Optimizing the detergent concentration conditions for immunoprecipitation (IP) coupled with LC-MS/MS identification of interacting proteins

摘要

Immunoprecipitation (IP) coupled with LC-MS/MS is a widely used method in proteomics research tonidentify proteins and to study protein-protein interactions. IP techniques allow purification of proteinsnof interest and reduce sample complexity before introduction to the mass spectrometer. Theneffectiveness of IP experiments is an important factor for identification of proteins and protein-proteinninteractions. In this paper, a variety of IP conditions were studied systematically to improve IP-basednprotein interaction identification capabilities. Low concentration detergent (around 0.05% NP40/PBS)nwas found to improve IP effectiveness by decreasing non-specific binding. However, highernconcentration detergent (e.g. 1% NP40/PBS) was detrimental. Furthermore, with lower proteinnconcentrations, the IP system showed lower tolerance to detergent. For example, with a detergentnconcentration higher than 0.05% NP40/PBS, IP experiments were unsuccessful with low proteinnconcentration (e.g. 0.28 mMADH). In some cases the observed results were even worse than the resultsnobtained without detergent. However, when the protein concentration was high (e.g. 1.12 mM ADH),nthis effect was not obvious and the high detergent (higher than 0.1%) experimental results were similarnto those from low detergent concentration experiments (around 0.05%). Another application of thisnstrategy to a more general system based on FLAG-Bacterial Alkaline Phosphatase (BAP) and anti-nFLAG antibody was also performed. These results also suggested that low detergent concentrationn(0.05% NP40) is helpful for IP experiments, especially for the experiments with low proteinnconcentrations. Considering that in most real applications, the proteins of interest are usually present innlow abundance, a low amount of detergent is recommended to be used. The optimized detergentnconcentration was determined to be 0.05% in these studies. However, the key result presented herenillustrates that both detergent and protein concentrations should be carefully considered when one isntrying to optimize IP prior to mass spectrometry experiments.