In-gel stable isotope labeling for relative quantification using mass spectrometry Academic Article uri icon

Overview

MeSH Major

  • Isotope Labeling
  • Mass Spectrometry

abstract

  • Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24-26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.

publication date

  • June 2006

Research

keywords

  • Academic Article

Identity

Language

  • eng

Digital Object Identifier (DOI)

  • 10.1038/nprot.2006.7

PubMed ID

  • 17406210

Additional Document Info

start page

  • 46

end page

  • 51

volume

  • 1

number

  • 1