Measurement of Fractional Synthetic Rates of Multiple Protein Analytes by Triple Quadrupole Mass Spectrometry

[摘要] 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-D3]-leucine (D3-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% D3-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.

[学科分类] 过敏症与临床免疫学