title: Time series proteome profiling.
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.
authors.affiliation: Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Formolo
authors.forename: Michelle
authors.initials: M
authors.lastname: Mintz
authors.forename: Asako
authors.initials: A
authors.lastname: Takanohashi
authors.forename: Kristy J
authors.initials: KJ
authors.lastname: Brown
authors.forename: Adeline
authors.initials: A
authors.lastname: Vanderver
authors.forename: Brian
authors.initials: B
authors.lastname: Halligan
authors.forename: Yetrib
authors.initials: Y
authors.lastname: Hathout
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000473:pathology; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D013312:Stress, Physiological; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
chemical_list: D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D020543:Proteome