Name: a protocol for the identification of protein-protein interactions based on 15n metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation
Uploaded: 2012-09-24T13:00:00
Duration: 14 min 44 s
Description: Watch this Scientific Journal Video about A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Aff

We thank Olivier Vallon for the antiserum against CF1&beta;. This work was supported by the Max Planck Society and grants from the Deutsche Forschungsgemeinschaft (Schr 617/5-1) and the Bundesministerium f&uuml;r Bildung und Forschung (Systems Biology Initiative FORSYS, project GoFORSYS).

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No conflicts of interest declared.

We have recently introduced two improvements to the QUICK approach: a crosslinking step for capturing transient protein-protein interactions (QUICK-X), and a control precipitation to normalize for unequal precipitation efficiencies6. Here we present a protocol containing two more improvements of QUICK: first, we replace SILAC5 for 15N metabolic labeling. The advantages are that 15N metabolic labeling is much cheaper than SILAC, if 15N is provided as simple inorganic salt. Furthermore, with 15N metabolic labeling QUICK can be applied to organisms prototrophic for all amino acids, like most plants, fungi and bacteria. And finally, arginine-to-proline interconversion inherent to SILAC5,6 does not present a problem for quantification of 15N labeled peptides. Examples for suitable tools for the quantitative evaluation of 15N proteomics data are MSQUANT12 or IOMIQS13. 
Second, we introduce affinity modulation as a means for specifically reducing the amount of proteins interacting with a given target protein in one sample versus another. The advantages of this approach are that it circumvents the construction of knockdown mutants, which for some model systems are difficult to generate or cannot be generated at all in case of essential target proteins. Moreover, it avoids misinterpretations caused by differential protein expression potentially occurring as a response of the cell to knocking-down a target protein: if other proteins are down-regulated as well and cross-react with the antiserum used for immunoprecipitation, they would be interpreted as true interaction partners of the target protein. At last, affinity modulation abolishes the need of finding a proper antibody-to-antigen ratio.
Although we apply our protocol to Chlamydomonas reinhardtii as model organism, it can easily be adapted to any other organism that can be grown in cell culture and is able to use ammonium or nitrate as nitrogen source. Affinity modulation of protein complexes by ATP/ADP may directly be applied to other chaperones whose interaction with substrates and cohort proteins depends on the ATP state, like the GroEL/HSP60/Cpn60 or HSP90 chaperone systems14,15, or to any other system where binding affinities are modulated by ATP. Affinity modulation should also work for cases where affinities between protein interactions are altered by specific drugs, like radicicol or geldanamycin in the case of HSP90 systems15. 
A clear limitation of our protocol is that it requires affinity-purified antibodies against a target protein known to be sensitive to a specific treatment/drug that modulates its affinity for partner proteins. Therefore, it is no high-throughput method.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>ProteinA Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>P3391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMP (Dimethyl pimelimidate) </td>
 <td>Sigma-Aldrich</td>
 <td>D8388</td>
 <td>Store desiccated at -20 &deg;C, dissolve directly before use</td>
 </tr>
 <tr>
 <td>Protease inhibitor (complete, EDTA-free)</td>
 <td>Roche Applied Science</td>
 <td>11873580001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ATP (Adenosin-5'-triphosphat)</td>
 <td>Carl Roth</td>
 <td>K054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Creatine phosphate</td>
 <td>Sigma-Aldrich</td>
 <td>27920</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Creatine phosphokinase</td>
 <td>Sigma-Aldrich</td>
 <td>C7886</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DSP Dithiobis [succinimidyl propionate]</td>
 <td>Thermo Sientific</td>
 <td>22585</td>
 <td>Store desiccated at 4 &deg;C</td>
 </tr>
 <tr>
 <td>15NH4Cl</td>
 <td>Cambridge isotope laboratories, Andover, MA</td>
 <td>NLM-467 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lys-C (Endoproteinase 
 Lys-C) </td>
 <td>Roche Applied Science</td>
 <td>11047825001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin beads (Poroszyme Immobilized Trypsin)</td>
 <td>Applied Biosystems</td>
 <td>2-3127-00</td>
 <td>Mix vigorously directly before use</td>
 </tr>
 <tr>
 <td>Empore C18 47 mm Disk</td>
 <td>Varian</td>
 <td>12145004</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a protocol for the identification of protein-protein interactions based on 15n metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

Watch this Scientific Journal Video about A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Aff

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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