To see whether Sbp1 binds to 5′UTRPab1 in the translation initiation pathway of the Pab1 mRNA, we developed a so-called “TRAP” assay. The assay uses molecules known to participate in initiation, including 5′UTRs, translational factors, and 40S ribosomal subunit, as a bait complex for an affinity pull-down procedure followed by mass spectrometry to identify the cellular proteins that interact with these intermediates in initiation. In our experimental design, proteins belonging to the class IV initiation factor family, which includes eukaryotic initiation factors eIF4E, eIF4G, eIF4A, and eIF4B, as well as eIF3 and eIF5, are not used in the bait. These proteins are known to participate in mRNA and 40S activation prior to the start-codon recognition (Jackson et al. 2010), thus their binding to bait complexes provides a valuable positive control for our pull-down results.