PMC

Tree of metabolically labeled life. Branch lengths are not proportional to evolutionary distance.

Qualitative proteomics work flow. Proteins are extracted, digested, and separated by strong cation exchange. Each strong cation exchange fraction is then analyzed by nano-LC-MS/MS. Peptide fragment spectra are used in a database search to identify the peptide sequence and the corresponding protein.

Strategies for quantitative proteomics. Stable isotopes can be incorporated at different stages of the quantitative work flow and are indicated in black. The methods are metabolic labeling (left), protein labeling (middle), and peptide labeling (right). Relative expression levels are obtained by mass spectrometry where the signal of the unlabeled peptide is compared with that of the labeled peptide.

Overview of typical mass spectra that are obtained from different metabolic labeling strategies. In SILAC (A), labeled arginine can be converted to proline, indicated in red in the peptide sequence, resulting in peaks that are 6 Da higher than the labeled peptide. If such peaks are formed in heavy nitrogen labeling (B), they appear at the same position as the ¹⁵N-labeled peptide because they are isobaric. The red colored isotope at m/z 410.73 is a product of suboptimal ¹⁵N labeling. Minor enrichment of ¹³C in SMIRP leads to quantifiable peptide signals (C), whereas higher incorporation is used to determine protein synthesis and degradation (D).