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

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Transcript

This proteomics workflow enables the unbiased identification and mapping of endogenous protein-protein interactions with subcellular resolution. This technique is particularly advantageous when investigating bait proteins that are dosage sensitive, low abundance, or have multiple subcellular localizations. This method could be applied to mapping endogenous protein interactions assuming there's a suitable affinity reagent available.

This is particularly important for many of the thousands of proteins that have unknown function, many of which have been linked with human disease. This method can be adapted to any cell line or tissue amenable to subcellular fractionation. Any protein with a suitable affinity reagent could be targeted.

A common result for affinity purification experiments is often no result. Really good reagents, collecting QC data throughout, and limiting sample handling are all really important. Know that you have a sample.

To begin, thaw the prepared cell pellets for 15 minutes in 1X pellet volume of cold buffer A with protease inhibitors or phosphatase inhibitors. Place the tube containing the cell pellet on a nutator at four degrees Celsius to aid in resuspension for 30 minutes. Then place the tube in a centrifuge at 2, 000 times g and four degrees Celsius for 10 minutes to pellet.

Decant the supernatant and resuspend the cells with 5X the packed cell volume with buffer A.Incubate on ice for 20 minutes. Next, pellet again at 2, 000 times g and four degrees Celsius for 10 minutes. Decant the buffer and resuspend with 2X original packed cell volume buffer A with protease inhibitors or phosphatase inhibitors.

Dounce about seven times with the loose pestle A.Centrifuge the lysate for 10 minutes at 2, 000 times g and four degrees Celsius. Carefully transfer the supernatant to a new tube and flash freeze it with liquid nitrogen. Store the lysate at minus 80 degrees Celsius.

Resuspend the pellet with 0.9X pellet volume of buffer B with protease inhibitors or phosphatase inhibitors and mix on a nutator for five minutes at four degrees Celsius. To lyse the nuclei, dounce 20 times with the titer pestle B.Mix the nuclear lysate on a nutator for 30 minutes at four degrees Celsius so that it is homogenous. Then centrifuge the nuclear lysate for 30 minutes at 21, 000 times g at four degrees Celsius.

Pipette off the supernatant and save as a soluble nuclear protein extract. To dialyze the soluble nuclear extract, first cut an appropriate length of 24 millimeter width dialysis tubing with an eight kilodalton molecular weight cutoff. Clamp one side of the tubing and load the nucleoplasm into the tubing.

After loading the lysate, clamp the other end and submerge into a clean glass container containing buffer C with protease inhibitors. Dialyze for three hours at four degrees Celsius. After that, transfer the dialyzed nuclear extract nucleoplasm into a tube and centrifuge at 21, 000 times g at four degrees Celsius for 30 minutes.

Transfer three 20 microliter aliquots of the nuclear extract to new microcentrifuge tubes for fractionation validation by western blot. Using pipette tips that have been cut at the tip, prepare a protein A/G bead mixture for each replicate by combining 12.5 microliter bead volume of Protein A Sepharose with 12.5 microliters of Protein G Sepharose in microcentrifuge tubes. Wash the protein A/G bead mixture two times with 300 microliters of IP buffer I.Spin the beads at 1, 500 times g at four degrees Celsius for one minute and decant the buffer.

Next, prepare the antibody protein A/G beads. To bind the antibody to the beads, add to the tube 300 microliters of IP buffer I and 10 micrograms of the desired antibody. Allow the bead-antibody mixture to rock on a nutator at four degrees Celsius overnight.

Now aliquot appropriate volumes of the nuclear lysates into low retention microcentrifuge tubes for one milligram of protein input per replicate. Spin the lysate at 16, 000 times g for 30 minutes and transfer the supernatant to a new tube. Add one microliter of Benzonase at 250 units per microliter for each milligram of the nuclear lysate and rock on a nutator at four degrees Celsius for 10 to 15 minutes.

To prepare beads for pre-clearing the lysate, add 12.5 microliters of each protein A and protein G beads to 1.5 milliliter low retention tubes. Wash two times with IP wash buffer I with protease inhibitors. Decant the buffer and add one milligram of the prepared nuclear lysate to the beads.

Incubate while rocking on a nutator for one hour at four degrees Celsius. Centrifuge the pre-cleared lysates at 1, 500 times g and four degrees Celsius for one minute. Then wash the antibody protein A or G beads two times with IP buffer I with protease inhibitors.

After centrifuging at 1, 500 times g and four degrees Celsius for one minute, decant the buffer. Now transfer the pre-cleared nuclear lysate onto the antibody protein A or G beads. Incubate while rocking on a nutator at four degrees Celsius for four hours.

Following the incubation, centrifuge at 1, 500 times g and four degrees Celsius for one minute. Transfer the supernatant into tubes labeled as the flow through for each replicate. Wash the antibody protein A or G beads four times with one milliliter of IP buffer II with protease inhibitors.

Then wash the beads two times with one milliliter of IP buffer I with protease inhibitors. Ensure that all buffer is removed after the last wash. Elute protein off the beads by incubating with 20 microliters of 0.1 molar glycine at pH 2.75 each for 30 minutes on a nutator.

Then spin at 750 times g and four degrees Celsius for one minute and pipette off the supernatant. Repeat this incubation with elution buffer a second time. After preparing the protein pellet, resuspend it with 30 microliters of SDS alkylation buffer.

Incubate the sample on a 95 degree Celsius heat block for five minutes. Then let it cool at room temperature for 15 minutes. Add 300 milliliters of UA solution and 30 microliters of 100 millimolar TCEP to each sample.

Load the solution onto a 30K centrifugal filter. After spinning the centrifugal filter at 21, 000 times g at room temperature for 10 minutes, keep the flow through in case there is a problem with the filter. After washing the filter according to the manuscript, add three microliters of one microgram per microliter lyse C resuspended in 0.1 molar Tris pH 8.5.

Fill the filters up to the 100 microliter mark with 0.1 molar Tris pH 8.5. Allow it to digest for one hour at 37 degrees Celsius while rocking on a nutator. Next, add one microliter of one microgram per microliter MS grade trypsin.

Mix gently and allow the trypsin to incubate with the sample overnight at 37 degrees Celsius while rocking on a nutator. In the morning, centrifuge multiple times at 21, 000 times g for 20 minutes to elute the peptides from the filter into a clean low retention microcentrifuge tube. After desalting the peptides using C18 spin columns, resuspend the lyophilized peptides in seven microliters of 0.1%TFA in 5%acetonitrile.

Sonicate the sample for three minutes to ensure that the peptides have been resuspended, then spin down at 14, 000 times g for 10 minutes. Load 15 to 30%of the sample onto the liquid chromatography mass spectrometry system for analysis. In this study, triplicate immunoprecipitations were analyzed from five milligrams of HeLa nuclear extract utilizing a bead-only control.

The bait DYRK1A ranked among the top three enriched proteins over the control which indicates the performed IP-MS experiment was reliable. A clear separation was shown between the high confidence interactors and greater than 95%of copurified proteins were identified as nonspecific. For the example DYRK1A dataset, IREF interaction partners presented FC-A values as low as 0.45 representing a very low enrichment over control.

Accordingly, many of these previously reported interactions are false negatives within this dataset by falling below the FC-A threshold of three. The calculated absolute copy number of each IREF interaction showed no correlation to the detection levels of an interaction partner by IP-MS. The combined use of FC-A greater than three and saint greater than 0.9 reduced the list of high confidence interactors to six proteins.

However, when applying an FC-A cutoff of greater than three in isolation, eight additional proteins were added to the network. The most crucial portion of this protocol is the selection of antibody reagent for immunoprecipitation. Western blot or Coomassie validation of selectivity is recommended prior to completing this workflow.

Supporting evidence of protein-protein interactions can be had from co-IP western blotting of identified interactors. Protein chemical cross-linking, though more complex, can reveal interaction domains of interactors. Using this method, my research group has uncovered novel functions for DYRK1A, a protein kinase required for mammalian brain development and linked with Alzheimer's disease neuropathology.

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Journal (Biochemistry)

Journal (Biochemistry)

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

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