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.


1
. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment 2 . Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice 3 . 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 Knockdown 4 , 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 accurate 5,6 . 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 15 N 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 protein 7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state 8 . We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE1 9 .

Video Link
The 3. After the last centrifugation step, remove supernatant and leave approximately 0.5 ml of phosphate buffer. Add 0.9 ml 0.5 M phosphate buffer (pH 7.4), 400 μl affinity purified primary antibodies (50 μl per IP) against the target protein (here HSP70B), and 16 μl antibodies against a control protein (here CF1β). Fill up with ddH 2 O to a total volume of 5 ml.
(Note that affinity-purified antibodies should be used to reduce contamination by unspecific IgGs, which interfere with nano-LC-MS analysis -for a protocol see Willmund et al. (2005) 10 . CF1β is precipitated as a loading control and was chosen because it is abundant and, after cell lysis, present in soluble and membrane fractions. Alternatively, levels of contaminating proteins may be used to normalize for unequal loading.) 4. Allow Protein A Sepharose beads to adsorb IgGs during a 1-hr Incubation at 25 °C on a tube roller (CAT RM5W, 36 rpm). 5. Centrifuge for 60 sec at 1,000 x g and 4 °C to pellet the Protein A Sepharose beads. Carefully remove the supernatant and resuspend the beads in 5 ml 0.1 M sodium borate buffer (pH 9.0). Repeat this step three times to thoroughly remove amines that would quench the crosslinker. 6. Weigh out 25.9 mg of fresh, solid dimethylpimelimidate and resuspend it in 5 ml 0.1 M sodium borate buffer (pH 9.0) to obtain a final concentration of 20 mM. Add this solution to the Protein A Sepharose beads. 7. Allow IgGs to cross-link to Protein A for 30 min at 25 °C on a tube roller. 8. Centrifuge for 60 sec at 1,000 x g and 4 °C to pellet the beads. Carefully remove the supernatant and resuspend the beads in 5 ml 1 M Tris-HCl (pH 7.5) to quench free crosslinker. Repeat this step once and incubate for 2 hr at 25 °C or 12-24 hr at 4 °C on a tube roller. 9. Optional: if Protein A Sepharose beads coupled to IgGs are not directly used for IP, storage for up to one week is possible. For this, centrifuge for 60 sec at 1,000 x g and 4 °C to pellet the beads, carefully remove the supernatant and resuspend beads in 5 ml 0.1 M phosphate buffer (pH 7.5) containing 0.02% sodium azide and store at 4 °C until further use.  1. Pellet the Protein A Sepharose beads containing coupled antibodies (from steps 1.8 or 1.9) by a 60-sec centrifugation at 1,000 x g and 4 °C, carefully remove the supernatant and resuspend beads in 4 ml Lysis buffer. Repeat this step twice to equilibrate beads in Lysis buffer. 2. Fill up to 8 ml with Lysis buffer and transfer 1 ml of the suspension to each of the eight 15-ml Falcon tubes containing soluble or membrane protein complexes from ATP-replete and ATP-deplete 14 N-and 15 N-labeled cells (from step 2.9 and 2.12). 3. Incubate for 2 hr at 4 °C on a tube roller to precipitate protein complexes. 4. Pellet the beads by a 60-sec centrifugation at 1,000 x g and 4 °C and discard the supernatants. Leave a small volume of liquid on top of the beads to facilitate transfer. 5. Transfer the beads from each Falcon tube to 1.5-ml conical tubes (Eppendorf tubes). To collect all remaining beads in the Falcon tubes, add another 0.8 ml Lysis buffer containing 0.1% Triton to each, vortex gently, centrifuge for 60 sec at 1,000 x g and 4 °C and transfer the buffer with residual beads to the Eppendorf tubes. Tube exchange is necessary to prevent contaminations from proteins adhering to the plastic walls. 6. Pellet the beads by a 15-sec centrifugation at 16,100 x g and 4 °C, carefully remove the supernatants and resuspend beads in 1.3 ml Lysis buffer containing 0.1% Triton. Repeat this step twice with lysis buffer containing Triton and twice with Lysis buffer lacking Triton to thoroughly wash the beads. Leave a small volume of liquid on top of the beads to facilitate transfer. 7. Again transfer the beads to fresh 1.5-ml Eppendorf tubes to remove proteins adhering to the plastic walls. Wash the old tubes with 1 ml Lysis buffer lacking Triton and transfer all residual beads to the fresh tubes. 8. Centrifuge for 15 sec at 16,100 x g and 4 °C, remove the supernatants first with a normal pipette, then remove any remaining supernatant completely with a 50-μl Hamilton syringe.

Sample Preparation for nano-LC-MS/MS
The interaction of CGE1 with HSP70B only in the ADP state is also observed in the MS analysis: in Figure 3, representative MS1 spectra of HSP70B and CGE1 peptides from precipitates generated with the HSP70B antiserum from soluble cell extracts are shown. In the experiment shown in Figure 3A, precipitates were from mixtures of 14 N-labeled extracts lacking ATP and 15 N-labeled extracts containing ATP. While the heavy and light labeled form of the HSP70B peptide were detected at equal intensities, only the light labeled form of the CGE1 peptide (from -ATP extracts) was found. In Figure 3B the same peptides from the anti-HSP70B precipitate derived from mixtures of reciprocally labeled soluble cell extracts are shown. Accordingly, this time only the heavy labeled form of the CGE1 peptide (from -ATP extracts) was detected, while this was again the case for both, light and heavy labeled HSP70B peptides.  15 N-labeled fraction was depleted from ATP. Accordingly, the ratio of intensities of heavy labeled (dark colors) to light labeled (light colors) peptides from the control protein (CF1β), the target protein (HSP70B) and non-specifically bound contaminants should be around one, while this ratio is expected to be very high for proteins specifically interacting with the target protein (CGE1). Click here to view larger figure.

Discussion
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 efficiencies 6 . Here we present a protocol containing two more improvements of QUICK: first, we replace SILAC 5  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 downregulated 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 systems 14,15 , 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 systems 15 .
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.

Disclosures
No conflicts of interest declared.