Abstract
Recent in vivo tracer studies demonstrated that targeted mass spectrometry (MS) on the Q Exactive Orbitrap could determine the metabolism of HDL proteins 100s-fold less abundant than apolipoprotein A1 (APOA1). In this study, we demonstrate that the Orbitrap Lumos can measure tracer in proteins whose abundances are 1000s-fold less than APOA1, specifically the lipid transfer proteins phospholipid transfer protein (PLTP), cholesterol ester transfer protein (CETP), and lecithin-cholesterol acyl transferase (LCAT). Relative to the Q Exactive, the Lumos improved tracer detection by reducing tracer enrichment compression, thereby providing consistent enrichment data across multiple HDL sizes from 6 participants. We determined by compartmental modeling that PLTP is secreted in medium and large HDL (alpha2, alpha1, and alpha0) and is transferred from medium to larger sizes during circulation from where it is catabolized. CETP is secreted mainly in alpha1 and alpha2 and remains in these sizes during circulation. LCAT is secreted mainly in medium and small HDL (alpha2, alpha3, prebeta). Unlike PLTP and CETP, LCAT’s appearance on HDL is markedly delayed, indicating that LCAT may reside for a time outside of systemic circulation before attaching to HDL in plasma. The determination of these lipid transfer proteins’ unique metabolic structures was possible due to advances in MS technologies.
Highlights
The metabolic properties of circulating apolipoproteins have been intensely studied [1,2,3], especially since elevated levels of LDL-cholesterol and VLDL-triglycerides, and low levels of HDL-cholesterol, are associated with cardiovascular risk [4,5,6]
We demonstrate that the Orbitrap Lumos can measure tracer in proteins whose abundances are 1000s-fold less than apolipoprotein A1 (APOA1), the lipid transfer proteins phospholipid transfer protein (PLTP), cholesterol ester transfer protein (CETP), and lecithin-cholesterol acyl transferase (LCAT)
Each enzyme exhibits unique metabolic properties — PLTP transfers from alpha2 HDL to larger alpha1 and alpha0 HDL; CETP associates with primarily alpha1 and alpha2 and stays on these particles until it is cleared; and LCAT mainly resides in alpha2, alpha3, and prebeta, but its appearance in these sizes is markedly delayed, when compared with PLTP and CETP
Summary
The metabolic properties of circulating apolipoproteins have been intensely studied [1,2,3], especially since elevated levels of LDL-cholesterol and VLDL-triglycerides, and low levels of HDL-cholesterol, are associated with cardiovascular risk [4,5,6]. The reliance on unit resolution readouts to measure low abundant 2HM3 deuterium tracer has limited most studies to total HDL protein pools, such as total apolipoprotein A1 (APOA1), the major structural protein of HDL. In 2016 we demonstrated that parallel reaction monitoring (PRM) performed on the high resolution/accuracy mass (HR/AM) quadrupole Orbitrap (Q Exactive) could differentiate D3-Leu’s 2HM3 ion from its natural M3 isotopolog and baseline ions for multiple HDL proteins, in up to 5 HDL sizes [13]. Due to the ability for HR/AM technology to detect low abundant tracer signals, the HDL protein-specific responses to a diet intervention were reported for the first time
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