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.
Highlights
Endogenous labeling with stable isotopes is used to study the metabolism of proteins in vivo
multiple reaction monitoring (MRM) relies on low-resolution readouts that do not readily permit precise quantification of tracer enrichment that is lower than 1%, which is common in apolipoprotein kinetics [5, 6]
We aim to extend further the scope of in vivo kinetics by exploiting the recently developed highresolution/accurate mass parallel reaction monitoring (HR/AM-PRM) method performed on the quadrupole Orbitrap mass spectrometer [7, 8]
Summary
Endogenous labeling with stable isotopes is used to study the metabolism of proteins in vivo. 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. Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring. Usually determines isotope enrichment by measuring the derivatized forms of D0 and trideuterated leucine (D3-Leu) [2, 3], a method with high cost and low sensitivity and specificity. We aim to extend further the scope of in vivo kinetics by exploiting the recently developed highresolution/accurate mass parallel reaction monitoring (HR/AM-PRM) method performed on the quadrupole Orbitrap mass spectrometer [7, 8].
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