Step 1 Co-immunoprecipitation
Timing: 1.5 days
All steps should be performed as quickly as possible, on ice and with pre-chilled buffers. It is very important not to use previously frozen or otherwise manipulated samples, as this will result in loss of interactors. It is also strongly advised to test the specificity and suitability of antibodies for immunoprecipitation with Western blotting first, and verify the specificity of the antibody for the bait by including appropriate controls such as identical tissues or cells not expressing the bait (for example using a gene knockout model). While mass spectrometry is exquisitely sensitive, it cannot correct for insufficient antibody specificity or affinity or for suboptimal performance of the immunoprecipitation. Thus the bait should be highly enriched as compared to input and the antibody should be highly specific for best results. In this regard, it is recommended to use only Sepharose beads that are sufficiently saturated with antibody and show better than 90% covalent coupling efficiency of antibodies to the beads. Saturation of ProteinA/G-Sepharose beads and covalent coupling efficiency can be easily checked by SDS-gel electrophoresis and Coomassie blue staining. Additionally, the optimal starting amount may need to be empirically determined for different bait proteins. A very general recommendation is 1x 108 cells for bait proteins that are equal to or less than 100 molecules/cell.
1.1. Rinse cells with at least 10 volumes (v/v) of 1 x DPBS. Remove excess 1 x DPBS by decanting or by vacuum suction and tapping on a paper towel.
1.2. Immediately add 1.5 ml of ice-cold TNI lysis buffer per 15 cm cell culture dish (TNI: 0.5% Igepal CA-630, 50 mM Tris [pH 7.5], 250 mM NaCl, 1 mM EDTA and 1x Complete ULTRA EDTA-free Protease Inhibitor cocktail, 1x PhosStop).
1.3. Incubate for 20 min on ice on an orbital shaker.
1.4. Scrape off cell lysate with a large cell scraper and transfer to a 1.5 ml microcentrifuge tube.
1.5. Sonicate the cell lysate for 3 min in a water bath sonicator operating at 55 kHz. Critical: Do not use a probe sonicator, this will result in loss of interactions.
1.6. Centrifuge for 30 min. (18,000 x g, 4 ˚C).
1.7. Preclear the cell lysate with an appropriate amount of Protein G Sepharose 4 Fast Flow beads (100 µl of 50% slurry in lysis buffer) for 2 h at 4 ˚C with head-over-head rotation. The volume of preclear beads is the same as the volume of antibody coupled beads. For volumes larger than 1.4 ml, pool the cell lysates and transfer into a 15 ml tube.
1.8. Centrifuge for 3 min, 500 x g, 4˚C.
1.9. Transfer supernatant to a new microcentrifuge tube containing the
appropriate amount of antibody-coupled sepharose slurry (e.g. 100 µl of 50% antibody slurry, equaling 200 µg of antibody for starting amount of ≈10 mg). Incubate over night with head-over-head rotation at 4 ˚C.
1.10. Centrifuge the binding reaction for 3 min at 500 x g, 4 ˚C.
1.11. Remove the supernatant, transfer the beads to a new tube and wash the
beads three times with 20 – 100 bead volumes of TNI lysis buffer.
1.12. Centrifuge for 3 min at 500 x g, 4 ˚C, carefully remove supernatant, and
wash beads two times with TN lysis buffer containing no Igepal CA-630.
1.13. Carefully remove all of the supernatant with an insulin syringe. Optional: Freeze beads for >1 h at –80 ˚C to increase yield.
PAUSE POINT: beads can be stored at -80˚C for up to two weeks.
Step 2 Elution of protein complexes and sample clean up
**Timing**: **60** **min**
2.1. Elute proteins twice with at least four to 10 bead volumes of elution buffer (0.2 M glycine, pH 2.3/ 0.5% Igepal CA-630) for 20 min, 37 ˚C, with shaking.
2.2. Combine the eluates and transfer to a new microcentrifuge tube.
Critical: Make sure to get no beads into the eluate, they might clog microcapillary columns and produce background signal later in the mass spectrometer.
2.3. Neutralize eluate with 1/10 vol (v/v) freshly prepared 1 M NH4CO3 .
2.4. Add 4 vol (v/v) Methanol to the eluate and vortex.
2.5. Add 1 vol (v/v) Chloroform and vortex well for 0.5 - 1 min.
2.6. Centrifuge for 10 min at 18,000 x g.
2.7. Remove the supernatant without disturbing the pellet.
Note: The pellet may be very tiny or hardly visible at all.
2.8. Wash pellet with 3 vol (v/v) Methanol.
2.9. Centrifuge for 10 min at 18,000 x g.
2.10. Remove the supernatant without disturbing the pellet.
PAUSE POINT: the pellet can be stored at -80˚C for up to four weeks.
Step 3 Digestion of eluted proteins
**Timing**: **15** **h**
3.1. Re-solubilize the methanol / chloroform precipitated proteins (pellet) in 100 mM Tris, pH 8.5, 0.2% Rapigest and sonicate for 1 h in a water bath sonicator.
3.2. Reduce cysteine bonds with 5 mM TCEP for 20 min.
3.3. Alkylate with 10 mM Iodoacetamide or Chloroacetamide for 30 min shielded from light.
3.4. Digest proteins with recombinant trypsin (30:1 ratio protein/trypsin) over night at 37˚C with shaking (e.g. in an Eppendorf Thermomixer).
3.5. Inactivate Rapigest by adding formic acid to 9% final concentration and incubate for at least 1 h, 37˚C, with shaking.
3.6. Reduce samples to near dryness in vacuo (approximately 45 min).
PAUSE POINT: Samples can be stored at -80˚C for up to four weeks.
Step 4 Mass spectrometric analysis of peptides
**Timing**: **1-2** **days**
This step may be carried out in a proteomics core facility or proteomics laboratory with MudPIT experience.
Resolubilize sample in a small amount of buffer A, load onto a preparative MudPIT column and perform a MudPIT experiment as described in 2,3.
Step 5 Data analysis
Timing: 2 days to several weeks
The task to confidently identify proteins from mass spectra is complex. Prior experience in mass spectrometry as well as in data analysis software is very strongly recommended.
Convert raw data with an appropriate converter (e.g. RawExtract) into a file format that is compatible with the database search engine (e.g. Sequest or ProLuCID).
Search MS/MS spectra against the appropriate protein database with a search engine such as ProLuCID to determine cross-correlation scores of acquired to in silico calculated spectra.
Filter search engine results to a low false discovery rate (FDR), for example using DTASelect2. Search engine results from MS/MS spectra should be combined for all biological replicates before assigning peptides to proteins and filtering the results. We also strongly recommend to conservatively filter the search results and adjust the false positive rate to a peptide false positive rate of less than 0.5% and a protein false positive rate of less than 1.0%.
Resolve protein identification ambiguities for example by using a gene centric approach. Redundant identifications can skew the statistical analysis towards genes identified with many protein isoforms.
Critical: The bait must be among the top 10 proteins with the highest number of spectral counts. Recommended minimum spectral counts for the bait protein is between 50 and 100 spectral counts per experimental replicate (SpC).
To distinguish specific protein interactors from non-specifically binding proteins, run CoPITgenerator according to the instructions (www.proteomicswiki.com) . CoPIT generator reports a rank-ordered list of interactors with the bait protein that can be further analyzed. A confidence score (P-value) is reported for every potential interactor that reflects its likelihood of interaction. A user defined P-value cutoff can be set to differentiate weak from strong interactors.
To compare interactomes, the P-value cutoff can be adjusted according to the ratio of bait in the different experimental conditions:
To identify differentially binding proteins in two different experimental conditions, calculate ratios rp for individual proteins (p) from the sum of all intensities per protein Ip and experiment condition e1 and e2 and normalize to the sum of all bait intensities (SpC) according to:
Calculate errors according to random error of measurement:
in order to find significantly different proteins for example with a cutoff of at least 2-fold difference plus one or two standard deviations.
Step 6 Network representation and data presentation
**Timing**: **2** **days**
The core interactome can be loaded into the Radial Topology Viewer (www.proteomicswiki.com) for visualization. The Radial Topology Viewer displays the bait at the center of the interactome network and arranges the interactors radially according to a user-defined distance. Interactors can be further grouped in multiple spokes based on a user-defined classification scheme, for example according to GO-terms.
Interactions between the identified interactors can be obtained with the GeneMANIA 2.2 Plugin in Cytoscape 2.8.2 5,6. Export connectivity information to a .txt or .csv file to load into Radial Topology Viewer. A step-by-step instruction how to obtain networks from GeneMania and Cytoscape is available in Morris et al., 2014 7.