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Time Series Proteome Profiling

  • Catherine A. Formolo
  • Michelle Mintz
  • Asako Takanohashi
  • Kristy J. Brown
  • Adeline Vanderver
  • Brian Halligan
  • Yetrib HathoutEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 694)

Abstract

This chapter provides a detailed description of a method used to study temporal changes in the endoplasmic reticulum (ER) proteome of fibroblast cells exposed to ER stress agents (tunicamycin and thapsigargin). Differential stable isotope labeling by amino acids in cell culture (SILAC) is used in combination with crude ER fractionation, SDS–PAGE and LC-MS/MS to define altered protein expression in tunicamycin or thapsigargin treated cells versus untreated cells. Treated and untreated cells are harvested at different time points, mixed at a 1:1 ratio and processed for ER fractionation. Samples containing labeled and unlabeled proteins are separated by SDS–PAGE, bands are digested with trypsin and the resulting peptides analyzed by LC-MS/MS. Proteins are identified using Bioworks software and the Swiss-Prot database, whereas ratios of protein expression between treated and untreated cells are quantified using ZoomQuant software. Data visualization is facilitated by GeneSpring software.

Key words

Time series Proteome profiling SILAC LC-MS/MS ER stress response Subcellular proteomics 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Catherine A. Formolo
    • 1
  • Michelle Mintz
    • 1
  • Asako Takanohashi
    • 1
  • Kristy J. Brown
    • 1
  • Adeline Vanderver
    • 1
  • Brian Halligan
    • 2
  • Yetrib Hathout
    • 1
    Email author
  1. 1.Center for Genetic Medicine ResearchChildren’s National Medical CenterWashingtonUSA
  2. 2.Bioinformatics, Human and Molecular Genetics CenterMedical College of WisconsinMilwaukeeUSA

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