Genome-wide Identification and Quantification of Protein Synthesis in Cultured Cells and Whole Tissues by Puromycin-Associated Nascent Chain Proteomics (PUNCH-P)

Nat Protoc. 2014 Apr;9(4):751-60. doi: 10.1038/nprot.2014.051. Epub 2014 Mar 6.

Abstract

Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or whole tissues by using a biotinylated derivative of puromycin, which becomes incorporated into nascent polypeptide chains by ribosome catalysis. In this method, termed puromycin-associated nascent chain proteomics (PUNCH-P), intact ribosome-nascent chain complexes are first recovered from cells by ultracentrifugation, followed by biotin-puromycin labeling of newly synthesized proteins, streptavidin affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unlike methods that require in vivo labeling, the sensitivity and coverage of PUNCH-P depend only on the amount of starting material and not on the duration of labeling, thus enabling the measurement of rapid fluctuations in protein synthesis. The protocol requires 3 d for sample preparation and analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotin / chemistry
  • Cells, Cultured
  • Chromatography, Liquid
  • Protein Biosynthesis / genetics*
  • Proteomics / methods*
  • Puromycin / chemistry*
  • Ribosomes / genetics
  • Tandem Mass Spectrometry / methods*
  • Ultracentrifugation

Substances

  • Puromycin
  • Biotin