Abstract
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.
Highlights
A combined multidimensional chromatography-mass spectrometry approach known as “multidimensional protein identification technology (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
We tagged a test set of 25 gene products involved in transcription and progression through mitosis and established a variant of the tandem affinity purification (TAP) protocol [13] that employs affinity chromatography on a 9E10 monoclonal antibody resin followed by elution with PreScission Protease and adsorption to nickel nitrilotriacetic acid (Ni-NTA) resin
Preliminary mass spectrometric analyses showed that the eluates from the 9E10 resin still retained a high level of contaminating protein background, and subsequent analyses were performed only on samples that were subjected to the complete TAP protocol
Summary
To construct pJS-HPM53H, a 940-bp fragment was PCR-amplified from pJS-TM53H (RDB1344) [9] with the primers HTM A and B (see supplemental Table I). This was used as a template to PCR-amplify a HPM tag containing a 670-bp fragment with the primers HPM C and D (see supplemental Table I), which replaced the XhoI-EcoRI restriction fragment of pJS-TM53H
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