Quantitative proteomics: Measuring protein synthesis using ¹⁵N amino acid labeling in pancreatic cancer cells

Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of ¹⁵N amino acids mixture for 72 h. During protein synthesis, the incorporation of ¹⁵N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of ¹³C and ¹⁵N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the"labeled" (new) and the "unlabeled" (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0% (control), 50%, and 33% ¹⁵N enriched media. These protein spots were digested and analyzed with matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of the same peptide from cells grown in 50% and 33% ¹⁵N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44 and 76%.