(129f) Noncanonical Amino Acids for Temporally-Resolved Proteomic Profiling

Noncanonical amino acids (ncAAs) can be incorporated into cellular proteins on a proteome-wide basis, allowing chemical modification of thousands of proteins simultaneously. Incorporation of ncAAs bearing bioorthogonal “clickable” functional groups in a well-defined pulse makes newly synthesized proteins easy to distinguish from old proteins (those produced pre-pulse). This approach allows selective analysis of the nascent proteome: labeled proteins can be conjugated to fluorescent probes for visualization, or to affinity tags for enrichment and identification by mass spectrometry. This method, termed bioorthogonal noncanonical amino acid tagging (BONCAT), is a powerful approach for measuring patterns of protein synthesis in response to biological cues.

We combine BONCAT with the quantitative proteomics technique pulsed stable isotope labeling with amino acids in cell culture (pSILAC), yielding a proteomic approach that can both enrich and quantify proteins synthesized during a ncAA pulse. The combined BONCAT-pSILAC approach provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. We use this method to quantify 1400 HeLa cell proteins produced during a 30 minute pulse, a time scale inaccessible to isotope labeling techniques alone. Potential artifacts due to the incorporation of ncAAs can be reduced to insignificant levels by limiting the extent of amino acid replacement. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods.