Review Affinity purification of protein complexes for analysis by multidimensional protein identification technology Charles A.S. Banks a , Stephanie E. Kong b , Michael P. Washburn a,c,⇑ a Stowers Institute for Medical Research, Kansas City, MO 64110, United States b Department of Biology, Rockhurst University, Kansas City, MO 64110, United States c Departments of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, United States article info Article history: Received 14 June 2012 and in revised form 10 September 2012 Available online 24 September 2012 Keywords: Quantitative proteomics Multidimensional protein identification technology Mass spectrometry Affinity tag Protein complex Normalized spectral abundance factor 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 part- ners, 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 iso- lation using affinity purification and will address issues that biochemists should bear in mind as they iso- late protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT) 1 . Protein complex analysis by mass spectrometry frequently involves the collabora- tive efforts of biochemists or biologists who purify protein complexes and proteomic specialists who ana- lyze 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 affin- ity 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 analyz- ing the resulting data. Ó 2012 Elsevier Inc. All rights reserved. Contents Introduction ...................................................................................................... 106 Foundations of protein complex purification ............................................................................ 106 Affinity tagging .................................................................................................... 106 Single-step purification using affinity tags .............................................................................. 107 FLAG immunoaffinity purification ....................................................................................... 107 Halo tag .............................................................................................................. 108 Peptides that bind streptavidin .......................................................................................... 108 Maltose binding protein ................................................................................................ 108 c-Myc epitope tag ..................................................................................................... 108 Polyhistidine tag ...................................................................................................... 108 Other affinity tags ..................................................................................................... 108 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.09.007 ⇑ Corresponding author. Address: Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, United States. E-mail address: mpw@stowers.org (M.P. Washburn). 1 Abbreviations used: MudPIT, multidimensional protein identification technology; MBP, maltose binding protein; GST, glutathione S-transferase; SBP, streptavidin binding peptide; TAP, tandem affinity purification; CBP, calmodulin-binding peptide; CBD, chitin-binding domain; SPA, sequential peptide affinity; CAM, chloroacetamide; SCX, strong cation exchange; RP, reverse phase; ESI, electrospray ionization; LTQ, Linear Trap Quadrupole. Protein Expression and Purification 86 (2012) 105–119 Contents lists available at SciVerse ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep