RATIONALE: Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo(a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS: Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [(2)H3]-leucine tracer into protein-derived peptides. RESULTS: We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS: The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [(2)H3]-leucine.
RATIONALE: Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo(a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS: Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [(2)H3]-leucine tracer into protein-derived peptides. RESULTS: We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS: The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [(2)H3]-leucine.
Authors: Haihong Zhou; Wenyu Li; Sheng-Ping Wang; Vivienne Mendoza; Raymond Rosa; James Hubert; Kithsiri Herath; Theresa McLaughlin; Rory J Rohm; Michael E Lassman; Kenny K Wong; Douglas G Johns; Stephen F Previs; Brian K Hubbard; Thomas P Roddy Journal: J Lipid Res Date: 2012-03-03 Impact factor: 5.922
Authors: Jennifer L Jenner; Leo J Seman; John S Millar; Stefania Lamon-Fava; Francine K Welty; Gregory G Dolnikowski; Santica M Marcovina; Alice H Lichtenstein; P Hugh R Barrett; Carl deLuca; Ernst J Schaefer Journal: Metabolism Date: 2005-03 Impact factor: 8.694
Authors: John C Price; William E Holmes; Kelvin W Li; Nicholas A Floreani; Richard A Neese; Scott M Turner; Marc K Hellerstein Journal: Anal Biochem Date: 2011-09-14 Impact factor: 3.365
Authors: Yaoyang Zhang; Stefan Reckow; Christian Webhofer; Michael Boehme; Philipp Gormanns; Wolfgang M Egge-Jacobsen; Christoph W Turck Journal: Anal Chem Date: 2011-02-11 Impact factor: 6.986
Authors: Michael S Bereman; Daniela M Tomazela; Hillary S Heins; Manuela Simonato; Paola E Cogo; Aaron Hamvas; Bruce W Patterson; F Sessions Cole; Michael J MacCoss Journal: Anal Bioanal Chem Date: 2012-04-14 Impact factor: 4.142
Authors: Michael E Lassman; Thomas McAvoy; Anita Y H Lee; Derek Chappell; Oitak Wong; Haihong Zhou; Gissette Reyes-Soffer; Henry N Ginsberg; John S Millar; Daniel J Rader; David E Gutstein; Omar Laterza Journal: Clin Chem Date: 2014-04-21 Impact factor: 8.327
Authors: Yi Pan; Haihong Zhou; Ablatt Mahsut; Rory J Rohm; Olga Berejnaia; Olga Price; Ying Chen; Jose Castro-Perez; Michael E Lassman; David McLaren; James Conway; Kristian K Jensen; Tiffany Thomas; Gissette Reyes-Soffer; Henry N Ginsberg; David E Gutstein; Michele Cleary; Stephen F Previs; Thomas P Roddy Journal: J Lipid Res Date: 2014-04-02 Impact factor: 5.922
Authors: Gissette Reyes-Soffer; Marianna Pavlyha; Colleen Ngai; Tiffany Thomas; Stephen Holleran; Rajasekhar Ramakrishnan; Wahida Karmally; Renu Nandakumar; Nelson Fontanez; Joseph Obunike; Santica M Marcovina; Alice H Lichtenstein; Nirupa R Matthan; James Matta; Magali Maroccia; Frederic Becue; Franck Poitiers; Brian Swanson; Lisa Cowan; William J Sasiela; Howard K Surks; Henry N Ginsberg Journal: Circulation Date: 2016-12-16 Impact factor: 29.690
Authors: Tiffany Thomas; Haihong Zhou; Wahida Karmally; Rajasekhar Ramakrishnan; Stephen Holleran; Yang Liu; Patricia Jumes; John A Wagner; Brian Hubbard; Stephen F Previs; Thomas Roddy; Amy O Johnson-Levonas; David E Gutstein; Santica M Marcovina; Daniel J Rader; Henry N Ginsberg; John S Millar; Gissette Reyes-Soffer Journal: Arterioscler Thromb Vasc Biol Date: 2017-07-20 Impact factor: 8.311