Validation of ApolipoProtein A-I Associated Lipoprotein Panel for the Prediction of Cholesterol Efflux Capacity Cory Bystrom (Presenter) Cleveland HeartLab

Authors: Zhicheng Jin, Timothy S. Collier, Cory Bystrom
Cleveland HeartLab, Cleveland, OH

We developed a rapid affinity method to enrich apolipoprotein A-I associated lipoproteins and utilized mass spectrometry-based approach for multiplex quantitation. Utilizing this technique, a multiprotein panel was developed which can be used to estimate cholesterol efflux capacity. The analytical workflow was validated and used in an 8 week longitudinal study. In normal healthy volunteers, predicted cholesterol efflux was stable.

Introduction

High Density Lipoprotein (HDL) cholesterol in serum is inversely associated with the risk of coronary artery disease (CAD). This association has given rise to intense interest in the structure and function of HDL. Cholesterol efflux capacity (CEC) has been identified as a key HDL function with the observation of an inverse correlation between CEC and CAD in multiple clinical studies.

To enable proteomic studies of HDL function, our group recently developed a rapid affinity approach for the isolation ApoA-I associated lipoproteins (AALP) from serum specimen. Coupled with a targeted MRM workflow, we quantified ApoA-I associated lipoproteins and developed a multiprotein panel which can be used to estimate cholesterol efflux. The high-throughput assay has been fully validated with figures of merit that indicate suitability for clinical studies.

Methods

Human serum was incubated with 15N-labeled His6-tagged ApoA-I, which associates with HDL allowing for enrichment by affinity chromatography. Ni-NTA micro affinity column (PhyTips) were used to isolate exogeneous ApoA-I and associated lipoproteins. After washing, the eluted proteins were digested with Lys-C at 37 °C for four hours. After digest, the peptide mixtures were spiked with stable isotope labeled internal standards and quantified using LC-MS/MS. The metal affinity enrichment protocol and LC-MS/MS quantification method were optimized and validated. Thirty apparently healthy volunteers were recruited to investigate analytical and biological variation over an eight-week period.

Results

To meet the requirement of a high-throughput assay, the rapid enrichment protocol was implemented on an automated liquid handler. After sample preparation, an automated multiplex LC-MS/MS system with 3 independent HPLC systems was deployed. Thirty-six peptide transitions were monitored for eight surrogate peptides from five target proteins. Dynamic MRM improved dwell time management due to co-eluting peptides. Three pooled and two unique individual serum samples with a wide range of protein composition were analyzed with four replicates each for 15 days, across 3 HPLC systems. The peptide quantification precision was excellent with total imprecision for ApoA-I, ApoC-I, ApoC-II, and ApoC-III below 10% CV. The lowest abundance protein ApoC-IV, present at levels 4 orders of magnitude lower than ApoA-I, had total imprecision below 14% CV. A longitudinal study showed that the overall variation (analytical and biological variation) for integrated cholesterol efflux prediction is below 15% for all individuals.

Conclusions & Discussion

We demonstrated that apolipoprotein A-I associated lipoproteins enrichment followed by mass spectrometry-based quantification is a robust method to quantify lipoproteins. The performance meets stringent analytical criteria for a multiprotein panel intended to be deployed in clinical studies. Longitudinal evaluation of a small cohort of subjects indicate that lipoprotein composition and predicted cholesterol efflux capacity appear to be stable over time.

10.1021/acs.jproteome.7b00816