Proteome analysis of puromycin-labeled nascent polypeptides

Emerging nascent polypeptides from ribosomes and their protein N-termini have significant impact on protein stability, folding, interaction and subcellular targeting. To profile elongating nascent polypeptides on a proteome-wide scale, here we present a streamlined protocol that combined a biochemical enrichment of puromycin-labeled nascent polypeptides and mass spectrometry-based quantitative proteomics. This chapter includes the detailed protocol for metabolic pulse labeling with puromycin and SILAC amino acids, immunoprecipitation for nascent polypeptides, fractionation and enrichment of N-terminal acetylated peptides as well as MS and data analysis. These protocols are illustrated using HeLa cells treated with cycloheximide, a protein synthesis inhibitor, but can be broadly applied to any cell culture systems, including primary cultures, or any treatments (e.g., drugs).