Proteome-wide analysis of elongating nascent polypeptide chains – CX4945 treatment

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 pre-incubated with either DMSO or a CK2 inhibitor CX4945 20 for 10 min, and subsequently, cells were pulse-labeled with puromycin and SILAC amino acids for 2 hr in the presence of DMSO or CX4945, and immunoprecipitation of puromycylated proteins was performed in triplicates.