Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue
Year: 2012
Type: article
Abstract: High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL) 1 1The abbreviations used are:SILACstable isotopic labeling with amino acids in cell cultureICPLisotope coded protein labelingIPimmunoprecipitationTAPtandem affinity purificationMSmass spectrometryDMEMDulbecco′s modified eagle mediumPBSphosphate buffered salineHClhydrochloric acidLCliquid chromatographyROSrod outer segmentsTFAtrifluoroacetic acidiTRAQisobaric tag for relative and absolute quantification. with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.
Source: Molecular & Cellular Proteomics
Authors: Andreas Vogt, Bettina Fuerholzner, Norbert Kinkl, Karsten Boldt, Marius Ueffing
Institutions: University of Tübingen, Helmholtz Munich
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Funder: European Commission
OpenAlex ID: W2050319125