Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

NOTE: All buffer compositions and protease mixtures are outlined in Table 1.

1. Preparation of cells

NOTE: A starting material of 1−10 mg nuclear lysate per replicate is desired for this immunoprecipitation mass spectrometry (IP-MS) approach. Cell quantities will be given for 1 mg of nuclear immunoprecipitations in triplicate plus triplicate controls.

1. If using an adherent cell line, grow the cells to 90% confluency in 3 x 15 cm dishes per replicate prior to harvesting.
   NOTE: It is recommended to perform a minimum of three replicate immunoprecipitations for bait and control conditions. This protocol will assume the use of ‘beads only’ controls, which control for abundant nonspecific interactions with the beads, starting at section 4. Other types of controls may be useful. These are described in depth in the discussion section.
   1. Wash plates 2x with phosphate buffered saline (PBS) and trypsinize cells using 3 mL of 0.25% trypsin per 15 cm plate. Spin down the cells at 1,200 x g for 5 min and decant the trypsin.
2. For suspension cells, grow to a similar scale/density to achieve 70−80 mg of total protein.
   1. Pellet cells at 1,200 x g and 4 °C for 5 min. Decant the media carefully.
      NOTE: Pellets can be combined during this step to enable efficient processing using the large-scale subcellular fractionation outlined in section 2.
3. Wash the cell pellet 2x with PBS + 5 mM MgCl₂ supplemented with protease inhibitors (PIs) and phosphatase inhibitors (PhIs) (see Table 1).
   NOTE: Cell pellets may be flash frozen in liquid nitrogen and stored at -80 °C until ready for fractionation.

2. Preparation of nuclear extract

NOTE: Protease and phosphatase inhibitors should be added to the fractionation buffers within 30 min of use.

1. If frozen, thaw the cell pellets for 15 min in 1x pellet volume of cold Buffer A + PIs/ PhIs. Place the cell pellet on a nutator at 4 °C to aid in resuspension while thawing. Otherwise, resuspend the cell pellet from step 1.3 into 1x pellet volume Buffer A + PIs /PhIs.
   1. Pellet at 2,000 x g and 4 °C for 10 min. Decant the buffer.
2. Suspend the cells with 5x the packed cell volume with Buffer A and incubate on ice for 20 min.
3. Pellet at 2,000 x g and 4 °C for 10 min. Decant the buffer and resuspend with 2x original packed cell volume Buffer A + PIs/ PhIs and dounce ~7x with “A”/loose pestle.
4. Centrifuge the lysate for 10 min at 2,000 x g and 4 °C.
5. Carefully pipette off the supernatant and flash freeze with liquid nitrogen. Store the lysate at -80 °C. The supernatant from this step is the cytosolic subcellular fraction.
   NOTE: The nuclear pellet can be saved during this step by flash freezing with liquid nitrogen and storing at -80 °C
6. Resuspend the pellet with 0.9x pellet volume of Buffer B + PIs/ PhIs and mix on a nutator for 5 min at 4 °C.
7. To lyse the nuclei, dounce 20x with a tighter pestle “B”.
8. Mix the nuclear lysate on a nutator for 30 min at 4 °C so that it is homogenous.
9. Centrifuge the nuclear lysate for 30 min at 21,000 x g at 4 °C. Pipette off the supernatant and save as a soluble nuclear protein extract.
   NOTE: Nuclease treatment of the resulting nuclear pellet allows for the recovery of a chromatin-associated protein fraction.
10. Dialyze the soluble nuclear extract against Buffer C + PIs for 3 h at 4 °C.
    1. Cut an appropriate length of 24 mm width dialysis tubing with an 8 kDa molecular weight cut off. Clamp one side of the tubing and load nucleoplasm into the tube. After loading the lysate, clamp the other end and submerge into a clean glass container containing Buffer C + PIs.
11. Centrifuge the dialyzed nuclear extract/nucleoplasm at 21,000 x g at 4 °C for 30 min. Aliquot 3x 20 µL volumes of nuclear extract for fractionation validation by western blot. The nuclear extract used for IP-MS analysis can be aliquoted and flash frozen in liquid nitrogen and stored at -80 °C, if needed.

3. Validation of subcellular fractionation

1. Complete a protein assay to determine the protein concentration of the nuclear lysate. A bicinchoninic acid protein assay provides sufficient sensitivity for downstream application.
2. Load 20 µg of both the cytosolic and nuclear fractions on an SDS-PAGE gel for the western blot analysis as previously described¹³. Skip lanes when loading to avoid mischaracterization of a sample.
3. Probe the western blot for p84 (THOC1) as a nuclear marker, and GAPDH as a cytosolic marker. Determine the extent of fractionation by the ratio of cytosolic marker in the nuclear fraction and vice versa.
   NOTE: Antibodies for other nuclear and cytosolic markers may be used.

4. Immunoprecipitation of endogenous nuclear bait protein

NOTE: It is recommended to use low retention tubes from this point on. This will reduce the nonspecific binding to the tubes during sample handling and avoid unnecessary loss of sample. Additionally, ensure that LCMS grade H₂O is used to prepare buffers for the remaining steps.

1. Prepare a protein A/G bead mixture for each replicate by combining 12.5 µL of bead volume for both protein-A and protein-G in microcentrifuge tubes. Store the bead stocks as a slurry containing 20% ethanol. Determine the concentration of beads within the slurry %(v/v) and pipette the necessary volume using a pipette tip that has been cut on the tip to ensure that the beads can enter the tip.
2. Wash the protein A/G bead mixture 2x with 300 µL of IP Buffer 1. Spin the beads at 1,500 x g at 4 °C for 1 min and decant buffer.
3. Prepare the antibody-protein A/G beads: To bind the antibody to the beads, add 300 µL of IP Buffer 1 and 10 µg of the desired antibody. Allow the bead/antibody mixture to rock on a nutator at 4 °C overnight. For bead-only controls, do not add any antibody.
   NOTE: A total of 10 µg of antibody per replicate can be used as a starting point, but the exact amount will need to be optimized for each individual antibody and scale of the lysate used in the experiment
4. Thaw the nuclear lysates from step 2.10 in a water bath and aliquot appropriate volumes into low retention microcentrifuge tubes for 1 mg protein input per replicate.
   1. Spin the lysate at 16,000 x g for 30 min and transfer the supernatant to a new tube.
   2. Add 1 µL of benzonase (250 units/µL) per 1 mg of the nuclear lysate and rock on a nutator at 4 °C for 10−15 min.
5. Prepare beads for preclearing the lysate. Add 12.5 µL of each protein A and protein G beads to 1.5 mL low retention tubes as in step 4.1. Wash 2x with IP Wash Buffer 1 + PIs and decant the buffer.
6. Add 1 mg of the nuclear lysate to the beads from step 4.5. Incubate while rocking on a nutator for 1 h at 4 °C.
   1. Centrifuge precleared lysates at 1,500 x g and 4 °C for 1 min.
   2. While nuclear lysates are incubating with beads in step 4.5.1, wash the antibody-protein A/G beads 2x with IP Buffer 1 + PIs. Centrifuge at 1,500 x g and 4 °C for 1 min and decant the buffer.
7. Transfer the precleared nuclear lysate from step 4.6.1 onto the antibody-protein A/G beads. Incubate while rocking on a nutator at 4 °C for 4 h. Centrifuge following the incubation at 1,500 x g and 4 °C for 1 min.
8. Transfer the supernatant into tubes labeled as the flow through for each replicate.
9. Wash the antibody-protein A/G beads with 1 mL of IP Buffer 2 + PIs. Centrifuge at 1,500 x g and 4 °C for 1 min, decant buffer, and repeat for a total of 3x.
10. Wash the beads 2x with 1 mL of IP Buffer 1+ PIs centrifuging as in the previous step. Ensure that all buffer is removed after the last wash.
11. Elute 2x with 20 µL of 0.1 M glycine (pH 2.75) for 30 min each. Ensure that the tubes is rocking during the incubation with the elution buffer. Spin at 750 x g and 4 °C for 1 min and pipette off the supernatant after each 30 min incubation.
    NOTE: While the low pH glycine method described here elutes most bait proteins, some antibody-antigen interactions require more stringent buffer conditions.
12. Flash freeze eluates in liquid nitrogen and store at -80 °C.

5. Sample preparation

NOTE: Insulin spiked into the immunoprecipitation elution samples aids in the recovery of proteins during trichloroacetic acid (TCA) precipitation and sample processing, which is important for low abundance endogenous bait proteins.

1. Thaw the eluates at room temperature if frozen.
   1. Place the samples on ice and add 10 µL of 1.0 mg/mL insulin for every 100 µL of eluate. Vortex and then immediately add 10 µL of 1% sodium deoxycholate. Vortex the sample again and add 30 µL of 20% TCA followed by one final vortex.
   2. Incubate the samples on ice for 20 min, then centrifuge at 21,000 x g at 4 °C for 30 min.
   3. Aspirate the supernatant and add 0.5 mL of acetone that has been prechilled to -20 °C. Vortex and then spin at 21,000 x g and 4 °C for 30 min. Repeat this step.
   4. Aspirate the supernatant and air dry the pellet remaining in the bottom of the tube.
2. Prepare the sample for mass spectrometry using a modified Filter Aided Sample Prep (FASP) method, optimized for reducing sample handling, as outlined below¹⁴.
   1. Resuspend the protein pellet from step 5.1.4 with 30 µL of SDS Alkylation Buffer (see Table 1). Incubate the sample on a 95 °C heat block for 5 min. Let it cool at room temperature for 15 min before preceding to the next step.
   2. Add 300 µL of UA solution and 30 µL of 100 mM TCEP to each sample. Load this solution onto a 30k centrifugal filter. Spin the centrifugal filter at 21,000 x g at room temperature for 10 min.
      NOTE: The bait protein and its putative interactors should be bound to the filter at this point. However, the flow through may be kept, in case there is a problem with the filter.
   3. Wash the filter with 250 µL of UA and centrifuge at 21,000 x g for 10 min. Decant the flow through and repeat for a total of 3x.
   4. Wash the filter with 100 µL of 100 mM Tris pH 8.5 and centrifuge at 21,000 x g for 10 min. Decant the flow through and repeat for a total of 3x.
   5. Add 3 µL of 1 µg/µL Lys C resuspended in 0.1 M Tris pH 8.5. Fill the filters up to the 100 µL mark and allow to digest for 1 h at 37 °C while rocking on a nutator.
   6. Add 1 µL of 1 µg/ µL MS grade trypsin. Mix gently and allow for the trypsin to incubate with the sample overnight at 37 °C while rocking on a nutator.
   7. Centrifuge at 21,000 x g for 20 min to elute the peptide from the filter.
      NOTE: Multiple rounds of centrifugation may be required to recover all the eluate. If this is not done, there is a potential for severe sample loss.
3. Desalt the peptides using C18 spin columns. Follow the protocol provided by the manufacturer.
4. Resuspend the lyophilized peptide in 7 µL of 0.1% TFA in 5% acetonitrile. Sonicate the sample for 3 min to ensure that the peptides have been resuspended. Spin down at 14,000 x g for 10 min.
5. Transfer the resuspended peptide into an appropriate sample vial to for loading onto the liquid chromatography–mass spectrometry (LC/MS) system.

6. LC/MS system suitability

NOTE: Due to the small scale and generally lower abundance of protein from affinity-purified samples, it is critical that the LC/MS platform operates at a maximal sensitivity and robustness.

1. Add 1 mL of LC/MS grade formic acid to 1 L of LC/MS grade water for mobile phase A, and add 1 mL of LC/MS grade formic acid to 1 L of LC/MS grade acetonitrile for mobile phase B.
2. Prepare or install a 75 µm fused-silica capillary column packed with <2 µm reversed-phase C18 resin that is ≥250 mm in length. Best results will be had with a direct injection of samples into the column.
3. Purge the Ultra Performance Liquid Chromatography (UPLC) system with fresh mobile phases. With a C18 column installed, establish a stable flow rate and electrospray with a suitable emitter (i.e., 20 µm id x 360 µm od pulled to a 10 µm tip). Maintain the column at 40−60 °C.
4. Test the overall LC/MS system performance by injecting a complex quality control standard, such as 100−200ng of a HeLa whole cell lysate tryptic digest. Elute with a suitable gradient for a complex sample (i.e., 2−3 h gradient elution time). Establish a baseline system performance of the peptide and protein identifications.
   NOTE: For best results, 3,000−5,000 or more protein identifications from 20,000−35,000 unique peptides will provide optimal performance for experimental samples.
5. For routine LC/MS system suitability, inject 100−200 fmol or less of a single protein digest standard, such as Bovine Serum Albumin (BSA). Elute with a short gradient (i.e., 20−30 min).
   NOTE: Multiple injections of a protein digest will help establish baseline LC/MS system performance, and repeat injection after each IP-MS sample provides a measure of system performance throughout the experiment and allows for the detection of instrument drift, which can bias label-free experiments. A baseline of the select individual peak intensities and peak shapes will inform on MS, LC, and column performance.
6. To avoid overloading the analytical column, load a small portion (15−30% of the total) of an experimental sample onto the column and separate using a gradient suitable for complex samples (i.e., 2−3 h). If the number of protein identifications is unsatisfactory, load all of the sample onto the column.
7. Run a single protein digest standard in between samples to monitor LC/MS system performance and sample carryover. Multiple standards may be required to reduce sample carryover depending on your samples.

7. Data Processing

1. Download the proteomics software package MaxQuant found at https://www.maxquant.org/.
   NOTE: This will be used to process the RAW MS data file from step 6.6 into data tables of protein IDs, gene names, and quantitative values associated with the identification of these for downstream analysis.
   1. Select Load within the Input Data subheader of the Raw Data tab. Open the file location where the MS raw files are stored and select raw files for each MS/MS run.
   2. Click on the Group-Specific tab and select Digestion. Within the enzyme list select LysC and click on the right arrow to add this enzyme into the list that will be used in the search. Next, select Instrument and ensure that the correct instrument type appears in the drop-down list at the top of the screen. Leave other group-specific search parameters on standard settings.
   3. Click on the Global Parameters tab and select Sequences. Add the appropriate FASTA file for the taxonomy that will be used in this search. Peptides will not be assigned properly if this is not done. For the human proteome, download the FASTA file from UniProt at https://www.uniprot.org/help/human_proteome.
   4. Within the Global Parameters tab, click on Protein Quantification. Within the Peptides for Quantification drop-down menu, select Unique + Razor.
      NOTE: MaxQuant offers alternate quantitation of proteins through intensity-based absolute quantification (iBAQ) and label-free quantitation (LFQ). However, peptide count information is sufficient for the downstream analysis in this protocol¹⁵.
   5. In the bottom left of the MaxQuant interface, select the number of processors to be used for the search. This will directly affect the length of time required for the run, so select as many as possible for this). Click Start in the bottom left of the screen to start the run. Select the Performans tab at the top of the screen to view the progress of the search.
2. When the run has completed, open the proteingroups.txt file in Perseus, a proteomics computation platform, or other spreadsheet program to view the data¹⁶.
   1. Use Perseus to remove common contaminants and hits to reversed protein sequences. Follow the detailed Perseus documentation at http://www.coxdocs.org/doku.php?id=perseus:user:use_cases:interactions.
      NOTE: Opening the proteingroups.txt file in analysis software (e.g., Excel) will automatically corrupt certain gene and protein names.
3. Analyze experimental data using the Contaminant Repository for Affinity Purification (CRAPome). Register an account at this repository http://crapome.org/ and follow the tutorial as needed^17,18.
   1. Use the Analyze Your Data workflow found on the CRAPome homepage. Select external controls that correspond to the affinity purification system used in this interaction experiment.
      NOTE: These controls can be used to calculate a second fold change enrichment that is useful for detecting common contaminants.
   2. Generate an input file from the proteingroups.txt output from MaxQuant using Perseus or a suitable spreadsheet application. Details for manual formatting can be found at http://crapome.org/?q=fileformatting. Alternatively, use the provided R script “export_CRAPomeSAINT_Input_File.R” to generate the SAINT/CRAPome input file. See README.txt in the Supplemental Coding Files.
   3. Run an analysis to determine fold-change enrichment and SAINT (Significance Analysis of INTeractome) probability for each bait protein in the immunoprecipitation. Ensure that 'User Controls’ is selected in the drop-down menu under FC-A, ‘CRAPome controls’ or ‘All Controls’ are selected in the FC-B drop-down and Probability Score is selected to generate SAINT probabilities. When the run has concluded, view the output that is available under ‘Analysis Results’ along with a job ID. Download the data matrix from the ‘Analysis Results’ for future plotting and data visualization.
4. Plot proteins as a function of FC-A (IPs vs. user controls) and SAINT probability by following the R-Scripts as provided in Supplemental Coding Files.
   NOTE: A set of R scripts is provided for producing plots of FC-A vs. SAINT probability and iBAQ vs. log₂ (protein abundance), colored by the adjusted p value range from empirical Bayes analysis of the label-free intensities. The details of the differential statistical analysis and plotting are found in README.txt and the R script “main_differential_analysis.R” in the Supplemental Coding Files.
5. Evaluate where known interacting proteins of the bait protein are ranked by FC-A and SAINT. Make a cutoff of FC-A > 3.00 and SAINT > 0.7 for single bait experiments in triplicate as a starting point.
   NOTE: Selection of cutoffs for a “high-confidence” interactor and a “low-confidence” interactor must be informed by biological information.

8. Data visualization

NOTE: There are many programs that can effectively visualize proteomics data (e.g., R, Perseus, Cytoscape, STRING-DB). Analyzing the connectivity between high-confidence hits, and functional enrichment of these interactors can be a useful strategy for prioritizing hits for further validation and functional characterization.

1. Download Cytoscape, an open source network visualization tool at https://cytoscape.org/download.html¹⁹.
2. Prepare an input file for interaction data as a tab delimited file formatted with three columns: bait (source node), prey (target node), type of interaction (edge type). This can be done in Perseus or any spreadsheet program of your choice.
3. Select the Import Table from File icon towards the top left of the program (designated by a downwards arrow and a matrix in the icon). Cytoscape will auto-populate the interaction data into a network ready for custom formatting and design.
4. Select the Style tab on the control panel for Cytoscape and click on the squares in the Def column to adjust the attribute for the entire network. Select specific nodes or edges in the network and then select the square within the Byp. column of the style menu to bypass the default settings and adjust only selected objects. Alternatively, click on the drop-down menu at the top of the style menu to view the preset network formats.
   NOTE: STRING-db protein-protein interaction data may be integrated into this network at this time either manually through the input file or through various enrichment tools available as plug-ins in Cytoscape, http://apps.cytoscape.org/²⁰. A recommended cytoscape plug-in for enrichment analysis is found at http://apps.cytoscape.org/apps/cluego²¹.
5. To increase the confidence in the dataset generated in this workflow, perform reciprocal IP-MS or IP-western experiments that target prey proteins of interest as the bait.