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

The overall goal of the following experiment is to identify proteins specifically interacting with a given target protein in cell extracts containing soluble and membrane proteins. First, achieve full metabolic labeling of chlamydomonas cells with nitrogen 14 and nitrogen 15 and generate cell lysates containing and lacking a TP.An optional cross-linking step helps to stabilize transient protein protein interactions. Next, separate the cell lysates into fractions, enriched insoluble and membrane proteins from which the target protein, its specific interaction partners.

A control protein and contaminating proteins are immuno precipitated and alluded. Now, mix the immuno precipitated proteins and triply digest and analyze them by tandem LCMS. The results distinguished between true and false interaction partners by showing different nitrogen, 14 nitrogen 15 peptide ratios for proteins specifically interacting with the target protein and equal nitrogen.

14 nitrogen 15 peptide ratios for the target protein, the control protein and contaminating proteins. The method of choice for the analyzing protein protein interactions is to immuno precipitate the protein of choice directly with its interaction. Partners from cell extracts where both occur at their native confirmation and concentrations.

However, when doing so, also contaminating proteins are precipitated mainly because they interact with the antibodies used as modern mass spectrometers are extremely sensitive. They will unequivocally also identify the contaminating proteins and thereby it is impossible to distinguish fault from true interaction partners. This problem is overcome by the technique shown here, which is based on 15 N metabolic labeling immunoprecipitation affinity modulation here mediated by the A TP state and quantitative mass spectrometry.

The technique will be shown by Stefan SCH Malinger, who is a senior PhD student in my laboratory. After labeling the cells for 10 generations transfer two aliquots each of nitrogen 14 and nitrogen 15 labeled chlamydomonas cells to 4G SA tubes harvest by centrifugation Resus, suspend the pellets in two milliliters of ice cold lysis buffer and to transfer the suspensions to 15 milliliter Falcon tubes collect the remaining cells with an additional one milliliter of lysis buffer each to each Eloqua. Add 50 microliters of 25 x protease inhibitor and 12.5 microliters of one molar magnesium chloride sonicate samples four times 20 seconds on ice for complete cell ISIS with 22nd breaks in between for cooling.

Prepare four six milliliter sucrose cushions in SW 41 TI thin wall tubes. Carefully lay the entire volume of the cell lysates onto the sucrose cushions. Balance with lysis, buffer and centrifuge to separate soluble protein complexes from membranes.

Transfer the soluble protein complexes from the top of the gradient into four 15 milliliter falcon tubes. Making sure to avoid transferring parts of the sucrose cushion. Then to each sample add 10%Triton X 100 to a final concentration of 0.5%Mix carefully and add lysis buffer to a total volume of seven milliliters.

For SDS page and immuno blood analyses. Set aside 70 microliters of each soluble cell extract to a fresh 1.5 milliliter conical tube and add 70 microliters of two XSDS sample buffer. Next, discard the sucrose cushions from the SW 41 TI thin wall tubes and add three milliliters of lysis buffer to each pellet.

Add one milliliter of 10%tritton X 100 to a final concentration of 2%After dissolving each pellet by sonication, add lysis buffer to a total volume of five milliliters and centrifuge. As before, harvest the top fractions of membrane protein complexes into 15 milliliter falcon tubes. Add lysis buffer containing 2%tritton X 100 to a final volume of seven milliliters.

Use 70 microliters of these samples for SDS page and immuno blood analyses. First equilibrate the antibody coupled protein. A sero beads with two four milliliter washes of lysis buffer dilute to eight milliliters and aliquot.

One milliliter of the suspension to each of the eight 15 milliliter falcon tubes containing soluble or membrane protein complexes from a TP replete and a TP depleted nitrogen 14 and nitrogen 15 labeled cells incubate for two hours at four degrees Celsius on a tube roller to precipitate protein complexes pellet the beads by centrifugation, discard the supernatants, leaving a small volume of liquid on top of the beads to prevent contamination from proteins adhering to the plastic walls. Transfer samples to fresh 1.5 milliliter conical tubes to collect all remaining beads in the falcon tubes. Add another 0.8 milliliters of lysis buffer containing 0.1%tritton to each vortex, gently centrifuge, and to transfer the buffer with residual beads to the einor tubes After several washing steps, centrifuge samples for 15 seconds at 16, 100 times G and four degrees Celsius.

Remove the sample supernatants first with a normal pipette. Then remove any remaining supernatant completely with the 50 microliter Hamilton syringe. To each sample, add 100 microliters of freshly prepared elution buffer and incubate in a thermo mixer at 800 RPM centrifuge the samples for 15 seconds at 16, 100 times G and 25 degrees Celsius.

Now transfer each supernatant to a fresh tube using a clean 50 microliter Hamilton syringe. Also add 50 microliters of elution buffer to the beads process as with samples before and pool the respective elates. Remember to reserve 30 microliters of all sample EITs for SDS page and immuno blood analyses.

Now combine the eluded precipitates from plus and minus A TP treated nitrogen 14 and nitrogen 15 labeled soluble and membrane proteins. Then perform reduction alkylation and initial high urea digest as described in the accompanying manuscript. Proceed to a triptych digest on a rotation wheel for at least 16 hours.

At 37 degrees Celsius. Pellet the tripsin beads with a five minute centrifugation at 16, 100 times G and four degrees Celsius. Transfer the supernatants to fresh two milliliter einor tubes.

Wash the old tubes with 50 microliters of 20 millimolar ammonium bicarbonate, 0.5%acetic acid and pool with the first supernatants. In order to prepare homemade C 18 tips for desalting the samples cut out two discs from emcor C 18 material with a syringe needle and insert them into a 200 microliter pipette tip. Then punch holes into the lids of four two milliliter einor tubes and insert the tips precondition the C 18 stage tips with 50 microliters of solution B centrifuge for three minutes at 800 G and 25 degrees Celsius.

Next, equilibrate the C 18 stage tips with 100 microliters of solution, a centrifuge for three minutes at 800 G and 25 degrees Celsius. Repeat this equilibration Step once more. Proceed to load 100 microliters of the sample supernatants from the triptych digestions on the C 18 stage tips centrifuge for three minutes at 800 G and 25 degrees Celsius.

Repeat loading the column for complete application of sample supernatants. After washing the C 18 stage tips, elute the tryptic peptides into a fresh 1.5 milliliter einor tube with 50 microliters of solution B centrifuge for three minutes at 800 G and 25 degrees Celsius. Repeat the elucian step once and then dry the peptides to completion in a speed VAC reus.

Suspend the dried peptides in 20 microliters of solution A and incubate for at least one hour on ice, interrupted by two 15 minute incubations in a sonicate bath centrifuge for 20 minutes at 16, 100 times G and four degrees Celsius and apply Senna to nano C-M-S-M-S HSP 70. Heat shock proteins are known to interact with specific cos chaperones and substrates only in the absence of a TP in these nitrogen 14 labeled cell extracts hs P 70 B and CGE one are almost exclusively localized to the soluble fraction independent of the A TP state. In contrast, CF one beta is localized to both the soluble and membrane enriched fractions.

Since sonication shears part of CF one beta from the membrane located A TP Synthes, it serves as loading control for both fractions. Similar amounts of H HSP 70 B were precipitated with the anti HSP 70 B antibodies from nitrogen 14 and nitrogen 15 labeled soluble extracts independent of the A TP state. In contrast, low levels of HS P 70 B protein precipitated from membrane fractions with slightly larger amounts originating from a TP depleted membrane fractions as compared to a TP replete fractions as expected affinity of CGE E one for hs.

P 70 B is context dependent. No CGE one was co precipitated with HS P 70 B.In a TP replete soluble or membrane fractions. The amounts of CGE one protein co precipitated with HS P 70 B from a TP depleted, soluble fractions and a TP depleted membrane fractions correlate quantitatively with the amount of precipitated HSP 70 B.The interaction of CGE one with HS P 70 B only in the A DP state is also observed in the analysis by mass spectrometry.

In this experiment, precipitates were prepared for mixtures of nitrogen 14 labeled extracts lacking a TP and nitrogen, 15 labeled extracts containing A TP.While the heavy and light labeled forms of the HS P 70 B peptide were detected at equal intensities only the light labeled form of the CGE one peptide from the A TP depleted extracts was detected. The same peptides from the anti HS P 70 D DB precipitate derived from mixtures of reciprocally labeled soluble cell extracts are shown here. Accordingly, only the heavy labeled form of the CGE one peptide in a TP depleted extracts was detected as well as both the light and heavy labeled HSP 70 B peptides.

After watching this video, you should have a good impression on how to perform a coun precipitation experiment with sample preparation for mass spectrometry analysis. Don't forget that contaminations with human protein, especially keratin will disturb your prophy measurements in the mass spectrometer and therefore safety precautions such as gloves, clean environment and LCMS grade regions are crucial while performing this procedure.