This article is available online at http://www.jlr.org 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 specifi city. Recently, proteomics-based triple quadrupole multiple reaction monitoring (MRM) permitted a more practical and highly specifi c multipeptide approach to in vivo kinetic studies ( 4, 5 ). However, MRM relies on low-resolution readouts (unit mass resolution) that do not readily permit precise quantifi cation of tracer enrichment that is lower than 1%, which is common in apolipoprotein kinetics ( 5, 6 ). Factors contributing to low precision include interference by not only the sister isotope 13C15N M3 ion but also background ions. In this study, 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 ). The HR/AM fragment ion scan feature has the potential to measure D3-Leu enrichment between 0.03% and 1.0%, a low incorporation range that is a consequence of a bolus-administered tracer, useful in revealing tracer-tracee relationships. (Nagoya, Japan; M.A.) and the National Institutes of Health [ R01HL107550 (M.A.); UL1 RR 025758-01 ; and R01HL095964 (F.M.S.)]. Abstract Endogenous labeling with stable isotopes is used

Background-Studying atherosclerotic calcification in vivo requires mouse models with genetic

We developed an automated quantification workflow for PRM-enabled detection of D3-Leu labeled apoA-I in HDL isolated from humans. Subjects received a bolus injection of D3-Leu and blood was drawn at seven time points over three days. HDL was isolated and separated into six size fractions for subsequent proteolysis and PRM analysis for the detection of D3-Leu signal from ~0.03 to 0.6 % enrichment. We implemented an intensity-based quantification approach that takes advantage of high resolution/accurate mass PRM scans to identify the D3-Leu 2HM3 ion from non-specific peaks. Our workflow includes five modules for extracting the targeted PRM peak intensities (XPIs): Peak centroiding, noise removal, fragment ion matching using Δm/z windows, nine intensity quantification options, and validation and visualization outputs. We optimized the XPI workflow using in vitro synthesized and clinical samples of D0/D3-Leu labeled apoA-I. Three subjects’ apoA-I enrichment curves in six HDL size fractions, and LCAT, apoA-II and apoE from two size fractions were generated within a few hours. Our PRM strategy and automated quantification workflow will expedite the turnaround of HDL apoA-I metabolism data in clinical studies that aim to understand and treat the mechanisms behind dyslipidemia.