title: Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry.
abstract: BACKGROUND
  Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS.
  
  
  METHODS
  Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject.
  
  
  RESULTS
  The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation.
  
  
  CONCLUSIONS
  The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.
authors.affiliation: Molecular Biomarker Laboratory, Merck Research Laboratories, Rahway, NJ, USA.
authors.forename: Anita Y H
authors.initials: AY
authors.lastname: Lee
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Yates
authors.forename: Marina
authors.initials: M
authors.lastname: Ichetovkin
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Deyanova
authors.forename: Katie
authors.initials: K
authors.lastname: Southwick
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Fisher
authors.forename: Weixun
authors.initials: W
authors.lastname: Wang
authors.forename: James
authors.initials: J
authors.lastname: Loderstedt
authors.forename: Nykia
authors.initials: N
authors.lastname: Walker
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhou
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhao
authors.forename: Carl P
authors.initials: CP
authors.lastname: Sparrow
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hubbard
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rader
authors.forename: Ayesha
authors.initials: A
authors.lastname: Sitlani
authors.forename: John S
authors.initials: JS
authors.lastname: Millar
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Hendrickson
mesh_terms: D016632:Apolipoprotein A-I / Q000032:analysis* / Q000096:biosynthesis; D053299:Apolipoprotein B-100 / Q000032:analysis* / Q000096:biosynthesis; D002853:Chromatography, Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D014176:Protein Biosynthesis; D055550:Protein Stability; D012680:Sensitivity and Specificity
chemical_list: D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100