Automation of PRM‐dependent D3‐Leu tracer enrichment in HDL to study the metabolism of apoA‐I, LCAT and other apolipoproteins

Abstract

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.

Metabolism of PLTP, CETP, and LCAT on multiple HDL sizes using the Orbitrap Fusion Lumos

“…Similarly, APOA1 enrichment variance decreased on the Lumos compared to the Q Exactive, but the relative differences in enrichment curves and resulting model fits were conserved between the two instruments' data (Supplemental Figure 4). This conservation is not unexpected since in previous studies using the Q Exactive tracer detection methods had been carefully optimized (13,29). On the other hand, the increase in sensitivity and precision in mass spectrometric measurements that are required to study, for example PLTP, CETP and LCAT metabolism, are why next generation quadrupole HR/AM instruments such as the Lumos are continually being designed (36).…”

“…HDL isolation, size fractionation and proteolysis. HDL sample preparation has been reported in great detail previously (13,14,29), but the salient steps are outlined here. For each participant, HDL was isolated from 8 to 14 timepoints post-D3-Leu infusion: The specific time points (listed in Supplemental Table 3) were chosen before data analysis and varied across participants due to sample availability (samples from this same clinical study were analyzed in pervious publications (1,14,29)).…”

“…We next compared PLTP alpha1 and CETP alpha2 enrichment data acquired on the Q Exactive and Lumos. We did not perform this inter-instrument enrichment comparison for LCAT, as we previously reported the Q Exactive-generated enrichment and metabolism of LCAT in alpha3 HDL, the size fraction in which the enzyme predominates (14,29). PLTP and CETP mainly reside in alpha1 and alpha2 HDL with PLTP more so in alpha1 and CETP in alpha2 ( Figure 1B) (14).…”

High‐density lipoproteins in high resolution: Will proteomics solve the paradox for cardiovascular risk?

“…Kinetics studies of HDL particles use stable isotope amino acid tracers such as tri‐deuterated‐leucine (D3‐Leu) , labeling endogenous apoA‐I as a surrogate for HDL metabolism. Unfortunately, the incorporation of D3‐Leu in apoA‐I is very low, limiting the sensitivity and accuracy of the traditional methods of GC‐MS or more recently, MRM , to analysis of total HDL only.…”

“…In the previous issue of this journal, the group presented an automated software tool for the detection and quantification of D3‐leu tracer enrichment . Using HRAM and PRM together with their open‐source software, named “extracted PRM peak intensity” (XPI) program, they were able to evaluate sources of technical noise and thus achieve automated quantification of very low tracer enrichment.…”