Figure 1. The ¹⁵N-labeling workflow for measuring the protein dynamics.

(A) After weaning, C57BL/6J mice were subjected to an exclusively ¹⁵N chow diet starting at P21 for a total duration of 12 weeks. ¹⁵N was incorporated into newly synthesized proteins. (B) In LC-MS/MS, ¹⁴N- and ¹⁵N-peptides of the same sequence co-elute (left panel). Regardless of where MS/MS is triggered (arrows pointing at random positions), the MS1 peptide signal intensities between the ¹⁴N and ¹⁵N channels reflect of their relative abundance (right panel). (C) Representative proteins showed different turnover rates. D. Among 543 lens proteins (blue dots) that were quantified via their ¹⁴N vs. ¹⁵N ratios (y-axis), there was a wide range of total protein abundance as estimated by MS/MS spectral counts (x-axis). Note that Calpain protease was long-lived, and the highly abundant α-, β- and γ-Crystallin proteins each had different levels of ¹⁴N and ¹⁵N.