Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry

Abstract

RATIONALE Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo (a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [2H3]-leucine tracer into protein-derived peptides. RESULTS We demonstrated that it is feasible to quantify the incorporation of [2H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [2H3]-leucine.

Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS

“…The use of proteotypic peptides involves an optimal proteolysis to obtain a full recovery; otherwise the concentrations could be underestimated. Using our protocol and an overnight trypsin digestion, the hydrolysis was complete for each target protein ( 6,(9)(10)(11). LC/MS/MS detector responses could also be altered by matrix effects.…”

“…For peptides containing two leucines (ApoA-I and ApoE), two M3 isotopomers could form and be detected simultaneously by the selected MRM transitions. Their proportions were assumed to be identical, as described previously ( 9,17 ), and the analytical signal obtained during labeled ApoA-I and ApoE detection was experimentally enhanced 2-fold by the two coeluted isotopomers. In addition, the peptide isotopologues of ApoA-I and ApoE, containing two labeled leucine residues, were not detected in our analytical conditions and were considered as negligible.…”

Empirical calibration of the clinopyroxene–garnet magnesium isotope geothermometer and implications

“…Magnesium isotopic analyses were performed at the Isotope Laboratory of the University of Arkansas, Fayetteville, following previously established procedures (Teng et al 2007Yang et al 2009;Teng and Yang 2014). A brief description is given here.…”

Quantification of Tau in Cerebrospinal Fluid by Immunoaffinity Enrichment and Tandem Mass Spectrometry

“…The assay employs a monoclonal antibody (mAb) to selectively enrich tau from routinely available volumes of CSF, followed by tryptic digestion to produce proteotypic peptide fragments that can be quantified by LC-MS/MS. The use of microflow chromatography, combined with a microfluidic device, provides enhanced analytical sensitivity with the robustness required for clinical assays (16 ).…”