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.

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 is available.

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Figure 1. Overview of the AP-MS procedure. In Scheme A, SDS-PAGE separates a purified protein complex, the gel slice is excised, the proteins are digested, and the peptides are extracted from the gel. Peptides arising from a single protein are either separated by HPLC and analyzed by ESI/MS or MS/MS, or they are analyzed by MALDI/TOF. In Scheme B, the entire protein complex is digested. Peptides arising from the digestion of multiple proteins are separated by HPLC and analyzed by ESI MS/MS. Database searching reveals the identity of the proteins in the complex. (RP) Reverse phase; (SCX) strong cation exchange.

After a protein complex has been purified, its individual components are determined by MS. Figure 1 illustrates two approaches that are commonly used. In Scheme A, the protein complex is separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Coomassie or silver staining (Silver Staining, Digestion, and Extraction of Peptides from an Acrylamide Gel for MS Analysis) is used to visualize the individual components. The individual stained protein bands are excised from the gel, the proteins are enzymatically digested into peptides, and the peptides are extracted (Silver Staining, Digestion, and Extraction of Peptides from an Acrylamide Gel for MS Analysis). The peptides from each protein can be analyzed by two approaches. In the first approach, the isolated peptides are fractioned in a single-phase column with high-pressure liquid chromatography (HPLC) and analyzed by electrospray ionization (ESI) MS. This generates a mass fingerprint of the peptides that comprise the unknown protein. Further structural data for peptides can be obtained by fragmenting the ionized peptides to generate a tandem mass spectrum. In the second approach, a mass fingerprint of the peptides is obtained by matrix-assisted laser desorption/ionization in combination with a time-of-flight instrument (MALDI/TOF) (HPLC Separation of Digested Proteins and Preparation for Matrix-Assisted Laser Desorption/Ionization Analysis). Briefly, the peptides are mixed with a matrix, and a laser is used to induce ionization for subsequent analysis in a TOF instrument. Both of these approaches generate a mass fingerprint that can be used to determine the original protein identity.

In Scheme B, the entire protein complex is digested by chemical agents or by proteases (Direct Enzymatic Digestion of Protein Complexes for MS Analysis). The mixture of peptides that results is more complex than that obtained from the digestion of a single protein, and is therefore best resolved using HPLC in combination with a biphasic column (HPLC Separation of Digested Proteins and Preparation for Matrix-Assisted Laser Desorption/Ionization Analysis). The biphasic column is packed with a strong cationic exchange (SCX) and a C18 resin, in a tandem arrangement. Peptides interact with the SCX resin, as they are positively charged in the acidic loading buffer and are eluted in small portions into the C18 resin by a mobile phase containing ammonium acetate. Similar to the single-phase column, peptides are eluted from the C18 resin into the mass analyzer with a gradient of acetonitrile. This process of eluting small amounts of peptides from the SCX resin into the C18 resin, and then into the mass analyzer, is repeated several times depending on the complexity of the protein complex. If the protein complex contains a larger number of components, a more gradual increase in ammonium acetate concentration can be used to elute smaller amounts of peptides from the SCX resin into the C18 resin. This will lead to a better separation of peptides. The identities of peptides from complex mixtures are best obtained by generating a tandem mass spectrum. Peptide identification from a mass fingerprint or a tandem mass spectrum is initiated by searching the most recent protein sequence libraries that are available. Several algorithms have been developed to assist in searching a peptide mass fingerprint or a tandem mass spectrum. Outputs from these searches reveal the components of the protein complex.