Premium
Validation of method using 2 H 2 O to measure rates of protein breakdown in myoblasts
Author(s) -
Watkins Molly,
Ehrlicher Sarah,
Khademi Shadi,
Reuland Danielle J.,
Miller Benjamin F.,
Hamilton Karyn L.
Publication year - 2013
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.27.1_supplement.1208.5
Subject(s) - cycloheximide , protein degradation , chemistry , protein biosynthesis , protein catabolism , biochemistry , amino acid
Rates of protein synthesis and breakdown are thought to relate to longevity in several organisms. Since assessing protein breakdown is important for research aimed at identifying interventions to prolong healthspan, an effective, practicable method for determining rates of protein breakdown is needed. We sought to validate a method for measuring protein breakdown in vitro using deuterium oxide ( 2 H 2 O) labeling of proteins and the subsequent loss of the label. C 2 C 12 myoblasts were grown in medium supplemented with 10% 2 H 2 O. After 5 passages, the 2 H 2 O solution was replaced with unlabeled media. Cycloheximide (CHX, 50μg/ml) was included to prevent reintegration of 2 H liberated from cell breakdown. In the treated cells, Rapamycin (Rap, 5nM) was utilized to stimulate protein breakdown. At 4hr, 8hr, 12hr, and 18hr, duplicate samples of each treatment group were collected. The samples were fractionated into mixed, cytoplasmic, and mitochondrial fractions and analyzed by gas chromatography‐mass spectroscopy. Initial analysis shows protein concentrations decreasing from the 0–18 hr time points before plateauing/slightly increasing. This suggests the 2 H 2 O labeling method could be used to examine how rates of protein degradation differ in long‐lived vs. non‐long‐lived organisms, or how rates differ for conditions related to lengthened healthspan. Supported by the APS Undergraduate Summer Research Fellowship.