title: Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.
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) 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.
authors.affiliation: Institute for Ophthalmic Research, Division of Experimental Ophthalmology and Medical Proteome Center, University of Tuebingen, D-72076 Tuebingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Vogt
authors.forename: Bettina
authors.initials: B
authors.lastname: Fuerholzner
authors.forename: Norbert
authors.initials: N
authors.lastname: Kinkl
authors.forename: Karsten
authors.initials: K
authors.lastname: Boldt
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002417:Cattle; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D005136:Eye Proteins; D046912:Multiprotein Complexes; D020543:Proteome; D020742:rhoA GTP-Binding Protein