Measuring protein synthesis by mass isotopomer distribution analysis (MIDA)

The measurement of protein kinetics by isotopic techniques has been hindere d by the long-standing difficulty of accurately measuring the isotope conte nt of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass iso topomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measu rements on a polymeric product, based on combinatorial probabilities of lab eled and unlabeled monomeric subunits, Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so t hat resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach fo r measuring protein synthesis by MIDA is described and tested here: First, in vitro, using a synthetic peptide present in human serum albumin; and the n, in vivo, for albumin synthetic rate in rats. A peptide contained in huma n serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymot rypsin proteolysis was synthesized by solid-phase peptide synthesis using k nown mixtures of natural abundance and [5,5,5-H-2(3)]leucine, Additionally, enriched and natural abundance peptides were mixed in vitro to simulate in vivo biosynthesis and to address problems of instrument accuracy, precisio n, and data management. The mass isotopomer patterns of the synthetic pepti des were analyzed using electrospray ionization (ESI) with both magnetic se ctor and quadrupole mass analyzers. The resolution of the magnetic sector w as superior to that of the quadrupole instrument, but accuracy and precisio n in the measurement of mass isotopomer abundances and kinetic parameters w ere comparable and both gave values close to those predicted. Next, rats we re infused with [5,5,5-H-2(3)]leucine intravenously, and a leucine-rich pep tide was isolated and purified from trypsin-digested rat serum albumin (RHP DYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector ins trument. Precursor pool enrichments and fractional synthetic rates (0.45 +/ - 0.03 day(-1), t(1/2) = 1.53 +/- 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichment s by MIDA is technically feasible and practical in vivo using proteolytical ly derived peptides and ESI-MS analysis. (C) 1999 Academic Press.