Abstract
Apolipoproteins are major structural and functional constituents of lipoprotein particles. As modulators of lipid metabolism, adipose tissue biology, and energy homeostasis, apolipoproteins may serve as biomarkers or potential therapeutic targets for cardiometabolic diseases. Mice are the preferred model to study metabolic disease and CVD, but a comprehensive method to quantify circulating apolipoproteins in mice is lacking. We developed and validated a targeted proteomics assay to quantify eight apolipoproteins in mice via proteotypic signature peptides and corresponding stable isotope-labeled analogs. The LC/MS/MS method requires only a 3 µl sample volume to simultaneously determine mouse apoA-I, apoA-II, apoA-IV, apoB-100, total apoB, apoC-I, apoE, and apoJ concentrations. ApoB-48 concentrations can be calculated by subtracting apoB-100 from total apoB. After we established the analytic performance (sensitivity, linearity, and imprecision) and compared results for selected apolipoproteins against immunoassays, we applied the method to profile apolipoprotein levels in plasma and isolated HDL from normocholesterolemic C57BL/6 mice and from hypercholesterolemic Ldl-receptor- and Apoe-deficient mice. In conclusion, we present a robust, quantitative LC/MS/MS method for the multiplexed analysis of eight apolipoproteins in mice. This assay can be applied to investigate the effects of genetic manipulation or dietary interventions on apolipoprotein levels in plasma and isolated lipoprotein fractions.
Highlights
Apolipoproteins are major structural and functional constituents of lipoprotein particles
Digestion kinetics of the eight investigated apolipoproteins, which are summarized in supplemental Fig. S2, confirmed that all selected signature peptides were released within 16 h of tryptic digestion
The presented method was suitable to quantify apolipoproteins in plasma and isolated lipoproteins (HDL) of WT C57/BL6 mice and hypercholesterolemic LDL receptor-deficient mouse (LDLR0) and apoE-deficient mouse (ApoE0) mice. This may be of particular interest, as profiling of apolipoprotein concentrations within lipoproteins may allow a more detailed view on pathophysiology associated with specific apolipoproteins
Summary
Apolipoproteins are major structural and functional constituents of lipoprotein particles. We present a robust, quantitative LC/MS/MS method for the multiplexed analysis of eight apolipoproteins in mice This assay can be applied to investigate the effects of genetic manipulation or dietary interventions on apolipoprotein levels in plasma and isolated lipoprotein fractions.—Wagner, R., J. Apolipoproteins like apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, and apoE that can associate with various lipoprotein classes are key players in lipid metabolism and may represent targets for the diagnosis and treatment of dyslipidemias and ASCVD [5,6,7]. The coupling of liquid chromatography and tandem mass spectrometry (LC/MS/ MS) facilitates simultaneous quantitation of multiple proteins from low volumes of plasma with high throughput [20,21,22] To this end, surrogate proteotypic peptides, which are unique to the investigated proteins, are generated by enzymatic digestion, followed by LC separation and MS detection. A corresponding method to determine apolipoprotein levels in plasma of mice has not been described so far
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
