title: Practical immunoaffinity-enrichment LC-MS for measuring protein kinetics of low-abundance proteins.
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.
authors.affiliation: Molecular Biomarkers and Diagnostics, michael_lassman@merck.com.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lassman
authors.affiliation: Molecular Biomarkers and Diagnostics.
authors.forename: Thomas
authors.initials: T
authors.lastname: McAvoy
authors.affiliation: Molecular Biomarkers-PPDM, and.
authors.forename: Anita Y H
authors.initials: AY
authors.lastname: Lee
authors.affiliation: Molecular Biomarkers and Diagnostics.
authors.forename: Derek
authors.initials: D
authors.lastname: Chappell
authors.affiliation: Molecular Biomarkers and Diagnostics.
authors.forename: Oitak
authors.initials: O
authors.lastname: Wong
authors.affiliation: Molecular Biomarkers-PPDM, and.
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Columbia University Medical Center, New York, NY;
authors.forename: Gissette
authors.initials: G
authors.lastname: Reyes-Soffer
authors.affiliation: 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.
authors.forename: Henry N
authors.initials: HN
authors.lastname: Ginsberg
authors.affiliation: Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA.
authors.forename: John S
authors.initials: JS
authors.lastname: Millar
authors.affiliation: Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rader
authors.affiliation: Clinical Pharmacology, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Whitehouse Station, NJ;
authors.forename: David E
authors.initials: DE
authors.lastname: Gutstein
authors.affiliation: Molecular Biomarkers and Diagnostics.
authors.forename: Omar
authors.initials: O
authors.lastname: Laterza
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D057230:Limit of Detection; D013058:Mass Spectrometry; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins
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