Robin Thomas (Presenter)
University of Minnesota
Authorship: Robin MK Thomas, Jesse C Seegmiller, Danni Li
University of Minnesota, Department of Laboratory Medicine and Pathology, 420 Delaware St. SE, Minneapolis, MN 55455
| Short Abstract High-density lipoprotein (HDL) consists of many apolipoproteins which play important roles in regulation of cholesterol transportation. Elevated cholesterol is a well-established risk factor for cardiovascular diseases. Individuals with cardiovascular risk factors have an increased risk of developing Alzheimer’s disease (AD); however cholesterol-lowering therapies do not appear to reduce AD risk. An analytical method to allow quantitative analysis of HDL will allow future investigation of HDL apolipoproteins as therapeutic targets for AD. We isolated HDL fractions in plasma specimens using ultracentrifugation. Following trypsin digestion, the fractions were assayed using LC-MS/MS, and quantified using peptide standard curves. Four HDL proteins (Apo-A1, Apo-A2, Apo-C2, and Apo-C3) were compared to immunoassay results with three indicating correlation. |
Long Abstract
Background
High-density lipoprotein (HDL) consists of many apolipoproteins that play important roles in various endogenous processes such as lipoprotein metabolism and regulation of cholesterol transportation. Elevated cholesterol is a well-established risk factor and therapeutic target for cardiovascular diseases. Individuals with cardiovascular risk factors also have increased risk for Alzheimer’s disease (AD). However, cholesterol-lowering therapies (i.e., statins) with proven utility in lowering cardiovascular risk do not appear to reduce risk for AD1, suggesting the need to identify novel therapeutic targets beyond cholesterol for AD. The objective of this study is to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of four apolipoproteins (Apo-A1, Apo-A2, Apo-C2, and Apo-C3) in HDL. These analytical methods will allow future investigation of HDL-associated apolipoproteins as potential therapeutic targets for AD.
Methods
Lipoprotein fractions, including HDL, were isolated from plasma by sequential ultracentrifugation. A HDL pool was made by combining individual HDL fractions, and was used for method development. Prior to trypsin digestion, HDL was diluted with 0.1M ammonium bicarbonate, denatured with 0.2 M dithiothreitol, and alkylated with 1M iodoacetamide.
Digested fractions were analyzed using a Shimadzu LC-20AD XR liquid chromatography system coupled to an AB Sciex 6500 Qtrap triple quadrupole mass spectrometer. Chromatography was performed with a Phenomenex C18 guard cartridge (4 x 3.0 mm) and a Phenomenex C18 analytical column (150 x 2.1 mm 2.6u 100Å) using a 30-minute linear gradient from 2% B to 95% B where mobile phase A is water, and mobile phase B is acetonitrile, both containing 0.1% formic acid. Standard curves were established using synthesized peptides representing tryptic peptides found in Apo-A1, Apo-A2, Apo-C2, and Apo-C3. Calibration curves from the tryptic peptides provided quantitative signatures for these proteins.
Results
We employed LC-MS/MS method to analyze Apo-A1, Apo-A2, Apo-C2, and Apo-C3 in eight HDL fractions isolated from plasma samples. These results were compared to those determined by a multiplex immunoassay (EMD Millipore, Temecula, CA). Linear regression analysis of the method comparison results yielded R2 values of 0.979, 0.716, 0.977, and 0.584 for Apo-A1, Apo-A2, Apo-C2, and Apo-C3, respectively. These results suggest correlation between the multiplex immunoassay and the LC-MS/MS method for quantitative analysis of Apo-A1, Apo-A2, and Apo-C2.
Conclusion
We have developed a LC-MS/MS method for simultaneous measurement of four apolipoproteins (Apo-A1, Apo-A2, Apo-C2, and Apo-C3) from plasma. In the future, we will include synthetic, heavy-labeled peptides as internal standards in the method.
References & Acknowledgements:
References
1. Stukas, S., Robert, J., Wellington, C.L. (April 1, 2014). High-Density Lipoproteins and Cerebrovascular Integrity in Alzheimer’s Disease. Cell Metabolism. Vol 19, Issue 4, p 574-591. Retrieved from http://www.sciencedirect.com/science/article/pii/S1550413114000096.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |