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 affinitybased 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 ( Classical antibody-based strategies to determine protein interactions have long been hampered by the fact that most binders exhibit unspecific binding. Immunoprecipitationsthe most widely used method-not only suffer from nonspecific binding because of compromised selectivity and specificity of the immunoglobulin, but also from nonspecific binding to the carrier beads. Because of this lack of specificity, a large proportion of reported protein interactions in the literature as well as in databases that gather interaction data are likely to be compromised by false positives. Furthermore, despite great advancements in sensitivity and accuracy of mass spectrometers and peptide separation techniques, mass spectrometry-based identifications usually fail to detect lowabundance members of protein complexes, medium affinity or transient binders. Several methods have tackled these problems. Tandem affinity purification (TAP) has resulted in an unprecedented specificity, concerning protein interaction data (1, 2). Yet this method is limited by the fact that recombinant expression of a TAP-fusion protein is required and additionally 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 (3-5). The main advantage of QUICK is that endogenous protein stoichiometries are the basis for immunoprecipitation, for the first time allowing one to accurately monitor protein interactions at endogenous protein concentrations from living cells and discriminate true positive from false positive interactions. Yet this method requires metabolic isotope labeling of whole organisms or reference cells, as described for SuperSILAC, to allow comparative analysis of two protein sets (6). Metabolic labeling, especially when applied to living organisms, requires feeding them with isotopic food (7-9). The procedure of la- 1 The abbreviations used are: SILAC, stable isotopic labeling with amino acids in cell culture; ICP...

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