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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
Published on: July 6, 2012
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Proteome analysis of puromycin-labeled nascent polypeptides
1RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan.
Methods in Enzymology
|September 24, 2025
Summary
This study introduces a streamlined protocol to profile nascent polypeptides using puromycin labeling and mass spectrometry. This method enables proteome-wide analysis of protein synthesis and N-termini, crucial for understanding protein function and targeting.
Area of Science:
- Proteomics
- Molecular Biology
- Biochemistry
Background:
- Nascent polypeptides emerging from ribosomes and their N-termini influence protein stability, folding, interactions, and cellular localization.
- Understanding these early stages of protein synthesis is critical for deciphering cellular mechanisms.
Purpose of the Study:
- To present a streamlined protocol for proteome-wide profiling of elongating nascent polypeptides.
- To enable detailed analysis of protein N-termini and their functional implications.
Main Methods:
- Biochemical enrichment of puromycin-labeled nascent polypeptides.
- Mass spectrometry-based quantitative proteomics.
- Metabolic pulse labeling with puromycin and Stable Isotope Labeling by Amino acids in Cell culture (SILAC).
- Immunoprecipitation, peptide fractionation, and N-terminal acetylation enrichment.
Main Results:
- A detailed, step-by-step protocol is provided for nascent polypeptide profiling.
- The protocol integrates puromycin labeling, SILAC, and mass spectrometry for quantitative analysis.
- Demonstrated applicability in HeLa cells treated with cycloheximide, adaptable to various cell systems and treatments.
Conclusions:
- The presented protocol offers a robust method for analyzing nascent polypeptides on a proteome-wide scale.
- This technique facilitates the study of protein N-termini and their role in protein biogenesis.
- The protocol is versatile and applicable across different cell culture models and experimental conditions.
