The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low prebeta-1 functionality (ABCA1-dependent cholesterol-efflux normalized for prebeta-1 concentration) and controls who had either low or high prebeta-1 functionality. Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC/MS. The average number of lipid molecules decreased from 422 in the large spherical alpha-1 particles to 57 in the small discoid prebeta-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in alpha-mobility but not in prebeta-1 particles. Prebeta-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and TG) indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with prebeta-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I.

Protein digestion coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) detection enables multiplexed quantification of proteins in complex biological matrices. However, the reproducibility of enzymatic digestion of proteins to produce proteotypic target peptides is a major limiting factor of assay precision. Online digestion using immobilized trypsin addresses this problem through precise control of digestion conditions and time. Because online digestion is typically for a short time, the potential for peptide degradation, a major source of measurement bias, is significantly reduced. Online proteolysis requires minimal sample preparation and is easily coupled to LC-MS/MS systems, further reducing potential method variability. We describe herein a method optimized for the multiplexed quantification of several apolipoproteins in human serum using on-column digestion. We highlight key features of the method that enhance assay accuracy and precision. These include the use of value-assigned serum as calibrators and stable isotope-labeled (SIL) peptide analogs as internal standards. We also comment on practical aspects of column switching valve design, instrument maintenance, tandem mass spectrometry data acquisition, and data processing.

Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.

Tandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC-MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC-MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC-MS/MS methods for other proteins.

PURPOSE: Apolipoprotein A-I (ApoA-I) and Apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form. EXPERIMENTAL DESIGN: Simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without pre-digestion reduction and alkylation, followed by liquid chromatography separation coupled with isotope dilution mass spectrometry (IDMS) analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric-tagging-IDMS amino acid analysis. RESULTS: The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intra-day CVs (N = 5) and <7% inter-day CVs (N = 21). The repeated analysis of inter-laboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels. CONCLUSIONS AND CLINICAL RELEVANCE: The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run.However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE: Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.

High density lipoprotein (HDL) cholesterol efflux function may be a more robust biomarker of coronary artery disease risk than HDL cholesterol (HDL-C). To study HDL function, apoB containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods (polyethylene glycol (PEG), dextran sulfate/MgCl2, heparin sodium/MnCl2 and LipoSep immunoprecipitant (IP)) on HDL subspecies composition, apolipoproteins and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2 and heparin sodium/MnCl2 did not change the size distribution of HDL subspecies but altered the quantity of a subset of apolipoproteins. LipoSep IP resulted in a shift in the HDL size distribution, but less so than PEG. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.