title: Analyzing the homeostasis of signaling proteins by a combination of Western blot and fluorescence correlation spectroscopy.
abstract: The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation.
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Yi-Da
authors.initials: YD
authors.lastname: Chung
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sinzinger
authors.forename: Petra
authors.initials: P
authors.lastname: Bovee-Geurts
authors.forename: Marina
authors.initials: M
authors.lastname: Krause
authors.forename: Sip
authors.initials: S
authors.lastname: Dinkla
authors.forename: Irma
authors.initials: I
authors.lastname: Joosten
authors.forename: Werner J
authors.initials: WJ
authors.lastname: Koopman
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002138:Calibration; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D019169:Jurkat Cells; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D051380:GRB2 Adaptor Protein; D011993:Recombinant Fusion Proteins