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2H‐labeling of amino acids (AA) following administration of 2H2O: Implications for quantifying proteome dynamics in vivo.
Author(s) -
Gilge Danielle,
Combs Whitney,
Bederman Ilya,
Wang Benlian,
Anderson Ver,
Previs Stephen
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1046-a
Subject(s) - chemistry , proteome , in vivo , alanine , amino acid , peptide , chromatography , protein turnover , blood proteins , biochemistry , protein biosynthesis , biology , microbiology and biotechnology
We developed the use of 2 H 2 O for measuring protein turnover ( Am J Physiol 288: E1277, 2005). The principle is that following the administration of 2 H 2 O, 2 H rapidly distributes in body water and equilibrates with the carbon‐bound hydrogens of AAs. The rate of protein synthesis is determined by measuring the incorporation of a 2 H‐labeled AA(s) into a protein(s) of interest. Our initial studies were focused on measuring the incorporation of 2 H‐alanine. We hypothesized that the method could be extended by using proteome‐based assays, e.g. measure the 2 H‐labeling of proteins and/or peptide fragments via MALDI‐TOF. In those cases, determination of protein turnover requires knowledge of the 2 H‐labeling of all AA precursors. We report here on the equilibration of 2 H in 20 proteogenic AAs isolated from plasma. Briefly, overnight fasted rats were injected with 2 H 2 O (~20 μl per g wt) and randomized to a fasted or fed group. Blood samples were collected at various intervals. The 2 H‐labeling of water and plasma AAs was determined using GC‐MS. Nutritional status modestly affects the equilibration of 2 H‐labeling, with slower (less complete) equilibration in the fed state. We found substantial incorporation of 2 H in most of the non‐essential AAs but minimal incorporation of 2 H in the essential AAs. These data can be used to calculate rate constants for determining protein turnover using MS analysis of tryptic peptides.