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.
Highlights
Classical antibody-based strategies to determine protein interactions have long been hampered by the fact that most binders exhibit unspecific binding
To purify the -tubulin protein complex, a monoclonal -tubulin antibody and a second polyclonal antibody directed against -tubulin was used, whereas controls were incubated with species-specific IgGs or an anti-RGS9 antibody as a nonspecific control antibody
isotope coded protein labeling (ICPL) increases the likelihood of identifying protein complex components by decreasing the quantitative inaccuracy especially once lowly abundant peptides are compared
Summary
Classical antibody-based strategies to determine protein interactions have long been hampered by the fact that most binders exhibit unspecific binding. Tandem affinity purification (TAP) has resulted in an unprecedented specificity, concerning protein interaction data [1, 2] This method is limited by the fact that recombinant expression of a TAP-fusion protein is required and hampered by the risk that exogenous expression of the bait protein of interest may result in an artificial change of stoichiometries. To circumvent these drawbacks, Selbach and Mann developed a quantitative immunoprecipitation, combined with RNAi (QUICK), using stable isotope labeling with amino acids in cell culture (SILAC) to gain improved selectivity [3,4,5].
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