MCAT Chemical and Physical Foundations of Biological Systems 3 — Flashcards

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Metabolic labeling of proteins can be used for insight into their turnover rates. Such labeling was used to follow the turnover of specific proteins from the single-celled green algae Chlamydomonas reinhardtii. To accomplish this labeling for proteins in the algae, growth medium was prepared with standard Tris-acetate-phosphate (TAP) medium in which nitrogen-containing components of the media had their nitrogen atoms replaced with 14N, allowing for normal algal cell growth. By mass spectrometry, it was determined that 15N was incorporated into 13 amino acids at approximately 98% labeling efficiency. The 14N- and 15N-labeled crude algal protein extracts were mixed with detergent-like sodium dodecyl sulfate and separated by size and charge using SDS-PAGE. The 55-kDa SDS-PAGE bands from the crude extracts were digested using trypsin, and 27 proteins were identified by LC-MS/MS.

Five of these proteins, present in both the labeled and unlabeled samples, exhibited peptide sequence confidence levels higher than 95% and protein sequence coverage higher than 25%. After the newly synthesized 15N-labeled free amino acids and proteins reached maximal 15N incorporation, turnover rates were determined after the cells were transferred into unlabeled TAP medium.

Turnover rates were measured in terms of fractional abundance (Rt), with 14N and 15N levels quantified using the peak magnitude on the MS/MS spectrum, for each peptide of three proteins of interest from the extracts (Figure 1).