TITLE: Low throughput protocol for immunoprecipitation followed by mass spectrometry of cells stably expressing an HA-tagged protein
AUTHORS: Harper    JW

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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)].</div></div>

[STEPS]
?. [Cell Harvest]
Wash plates 2x with cold PBS, then add  per plate.
[ PBS]
?. [Cell Harvest]
Gently pipette up and down to dislodge cells and homogenize or gently scrape.
?. [Cell Harvest]
Transfer to 15 mL conical tube and pellet cells , discard sup.
Centrifuge: 3000 33, 5 min
?. [Cell Harvest]
Add   and transfer to 2 mL tube.
[PBS]
?. [Cell Harvest]
Spin, aspirate PBS, and snap freeze pellet.
?. [Cell Harvest]
Store at  or use immediately.
-80 °C
?. [Cell Harvest]
Quick thaw frozen 293T pellets in 2mL tubes in  water bath for approximately .
37 °C
?. [Cell Harvest]
Transfer to ice metal block and add .
[MCLB]
?. [Cell Harvest]
Once pellet is thawed, pipette up and down to resuspend and tumble tubes for   in the cold room at   to lyse cells.
4 °C
?. [Cell Harvest]
Spin at  in pre-chilled bench-top centrifuges to clear lysate.
Centrifuge: 16.1 1324, 20 min
?. [Cell Harvest]
Reserve  -  for QC protein assay and western blot of input, if desired.
25 µl
[lysate]
?. [Cell Harvest]
Carefully transfer remaining supernatant to fresh 1.5 mL tube containing .
[washed HA bead slurry]
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.
?. [Cell Harvest]
Incubate cleared lysate with beads for  with tumbling in the cold room at .
4 °C
?. [Cell Harvest]
Spin samples  to pellet beads in centrifuge.
Centrifuge: 3000 33, 1 min, 4 10
?. [Cell Harvest]
Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!)
?. [Cell Harvest]
Add  to each tube, and gently resuspend beads by shaking.
[MCLB]
?. [Cell Harvest]
Repeat spin/wash step 3 more times for a total of 4 x 1 mL washes with detergent present:
?. [Cell Harvest]
Perform 3 x 1 mL washes in the absence of detergent 50mM Tris/150mM NaCl, without NP-40:
?. [Cell Harvest]
Carefully aspirate remaining wash buffer. Use gel loading tip and pipettor to remove as close to beads as possible.
Can use P3111 capillary tips which are smaller than the agarose resin, but not entirely necessary, beads do not have to be dry.
?. [Cell Harvest]
Add  (HA peptide elution buffer + ).
[elution buffer]
[HA peptide]
?. [Cell Harvest]
Incubate in shaker at  (gentle shaking) for .
37 °C
?. [Cell Harvest]
Collect bead eluate by centrifuging .
Centrifuge: 3000 33, 1 min
?. [Cell Harvest]
Repeat elution with equal volume of HA peptide; incubate second elution for 15 min:
?. [Cell Harvest]
Transfer eluate to labeled 1.5mL tubes; freeze at .
-80 °C
?. [Cell Harvest]
Proceed to TCA precipitation.
?. [TCA Precipitation]
The following steps constitute the TCA precipitation/acetone wash and trypsinization in preparation for analysis by mass spectrometry: Can perform TCA precipitation  at  or for .
4 °C
on ice
?. [TCA Precipitation]
Add  to samples (assuming 2 x ), vortex to mix, thengently spin to ensure TCA is not in tube caps.
[neat TCA]
[elution]
?. [TCA Precipitation]
Spin max speed ; carefully aspirate all but .
Centrifuge: 13000 33, 30 min, 4 10
[sample]
?. [TCA Precipitation]
Wash pellet with  cold  made in HPLC grade water.
1 mL
[TCA]
?. [TCA Precipitation]
Spin max speed , vacuum as in Step 2.
?. [TCA Precipitation]
Wash with .
[cold Acetone]
?. [TCA Precipitation]
Spin max speed , vacuum.
?. [TCA Precipitation]
Repeat Acetone wash 2 more times (3 acetone washes total):
Do not reduce to 2 washes. TCA tracks along and samples do not reach basic pH in 200mM EPPS digest buffer)
?. [TCA Precipitation]
Air dry or use speedvac to dry pellet for digest, must be completely dry, as acetone can cause peptide modifications.
DO NOT HEAT.
?. [Trypsin Digestion]
Resuspend dried pellet in  (digest buffer).
[200mM EPPS (pH 8.5)/10% Acetonitrile]
?. [Trypsin Digestion]
Spot check with  for a couple of samples to ensure .
[sample]
?. [Trypsin Digestion]
Add  (Thermo). Stock is  in  (measure to confirm 20L for each tube). This is , make a master mix of trypsin digest buffer and add  to each sample.
[ trypsin per sample]
20 ng
20 µl
40 µl
Do not vortex, as this can dislodge the pellet.
?. [Trypsin Digestion]
Incubate at  for  (warm room or thermomixer, can shake gently).
37 °C
?. [Trypsin Digestion]
Acidify with 2 digest volumes of  in HPLC grade water. (For 40L digest, add .)
[formic acid]
[5% formic acid]
?. [Trypsin Digestion]
Spot check pH for a couple of samples to ensure .
?. [Trypsin Digestion]
Proceed to stage tip followed by analysis by mass spectrometry.
?. [Cell Harvest]
(Wash 1/3): Spin samples  to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add  to each tube, and gently resuspend beads by shaking.
Centrifuge: 3000 33, 1 min, 4 10
[MCLB]
?. [Cell Harvest]
(Wash 2/3): Spin samples  to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add  to each tube, and gently resuspend beads by shaking.
Centrifuge: 3000 33, 1 min, 4 10
[MCLB]
?. [Cell Harvest]
(Wash 3/3): Spin samples  to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add  to each tube, and gently resuspend beads by shaking.
Centrifuge: 3000 33, 1 min, 4 10
[MCLB]
?. [Cell Harvest]
(Wash 1/3): Spin samples  to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add  to each tube, and gently resuspend beads by shaking.
Centrifuge: 3000 33, 1 min, 4 10
[MCLB (without NP-40)]
?. [Cell Harvest]
(Wash 2/3): Spin samples  to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add  to each tube, and gently resuspend beads by shaking.
Centrifuge: 3000 33, 1 min, 4 10
[MCLB (without NP-40)]
?. [Cell Harvest]
(Wash 3/3): Spin samples  to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add  to each tube, and gently resuspend beads by shaking.
Centrifuge: 3000 33, 1 min, 4 10
[MCLB (without NP-40)]
?. [Cell Harvest]
Add  (HA peptide elution buffer + ).
[elution buffer]
[HA peptide]
?. [Cell Harvest]
Incubate in shaker at  (gentle shaking) for .
37 °C
?. [Cell Harvest]
Collect bead eluate by centrifuging .
Centrifuge: 3000 33, 1 min
?. [TCA Precipitation]
(Wash 1/2): Wash with . Spin max speed , vacuum.
[cold Acetone]
?. [TCA Precipitation]
(Wash 2/2): Wash with . Spin max speed , vacuum.
[cold Acetone]