Topic Introduction

Overview of Affinity Purification in Combination with Mass Spectrometry

This introduction was adapted from "Identification of Novel Protein Complexes and Protein-Protein Interactions by Mass Spectrometry," Chapter 18, in Protein-Protein Interactions 2nd ed. (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

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A basic outline of the AP-MS procedure is shown in Figure 1 . Protein complexes can be isolated by several different approaches. For example, a protein can be tagged with an epitope such as Flag or TAP and then overexpressed in a target cell, allowing the interacting proteins to be purified. Similarly, epitope tags can be homologously recombined into the endogenous locus ("knocked-in"), allowing protein complexes containing the tagged proteins to be isolated at their natural expression level. A second approach is the coimmunoprecipitation of interacting proteins without ectopic expression. This is possible if an antibody against a target protein . . . [Full Text of this Article]

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