein between the first and second cell populations, the modulation produces a difference in the relative abundance of a lipid of interest in the respective first and second cell populations;
- (iv) isolating a lipid fraction from each of the first and second samples, the lipid fraction from each of the samples including the respective biological lipid produced therein;
- (v) combining at least a portion of the lipid fraction from the first sample with at least a portion of the lipid fraction from the second sample to form a combined lipid fraction, the combined lipid fraction comprising the biological lipids from each of the samples;
- (vi) subjecting the isolated lipids in the combined sample to mass-spectrometry to produce mass spectrometry data;
- and
- (vii) determining the relative abundance of a lipid of interest in the first sample compared to the second sample using the mass spectrometry data.
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