Michael E Lassman1, Thomas McAvoy2, Anita Y H Lee3, Derek Chappell2, Oitak Wong2, Haihong Zhou3, Gissette Reyes-Soffer4, Henry N Ginsberg5, John S Millar6, Daniel J Rader6, David E Gutstein7, Omar Laterza2. 1. Molecular Biomarkers and Diagnostics, michael_lassman@merck.com. 2. Molecular Biomarkers and Diagnostics. 3. Molecular Biomarkers-PPDM, and. 4. Columbia University Medical Center, New York, NY; 5. Molecular Biomarkers and Diagnostics, Molecular Biomarkers-PPDM, and Clinical Pharmacology, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Whitehouse Station, NJ; Columbia University Medical Center, New York, NY; Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA. 6. Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA. 7. Clinical Pharmacology, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Whitehouse Station, NJ;
Abstract
BACKGROUND: For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples. METHODS: Human participants (n = 39) were infused with [(2)H(3)]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment. RESULTS: The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions. CONCLUSIONS: The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9.
BACKGROUND: For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples. METHODS:Humanparticipants (n = 39) were infused with [(2)H(3)]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment. RESULTS: The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions. CONCLUSIONS: The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9.
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