A rapid method for cross‐species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra‐performance liquid chromatography/tandem mass spectrometry

Abstract

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

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

“…We confi rmed that LC/MS/MS is able to accurately quantify plasma proteins, as previously published ( 6,8,10,11 ). We measured similar ApoB100 and ApoA-I concentrations whatever the analytical method previously employed.…”

“…We measured similar ApoB100 and ApoA-I concentrations whatever the analytical method previously employed. For the other Apos, the concentrations were in the same range compared with other studies involving either LC/MS/MS ( 6,8,11 ) or conventional methods ( 5, 14, 15, Kinetic parameters of Apos were also similar with both methods for ApoB100 and ApoA-I. Compared with previous reported data, the results obtained for ApoA-II, ApoC-II, ApoC-III, and ApoE were in similar ranges ( Table 2 , supplementary Table 2).…”

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

“…As is shown, we were able to measure the incorporation of 2 H and 18 O into peptides derived from apoE, apoB, apoA4, apoC3, apoA2, and apoA1. Regardless of whether mice were given 2 H 2 O or H 2 18 O, we CA) was spiked into plasma; 10 µl 10% sodium deoxycholate was added prior to reduction, alkylation, and tryptic digestion ( 16 ). These conditions allowed for the complete digestion of apoB; therefore, the apoB peptide concentration refl ected the apoB protein concentration (apoB peptide concentration was calculated using peak area ratio between the sample and internal standard).…”

Rapid Development of Atherosclerosis in the World’s Smallest Microminipig Fed a High-Fat/High-Cholesterol Diet

“…Rabbits, which have a similar lipid metabolism to humans and are very sensitive to HcD with respect to the induction of atherosclerosis, are the next most often used animals. LDLr gene-mutated Watanabe heritable hyperlipidemic rabbits are good models of human familial hypercholesterolemia 4) . The recent development of transgenic rabbits has clarified the effects of various specific genes on the development of atherosclerosis 5,6) .…”

“…A multiple reaction monitoring (MRM) method was developed for apoA1 and apoB48/100 4) . First, the chylomicron The recombinant adenoviral plasmid was purified and then transfected into 293A cells.…”