Metabolic labeling with stable isotope nitrogen ((15)N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii

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 (15)N-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 (15)NH(4)(15)NO(3) and ((14)NH(4))(6)Mo(7)O(24).4H(2)O was replaced with Na(2)MoO(4).2H(2)O. This medium designated (15)N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful (15)N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from (14)N- and (15)N-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 CF(1) alpha and beta subunits, the mitochondrial protein (F(1)F(0) ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized (15)N-labeled free amino acids and proteins obtained maximum incorporation of the (15)N-label, turnover rates were determined after transfer of cells into (14)N-TAP medium. The t(½) values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the (15)N-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.