Global analysis of cellular protein translation by pulsed SILAC

Abstract

Current methods for system-wide gene expression analysis detect changes in mRNA abundance, but neglect regulation at the level of translation. Pulse labeling with stable isotopes has been used to measure protein turnover rates, but this does not directly provide information about translation rates. Here, we developed pulsed stable isotope labeling by amino acids in cell culture (pSILAC) with two heavy isotope labels to directly quantify protein translation on a proteome-wide scale. We applied the method to cellular iron homeostasis as a model system and demonstrate that it can confidently identify proteins that are translationally regulated by iron availability.

Proteome-Wide Measurement of Protein Half-Lives and Translation Rates in Vasopressin-Sensitive Collecting Duct Cells

“…Recent advances in protein mass spectrometry using a technique called dynamic stable isotope labeling by amino acids in cell culture (SILAC) allow proteome-wide measurement of protein half-lives 14 and relative translation rates. 15 Here, we use the dynamic SILAC approach in mpkCCD cells to carry out large-scale measurements of protein half-lives and translation rates with and without vasopressin. This study provides the first proteome-wide profile of protein half-lives in a non-neoplastic epithelial cell and presents a database of protein half-lives for 4403 proteins expressed in mpkCCD cells.…”

SILAC for biomarker discovery

“…Finally, due to the incorporation of multiple labels per analyte, even very highly enriched isotopes result in partial labeling. For example, consider a typical peptide with 20 nitrogen atoms: even 99% pure 15 N will result in a labeling efficiency of only 82% (0.99 20 ), and this efficiency is further reduced for peptides with greater nitrogen content. These disadvantages pose a significant technical hindrance to biomarker discovery.…”

“…Moreover, since SILAC is a metabolic labeling method, it can also be employed to quantify protein dynamics by measuring the rate of isotope incorporation. For example, dynamic SILAC can quantify protein turnover and pulsed SILAC can measure changes in protein synthesis on a proteome-wide scale [20][21][22].…”

Quantitative proteomics for the analysis of spatio-temporal protein dynamics during autophagy

“…Kinetic profiles of 1,486 proteins were recorded suggesting an ordered degradation of cellular components during starvation-induced autophagy. 16,86 Quantitative MS relying on pulse and pulse-chase labeling has also been successfully used for the description of protein turnover, 87 synthesis, 88,89 and degradation. 90 These techniques should also be applicable to investigate global protein dynamics during autophagy.…”