MSACL 

26. Quantitation of Apolipoproteins in Human Serum Using Liquid Chromatography Mass Spectrometry
Mon 4:42 PM - PosterSplash Track 3
Sean Agger
University of Washington
Sean A. Agger, Luke C. Murey, Andrew N. Hoofnagle

Department of Laboratory Medicine, University of Washington, Seattle, WA
Introduction

Apolipoproteins are slowly becoming important screening biomarkers for predicting cardiovascular risk in the general population. Some studies suggest that they may be stronger predictors than traditional serum lipid measurements. Of the known apolipoproteins, apolipoprotein B (apoB) is of interest clinically due to its association with atherogenic particles. On the other hand, apoA-I, the most abundant protein in high density lipoproteins is associated with atheroprotection via its reverse cholesterol transport and anti-inflammatory capabilities. The ratio of the two may be even more sensitive and specific than either one alone. There are currently no clinically validated assays to simultaneously measure more than one protein in human serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS). We set out to determine if apoB/apoA-I could be reliably quantified in a multiplexed assay.

Methods

Peptides were originally selected using shotgun proteomics after digesting density gradient ultracentrifuge purified HDL and LDL. The most abundant peptides for ApoA-I and ApoB were identified for multiple reaction monitoring. Isotopically labeled internal standards were synthesized for the selected tryptic peptides Apo A1 (VQPYLDDFQK) and Apo B (SVSLPSLDPASAK). A set of Apo A1 and Apo B calibrators were developed from commercially purified protein (Gen Way Biotech, San Diego, CA) which were then matrix matched with fetal bovine serum by adjusting total protein to 60 g/L, levels expected in human serum. A tryptic digest of calibrators and patient samples were analyzed by LC/MS/MS at a flow rate of 0.4 mL/min.

Results

A calibration curve was generated from peak area ratios to the labeled peptide with Pearson correlation coefficient (r2) of 0.95 for Apo 1 and 0.92 for Apo B. Within-run CV were between 23-51% for ApoA-I calibrators and 23-70% for AboB. The correlation between the tandem mass spectrometric method and nephelometry (Dade Behring, BNII) was good with Pearson correlation coefficient (r2) of 0.95 for apoA-I and 0.90 for apoB.

Conclusion

In this work we describe great strides towards making a quantitative mass spectrometry assay for predicting CVD risk. This new method will allow for quantitation of Apo A1 and Apo B in human plasma. It also has the ability to multiplex apolipoproteins allowing for time and cost savings compared to our laboratory’s current methodology.

Email: [email protected]