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Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry

Lee, Anita Y H ; Yates, Nathan A ; Ichetovkin, Marina ; Deyanova, Ekaterina ; Southwick, Katie ; Fisher, Timothy S ; Wang, Weixun ; Loderstedt, James ; Walker, Nykia ; Zhou, Haihong ; Zhao, Xuemei ; Sparrow, Carl P ; Hubbard, Brian K ; Rader, Daniel J ; Sitlani, Ayesha ; Millar, John S ; Hendrickson, Ronald C

Clinical chemistry, March 2012, Vol.58(3), pp.619-27 [Peer Reviewed Journal]

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  • Title:
    Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry
  • Author: Lee, Anita Y H ; Yates, Nathan A ; Ichetovkin, Marina ; Deyanova, Ekaterina ; Southwick, Katie ; Fisher, Timothy S ; Wang, Weixun ; Loderstedt, James ; Walker, Nykia ; Zhou, Haihong ; Zhao, Xuemei ; Sparrow, Carl P ; Hubbard, Brian K ; Rader, Daniel J ; Sitlani, Ayesha ; Millar, John S ; Hendrickson, Ronald C
  • Description: 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. 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. 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. 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.
  • Is Part Of: Clinical chemistry, March 2012, Vol.58(3), pp.619-27
  • Identifier: E-ISSN: 1530-8561 ; PMID: 22249652 Version:1 ; DOI: 10.1373/clinchem.2011.172429
  • Subjects: Protein Biosynthesis ; Apolipoprotein A-I -- Analysis ; Apolipoprotein B-100 -- Analysis
  • Language: English

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