Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast

A combined multidimensional chromatography- mass spectrometry approach known as MudPIT enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS- polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty- five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification ( TAP) tag. TAP- MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/ Snf2p, the catalytic subunit of the Swi/ Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP- MudPIT analysis of Rtt102p revealed subunits of both the Swi/ Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/ cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process ( i. e. pathway proteomics) by systematic application of TAP- MudPIT.