Owing to their preeminent biological functions, the repertoire of expressed 
RNA-binding proteins (RBPs) and their activity states are highly informative 
about cellular systems. We have developed a novel and unbiased technique, called 
interactome capture, for identifying the active RBPs of cultured cells. By 
making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, 
covalently bound proteins are captured with oligo(dT) magnetic beads. After 
stringent washes, the mRNA interactome is determined by quantitative mass 
spectrometry (MS). The protocol takes 3 working days for analysis of single 
proteins by western blotting, and about 2 weeks for the determination of 
complete cellular mRNA interactomes by MS. The most important advantage of 
interactome capture over other in vitro and in silico approaches is that only 
RBPs bound to RNA in a physiological environment are identified. When applied to 
HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome 
capture can also be broadly used to compare different biological states, 
including metabolic stress, cell cycle, differentiation, development or the 
response to drugs.