Dataset Information

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring.

ABSTRACT: Endogenous labeling with stable isotopes is used to study the metabolism of proteins in vivo. However, traditional detection methods such as GC/MS cannot measure tracer enrichment in multiple proteins simultaneously, and multiple reaction monitoring MS cannot measure precisely the low tracer enrichment in slowly turning-over proteins as in HDL. We exploited the versatility of the high-resolution/accurate mass (HR/AM) quadrupole Orbitrap for proteomic analysis of five HDL sizes. We identified 58 proteins in HDL that were shared among three humans and that were organized into five subproteomes according to HDL size. For seven of these proteins, apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE, and apoM, we performed parallel reaction monitoring (PRM) to measure trideuterated leucine tracer enrichment between 0.03 to 1.0% in vivo, as required to study their metabolism. The results were suitable for multicompartmental modeling in all except apoD. These apolipoproteins in each HDL size mainly originated directly from the source compartment, presumably the liver and intestine. Flux of apolipoproteins from smaller to larger HDL or the reverse contributed only slightly to apolipoprotein metabolism. These novel findings on HDL apolipoprotein metabolism demonstrate the analytical breadth and scope of the HR/AM-PRM technology to perform metabolic research.

SUBMITTER: Singh SA

PROVIDER: S-EPMC4808760 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring.

Singh Sasha A SA Andraski Allison B AB Pieper Brett B Goh Wilson W Mendivil Carlos O CO Sacks Frank M FM Aikawa Masanori M

Journal of lipid research 20160209 4

Endogenous labeling with stable isotopes is used to study the metabolism of proteins in vivo. However, traditional detection methods such as GC/MS cannot measure tracer enrichment in multiple proteins simultaneously, and multiple reaction monitoring MS cannot measure precisely the low tracer enrichment in slowly turning-over proteins as in HDL. We exploited the versatility of the high-resolution/accurate mass (HR/AM) quadrupole Orbitrap for proteomic analysis of five HDL sizes. We identified 58 ...[more]

PMID: 26862155

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Project description:High-density lipoprotein (HDL), a lipid nanoparticle containing many different low abundance proteins, is an attractive target for clinical proteomics because its compositional heterogeneity is linked to its cardioprotective effects. Selected reaction monitoring (SRM) is currently the method of choice for targeted quantification of proteins in such a complex biological matrix. However, model system studies suggest that parallel reaction monitoring (PRM) is more specific than SRM because many product ions can be used to confirm the identity of a peptide. We therefore compared PRM and SRM for their abilities to quantify proteins in HDL, using (15)N-labeled apolipoprotein A-I (HDL's most abundant protein) as the internal standard. PRM and SRM exhibited comparable linearity, dynamic range, precision, and repeatability for protein quantification of HDL. Moreover, the single internal standard protein performed as well as protein-specific peptide internal standards when quantifying 3 different proteins. Importantly, PRM and SRM yielded virtually identical quantitative results for 26 proteins in HDL isolated from 44 subjects. Because PRM requires less method development than SRM and is potentially more specific, our observations indicate that PRM in concert with a single isotope-labeled protein is a promising new strategy for quantifying HDL proteins in translational studies.HDL, a complex matrix composed of lipids and proteins, is implicated in cardioprotection. Its cholesterol content correlates inversely with cardiovascular disease and it is the current metric to assess cardiovascular risk. However, the cholesterol content does not capture HDL's complexity and heterogeneity. Devising metrics that better capture HDL's cardioprotective effects, we developed an optimized method for quantification of HDL proteome, using PRM in concert with a single labeled protein as internal standard. The availability of a method that increases sample throughput without compromising the reproducibility, sensitivity, and accuracy could therefore point to better risk assessment for CVD or other diseases.

Dataset Information

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring.

Publications

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring.

Similar Datasets

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