Background: The length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of N-15-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii. Results: To achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which(14)NH(4)Cl was replaced with (NH4)-N-15 (NO3)-N-15 and ((NH4)-N-14)(6)Mo7O24.4H(2)O was replaced with Na2MoO4.2H(2)O. This medium designated N-15-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful N-15 incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from N-14- and N-15-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized N-15-labeled free amino acids and proteins obtained maximum incorporation of the N-15-label, turnover rates were determined after transfer of cells into N-14-TAP medium. The t(1/2) values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the N-15-fractional abundance over time. Conclusion: We describe a more rapid and non-radioactive method to measure free amino acid and protein turnover. Our approach is applicable for determination of protein turnover for various proteins, which will lead to a better understanding of the relationship between protein lifetime and functionality.

• 出版日期2014-3-3