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

Abstract

those of the entire lipoprotein metabolism ( 1, 3 ). The incorporation of a labeled amino acid into the protein during its synthesis is subsequently measured over time and analyzed with a compartmental model to obtain the kinetic data ( 4, 5 ). The reference method to measure tracer enrichments involves isolation of the Apos by gel electrophoresis followed by acid hydrolysis to obtain unlabeled and labeled amino acids. Then, the amino acids are derivatized for GC/ MS analysis ( 3 ). These approaches are limited to one or a small number of relatively abundant Apos and remain a timeconsuming process. Kinetic studies are therefore mainly focused on the most abundant structural Apos (ApoA-I and ApoB100) and less on others, although they have a central role in lipid metabolism (ApoC-II, ApoC-III and ApoE) ( 6 ).The combination of proteomic tools, such as enzymatic proteolysis and liquid LC/MS/MS, has appeared recently to be a powerful tool to study plasma proteins ( 7-11 ). Although promising, one analytical challenge is to use this method in a single run analysis for the determination of concentrations and tracer enrichments of a signifi cant set of plasma proteins with large differences in molecular mass or abundances ( 6, 11 ).We recently published an LC/MS/MS method to simultaneously measure the concentration, tracer enrichment, and average size of Apo(a) ( 9, 12 ). In the present study, we aimed to describe the development and the validation of a multiplexed LC/MS/MS method, performing in a single run the enrichment measurements and the quantifi cation of six major human Apos (ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE) in plasma samples obtained from a stable isotope kinetic study in humans. Lipoprotein kinetic studies using radioactive or stable isotope tracers have been performed for years in humans to gain a better understanding of the mechanisms involved in lipid metabolism disturbances and related diseases ( 1, 2 ). Endogenous labeling with an amino acid tracer of Apo, a main component of lipoproteins, is commonly used, assuming the kinetics of Apos represent a good estimate of This work was supported by the Biogenouest CORSAIRE core facility.

Kinetics of plasma apolipoprotein E isoforms by LC-MS/MS: a pilot study

“…Lipoprotein turnover can be assessed in vivo by measuring the incorporation of an injected tracer, usually 2 H 3 -leucine, in apolipoproteins over time, allowing the determination of lipoprotein kinetic parameters such as their production rates (PRs) and fractional catabolic rates (FCRs) (9). This approach has been improved by new analytical techniques involving enzymatic proteolysis and LC-MS/MS (10,11). LC-MS/MS is a powerful tool to simultaneously quantify several plasma proteins even at low concentrations (12,13).…”

“…Apolipoproteins (apoA-I, apoB100, apoC-II, apoC-III, and apoE) were analyzed in plasma, lipoprotein fractions, and concentrated lipoprotein fractions using a validated multiplexed assay involving trypsin proteolysis and the subsequent analysis of proteotypic peptides by LC-MS/MS (10). The method was updated for the quantification of apoE isoforms as described previously (2).…”

“…2 H 3 -leucine enrichments were assessed in apoE isoforms in plasma and concentrated lipoprotein fractions, as previously validated (10,14). Enrichments were calculated as described previously from unlabeled (M0) and 2 H 3 -leucine-labeled (M3) peptides (10,17). Briefly, the isotope ratio (IR), corresponding to the M3/ M0 percent ratio (%), was divided by the number of leucine residues in the peptide sequence.…”

Unbiased and targeted mass spectrometry for the HDL proteome

“…4a). In the last few years the metabolism of proteins such as apo(a), apoB, PCSK9, and CETP isolated from plasma or VLDL/LDL [27, 34, 54, 63, 64], or a subset of the classical apolipoproteins from HDL [52] have been studied in humans using MRM on the triple quadrupole platform (Fig. 4a).…”

Current Trends and Future Perspectives of State-of-the-Art Proteomics Technologies Applied to Cardiovascular Disease Research

“…As a consequence of this detection constraint, most apolipoprotein metabolism studies have been limited to either the quickly turning over apoB (~10% enrichment) or to the total HDL plasma pool (up to 2% peak enrichment depending on the labeling strategy). 68 Singh et al from our laboratory, however, demonstrated the ability of PRM to measure <1% tracer enrichment in not only apoA-I from 5 HDL size fractions, but also 6 additional HDL apolipoproteins. 72 Figure 7B demonstrates a PRM scan of the apoA-I peptide, THLAPYSDELR.…”