Applicability of Tandem Affinity Purification MudPIT to Pathway Proteomics in Yeast

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 ( To understand the function of a protein, it is crucial to characterize its physical environment: what other proteins is it interacting with under various conditions? Traditionally, this question has been addressed by biochemical fractionation of cell extracts under mild conditions and subsequent identification of the members of a purified protein complex by immunoblotting or peptide sequencing.Primed by the dawning of the post-genomic era, genomewide yeast two-hybrid interaction screens (1, 2) and protein chip-based methods (3) have supplemented traditional purification and identification techniques, allowing broader insight into the interaction networks that constitute a functional cell. Both of these approaches require the creation and maintenance of libraries of tagged proteins and in the case of protein chips the daunting task of purifying and spotting them under conditions that preserve their activity. The potential for detecting nonphysiological protein-protein interactions and the necessity to piece together interaction networks from a catalog of resulting binary interactions further complicate these approaches.Developed in parallel with two-hybrid and protein chip technologies, mass spectrometry of protein complexes purified through single or tandem affinity steps eliminates the need for complex-specific immunochemicals and enables analysis of very small amounts of sample on a proteome-wide scale (4,5). This approach can be performed under more physiological conditions and substitutes whole-complex analysis for the reconstruction of interaction networks from binary interaction data. However, the Gavin et al. (4) and Ho et al. (5) studies employed SDS-PAGE to separate affinity-purified protein mixtures prior to mass spectrometric analysis, thereby encountering the problems linked to this technique including: limitations of dynamic range of detection, considerable sample parallelization, variable elution efficiency of peptides from the polyacrylamide matrix, and potential selection against proteins with properties that impede analysis by SDS-PAGE (e.g. unusually high or low molecular mass, diffuse migration, comigration with contaminants, and poor binding to stain).To circumvent these problems, McCormack et al. (6) demo...

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

“…Methods using LC-MS/MS allow direct identification of peptides (31,35). The MS/MS analysis of Ste2p-Bio-DOPA 1 -␣-factor cross-linked product confirmed that the region Ser 251 -Met , eight b ions representing the N terminus and eight y ions representing the C terminus were identified.…”

Mechanisms of ATP dependent chromatin remodeling

“…Recently, Rtt102p was identified as the newest subunit of both SWI/SNF and RSC complexes by MudPIT or mass spectrometry analysis [17]. The loss of RTT102 created similar phenotypes consistent with the loss of other SWI/SNF subunits [18]. The role of SWI/SNF by all indications is far reaching.…”

Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

“…Two-step purification based on high-affinity interactions of the TAPtag with tag-specific binding resins result in highly purified protein complexes that can be analyzed by mass spectrometry. A number of tandem-affinity tags have been developed [1][2][3][4][5][6]. Here, we describe the procedures for the isolation of protein complexes under native purification conditions using the original TAP-tag developed by the Seraphin group [1,7] ( Fig.…”