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Harvard Team Uses PRM-MS to Improve Study of Protein Kinetics in Human HDL


NEW YORK (GenomeWeb) – A team led by researchers at Harvard Medical School has applied parallel-reaction monitoring mass spec to the study of apolipoprotein kinetics in human HDL.

Detailed in a paper published last week in the Journal of Lipid Research, the PRM approach enabled researchers to more thoroughly investigate the protein components of HDL and generate new insights into its metabolism, Harvard researcher Masanori Aikawa, senior author on the paper, told GenomeWeb.

HDL is of great interest to cardiovascular researchers and clinicians owing to findings that a low HDL level is a risk factor for coronary heart disease. However, noted Aikawa, who leads Brigham and Women's Hospital's Center for Interdisciplinary Cardiovascular Sciences, to date, no drug aimed at raising patient HDL levels has managed to reduce cardiovascular risk in clinical trials.

A likely reason for these failures, he said, is the fact that HDL levels are typically assessed by measuring either HDL cholesterol content or HDL apolipoprotein A-I content, neither of which fully account for the complexity of the particle, which consists of a large and diverse number of proteins and lipids.

Broadly speaking, HDL is thought to contribute to reduced cardiovascular risk by aiding the transport of cholesterol from, for instance, atherosclerotic plaques to the liver. However, Aikawa said, different HDL subfractions likely have different functions and behaviors, and simply measuring overall HDL cholesterol or apo A-I levels doesn't provide information about those different subfractions.

"Certain subfractions of HDL might contribute to reverse cholesterol transport or the reduction of cholesterol in atherosclerotic plaques," he said. "But the problem is you don't measure those [specific] subfractions in clinical studies, so therefore you don't know which subfraction is raised by [a given] drug. So this may make it hard to assess new HDL raising drugs. HDL metabolism is probably more complex than we think."

To get a more in depth look at HDL metabolism, researchers have used stable isotopes to label proteins of interest, which allows them to track their kinetics in vivo. To measure these labeled proteins, researchers have used approaches including gas chromatography mass spec and multiple-reaction monitoring mass spec. These methods have limitations, however. GC-MS, the study authors note, is relatively expensive and offers low sensitivity and specificity. MRM-MS, they said, has recently proven a more effective approach, but it struggles with precise quantitation of the labeled molecules due to interference from sister isotopes and background ions.

Aikawa and colleagues including HMS researcher Frank Sacks sought to improve the performance of such kinetics studies by performing targeted quantitation on a high-resolution mass spec instrument, employing PRM-MS.

PRM is essentially a variety of data-independent mass spec in which the mass spectrometer, rather than analyzing the full range of a sample, is trained on a more targeted mass and time window. Several years ago, researchers including Bruno Domon, head of the Luxembourg Clinical Proteomics Center, and Josh Coon of the University of Wisconsin, Madison, began exploring PRM on high-resolution instruments like Thermo Fisher Scientific's Q Exactive as an alternative to traditional triple quadrupole-based targeted protein quantitation.

Such an approach has various potential advantages. For instance, because their analyzers are able to collect data on a wide range of ions, high-resolution machines could allow for easier assay development and better specificity.

In a triple quad-based SRM assay, the first quadrupole isolates a target precursor ion, which is then fragmented in the second quadrupole, after which a set of preselected product ions are detected in the third quadrupole. By contrast, PRM approaches use the upfront quadrupole of a Q-TOF or Q Exactive machine to isolate a target precursor ion, but then monitor not just a few but all of the resulting product ions.

The larger number of product ions monitored via PRM should improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. The instrument's high resolution can also reduce the effects of co-isolating background peptides.

Additionally, because researchers don't have to determine upfront what the best transitions to monitor will be, assay development time is significantly reduced.

The researchers first used mass spec to characterize the mix of different apolipoproteins across various HDL subfractions, finding that the HDL proteome could be divided in five different sub-proteomes, each of which is linked to a different HDL subfraction. They then performed a kinetic analysis of seven apolipoproteins, measuring labeled versions of these proteins via HRM-MS on a Thermo Fisher Scientific Q Exactive instrument, allowing them to track the kinetics of these seven proteins across the five HDL subfractions.

"We have learned from previous studies that some HDL particles are not very functional," he said. "And what we found in this study is that each subfraction of HDL seems to have a unique set of proteins, which might be associated with the function of each of those subfractions."

This could have implications for the development of therapies targeting HDL levels, Sacks said. "If a certain subfraction contains the proteins that stimulate cholesterol transport out of atherosclerosis, for instance, you might want to raise the level of that particular subfraction.

Aikawa said that the time requirements of the PRM-MS method, and mass spec analysis in general, make it difficult to incorporate into large-scale cardiovascular outcome trials looking at thousands of subjects. He added, however, that he thought the approach they established could prove useful in relatively small clinical studies testing the effects of specific compounds on HDL metabolism.

Beyond the implications for clinical work, the study also identified a potentially novel mechanism in HDL metabolism, Sacks said. Conventional models of HDL metabolism hold that an HDL subfraction named pre-beta is released by the liver into the bloodstream and then collects lipids, becoming, in turn, the larger subfractions alpha3, alpha2, and alpha1.

The Harvard researchers' findings suggest, however, that most of these larger subfractions actually come directly out of the liver.

"This may not have direct clinical implications, but it provides new insight into HDL metabolism," he said.