title: Applying SILAC for the differential analysis of protein complexes.
abstract: Pull-downs based on tag fusion proteins as well as immunoprecipitations (IP) are widely used methods to analyze protein interactions. Selectivity and specificity of both methods are compromised by nonspecific binding to the capture agent or carrier beads thereby generating false positives. Here, we provide a method combining stable isotope labeling of amino acids in cell culture (SILAC) with affinity purification, coupled to quantitative tandem mass spectrometry. It permits the analysis of protein interactions with high sensitivity, while being able to discriminate contaminants and nonspecific binders. Besides pruning out contaminants, high-resolution MS data combined with quantitative proteomics software allow the comparative analysis of protein interaction patterns of different protein variants, for example mutated versus normal protein variant or of regulatory changes in a given protein complex due to different states of activity.
authors: Division of Experimental Ophthalmology and Medical Proteome Center, Center of Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Roentgenweg 11, 72076, Tübingen, Germany, karsten.boldt@uni-tuebingen.de.
authors: Karsten
authors: K
authors: Boldt
authors: Christian J
authors: CJ
authors: Gloeckner
authors: Yves
authors: Y
authors: Texier
authors: Felix
authors: F
authors: von Zweydorf
authors: Marius
authors: M
authors: Ueffing
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D016923:Cell Death; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D000596:Amino Acids; D011506:Proteins