Sep 06, 2021
Low throughput protocol for immunoprecipitation followed by mass spectrometry of cells stably expressing an HA-tagged protein
- Harper JW1
- 1Harvard Medical School
Protocol Citation: Harper JW 2021. Low throughput protocol for immunoprecipitation followed by mass spectrometry of cells stably expressing an HA-tagged protein. protocols.io https://dx.doi.org/10.17504/protocols.io.bqpfmvjn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 13, 2020
Last Modified: May 31, 2024
Protocol Integer ID: 45511
Keywords: immunoprecipitation, mass spectrometry, protein complexes, HA-tagged protein, ASAPCRN, bioplex protein interaction network, protein analysis of protein complex, protein complex, ha epitope into the gene, identifying protein, tagged protein analysis, protein, low throughput protocol for immunoprecipitation, other protein, mass spectrometry of cell, expressed lentivirus, protein of interest, epitope tag, ha epitope, hemagglutinin, mass spectrometry, epitope, immunoprecipitation, cell, huttlin et al cell
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Abstract
Analysis of protein complexes by mass spectrometry provides a powerful approach for identifying proteins that associate with other proteins. Frequently, this can be done by expressing the protein of interest with an epitope tag, such as a Hemagglutinin-A (HA) epitope, using either a stably expressed lentivirus or by gene editing the HA epitope into the gene of interest. The protocol has been used extensively to create the Bioplex protein interaction network [Huttlin et al Nature. 545:505-509 (2017); Huttlin et al Cell, 162: 425-440 (2015)].
Attachments
Materials
MCLB Stock:
- 50 millimolar (mM) Tris, pH 7.5
- 150 millimolar (mM) NaCl
- 0.5 % NP40
*Note: Made in batches of 0.5 L at 1x, filtered and stored at 4 °C
MCLB + Roche protease inhibitor tablets + DTT:
- cOmplete™ Protease Inhibitor CocktailRocheCatalog #4693116001
- 1 millimolar (mM) DTT
- Make 30 mL MCLB with inhibitors and DTT for first wash or diluting samples if necessary.
30 mL 1M DTT , 3 mini protease inhibitor tablets.
50 % Bead slurry:
- Prepare slurry of anti-HA beads, mouse monoclonal 12CA5 from Sigma, in a 1.5 mL tube.
- Spin beads gently at 3000 rpm, 00:00:30 to pellet.
- Remove buffer and wash with 3x 1 mL MCLB (no inhibitors) .
- Can store at 4 °C for several days.
HA elution buffer:
- Can use either 50 millimolar (mM) Tris pH 7.5 /150 millimolar (mM) NaCl or use PBS.
- PBS has been used for high throughput purposes.
HA peptide for elution:
- 250 µg/mL HA peptide dissolved in HA elution buffer.
- Crude HA peptide from Bio-Synthesis Inc: Sequence: H2N-YPYDVPDYA-CO2H
TCA precipitation:
- Neat TCA
- 10 % TCA in HPLC grade water
- Acetone
Trypsin Digestion:
- Digest Buffer: 200 millimolar (mM) EPPS, pH 8.5 /10 % Acetonitrile
- Trypsin (Thermo)
- 5 % formic acid in HPLC grade water
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
This protocol is for 293T cells but has been used broadly for many cell types. Typically, cells used have the proteins to be immunoprecipitated have stably expressed HA-tagged proteins via lentivirus vectors, or proteins fused with an HA epitope using gene editing.
Cell harvest:
- 2 x 15cm plate or 5 x 10cm plates per IP.
- Include HA-tagged GFP bait as a control.
Cell Harvest
17h 15m 3s
Wash plates 2x with cold PBS, then add 5 mL PBS per plate.
Gently pipette up and down to dislodge cells and homogenize or gently scrape.
Transfer to 15 mL conical tube and pellet cells 3000 rpm, 00:05:00 , discard sup.
Add 1 mL PBS and transfer to 2 mL tube.
Spin, aspirate PBS, and snap freeze pellet.
Store at -80 °C or use immediately.
Quick thaw frozen 293T pellets in 2mL tubes in 37 °C water bath for approximately 00:00:03 .
3s
Transfer to ice metal block and add 1.2 mL MCLB .
Once pellet is thawed, pipette up and down to resuspend and tumble tubes for 00:20:00 in the cold room at 4 °C to lyse cells.
20m
Spin at 16.1 rcf, 00:20:00 in pre-chilled bench-top centrifuges to clear lysate.
Reserve 25 µL -50 µL lysate for QC protein assay and western blot of input, if desired.
Carefully transfer remaining supernatant to fresh 1.5 mL tube containing 40 µL washed HA bead slurry .
Note
Use 1.5 mL tubes, not 2 mL for the IP, as the conical shape is more ideal for pelleting the beads/washing in subsequent steps.
Incubate cleared lysate with beads for 03:00:00 with tumbling in the cold room at 4 °C .
3h
Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge.
Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!)
Add 1 mL MCLB to each tube, and gently resuspend beads by shaking.
Repeat spin/wash step 3 more times for a total of 4 x 1 mL washes with detergent present:
(Wash 1/3): Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1 mL MCLB to each tube, and gently resuspend beads by shaking.
(Wash 2/3): Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1 mL MCLB to each tube, and gently resuspend beads by shaking.
(Wash 3/3): Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1 mL MCLB to each tube, and gently resuspend beads by shaking.
Perform 3 x 1 mL washes in the absence of detergent 50mM Tris/150mM NaCl, without NP-40:
(Wash 1/3): Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1 mL MCLB (without NP-40) to each tube, and gently resuspend beads by shaking.
(Wash 2/3): Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1 mL MCLB (without NP-40) to each tube, and gently resuspend beads by shaking.
(Wash 3/3): Spin samples 3000 rpm, 4°C, 00:01:00 to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1 mL MCLB (without NP-40) to each tube, and gently resuspend beads by shaking.
Carefully aspirate remaining wash buffer. Use gel loading tip and pipettor to remove as close to beads as possible.
Note
Can use P3111 capillary tips which are smaller than the agarose resin, but not entirely necessary, beads do not have to be dry.
Add 100 µL elution buffer (HA peptide elution buffer + 250 µg/mL HA peptide ).
Incubate in shaker at 37 °C (gentle shaking) for 00:30:00 .
30m
Collect bead eluate by centrifuging 3000 rpm, 00:01:00 .
Repeat elution with equal volume of HA peptide; incubate second elution for 15 min:
Add 100 µL elution buffer (HA peptide elution buffer + 250 µg/mL HA peptide ).
Incubate in shaker at 37 °C (gentle shaking) for 00:15:00 .
15m
Collect bead eluate by centrifuging 3000 rpm, 00:01:00 .
Transfer eluate to labeled 1.5mL tubes; freeze at -80 °C .
Proceed to TCA precipitation.
TCA Precipitation
17h 15m 3s
The following steps constitute the TCA precipitation/acetone wash and trypsinization in preparation for analysis by mass spectrometry: Can perform TCA precipitation Overnight at 4 °C or for 00:45:00 On ice .
45m
Add 55 µL neat TCA to samples (assuming 2 x 100 µL elution ), vortex to mix, then
gently spin to ensure TCA is not in tube caps.
Spin max speed 13000 rpm, 4°C, 00:30:00 ; carefully aspirate all but 30 µL sample .
Wash pellet with 1 mL cold 10 % TCA made in HPLC grade water.
Spin max speed 00:15:00 , vacuum as in Step 2.
15m
Wash with 1 mL cold Acetone .
Spin max speed 00:10:00 , vacuum.
10m
Repeat Acetone wash 2 more times (3 acetone washes total):
Note
Do not reduce to 2 washes. TCA tracks along and samples do not reach basic pH in 200mM EPPS digest buffer)
(Wash 1/2): Wash with 1 mL cold Acetone . Spin max speed 00:10:00 , vacuum.
10m
(Wash 2/2): Wash with 1 mL cold Acetone . Spin max speed 00:10:00 , vacuum.
10m
Air dry or use speedvac to dry pellet for digest, must be completely dry, as acetone can cause peptide modifications.
Note
DO NOT HEAT.
Trypsin Digestion
17h 15m 3s
Resuspend dried pellet in 40 µL 200mM EPPS (pH 8.5)/10% Acetonitrile (digest buffer).
Spot check with 0.2 µL sample for a couple of samples to ensure 8.5 .
Add 100 ng trypsin per sample (Thermo). Stock is 20 ng in 20 µL (measure to confirm 20L for each tube). This is 1 µg/µL , make a master mix of trypsin digest buffer and add 40 µL to each sample.
Note
Do not vortex, as this can dislodge the pellet.
Incubate at 37 °C for 06:00:00 (warm room or thermomixer, can shake gently).
6h
Acidify with 2 digest volumes of 5 % formic acid in HPLC grade water. (For 40L digest, add 80 µL 5% formic acid .)
Spot check pH for a couple of samples to ensure 2 .
Proceed to stage tip followed by analysis by mass spectrometry.