title: Affinity purification of protein complexes for analysis by multidimensional protein identification technology.
abstract: Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.
authors.affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
authors.forename: Charles A S
authors.initials: CA
authors.lastname: Banks
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Kong
authors.forename: Michael P
authors.initials: MP
authors.lastname: Washburn
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003902:Detergents; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006639:Histidine; C085706:FLAG peptide