Proteome-wide analysis of elongating nascent polypeptide chains – Nt-acetylation analysis

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. We combine quantitative proteomics, pulse labeling with puromycin, and stable isotope-labeled amino acids to analyze thousands of NPCs. Our approach enables global analyses of translational responses and co-translational modifications, providing a framework for dissecting co-translational regulations proteome-wide. HeLa cells were treated with either CHX or DMSO for 2 hr in the presence of puromycin and corresponding SILAC amino acid pairs. By combining pSNAP with low pH strong cation exchange (SCX) chromatography, we enriched Nt-acetylated peptides that were eluted earlier in SCX fractionation due to the loss of positive charge at their N-terminal ends. Immunoprecipitation of puromycylated proteins was performed in triplicates.