1. Dissolve 100 μg of the ligand of interest in 50 μL of 25 mM
HEPES (pH 8.2) in an Eppendorf tube (ligand sample).
2. Dissolve 100 μg of the control ligand in 50 μL of 25 mM
HEPES (pH 8.2) in an Eppendorf tube (control sample).
3. If no control ligand is available, quench TRICEPS with 100 μg
of glycine in 50 μL of 25 mM HEPES (pH 8.2).
4. Add 0.5 μL of the TRICEPS solution to each of the ligand and
control sample and mix them thoroughly by pipetting.
5. Incubate the mixture at room temperature under constant
gentle agitation for 90 min.
3.4.2 Receptor Capture
(All Steps Performed at
4
C)
1. Collect 2 Â 10
7 cells with gentle scraping (for adherent cells) or
centrifugation (for suspension cells) (see Notes 19 and 20).
2. Wash cells once with 50 mL PBS pH 6.5 in a 50 mL
centrifuge tube.
3. Centrifuge the cells at 300 Â g for 5 min at 4
C, discard the
supernatant, and resuspend the cells in the sodium (meta)
periodate buffer.
4. Incubate cells at 4
C in the dark for 15 min, under constant
gentle agitation.
5. Centrifuge the cells at 300 Â g for 5 min at 4
C, discard the
supernatant, and wash cells with 50 mL of PBS pH 7.4.
6. Centrifuge cells at 300 Â g for 5 min at 4
C and resuspend cells
in 20 mL 5-methoxyanthranilic acid buffer.
7. Split the cell suspension into two 15 mL centrifuge tubes.
8. Add the TRICEPS-coupled ligand of interest to one of the
tubes and the coupled control ligand to the other.
9. Incubate ligands with cells for 90 min at 4
C, under constant
gentle agitation.
10. Centrifuge the cells at 300 Â g for 5 min at 4
C and wash cells
with 10 mL PBS pH 7.4.
11. Centrifuge the cells at 300 Â g for 5 min at 4
C, remove the
supernatant, and freeze the cell pellets.
3.4.3 Cell Lysis and
Protein Enrichment
1. Resuspend the cell pellets in 800 μL of lysis buffer and transfer
the lysates to two Eppendorf tubes.
2. Sonicate the lysates using three 30 s sonication pulses and
remove debris by centrifugation at 16,000 Â g for 10 min.
3. Wash 200 μL alkyne agarose (per replicate) with 1.8 mL deionized water and add to the lysates.
4. Add 1 mL of 2Â click chemistry mastermix and incubate the
samples for 18 h (Æ2 h) at room temperature under gentle
agitation.
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Kyle S. Feldman et al.
HEPES (pH 8.2) in an Eppendorf tube (ligand sample).
2. Dissolve 100 μg of the control ligand in 50 μL of 25 mM
HEPES (pH 8.2) in an Eppendorf tube (control sample).
3. If no control ligand is available, quench TRICEPS with 100 μg
of glycine in 50 μL of 25 mM HEPES (pH 8.2).
4. Add 0.5 μL of the TRICEPS solution to each of the ligand and
control sample and mix them thoroughly by pipetting.
5. Incubate the mixture at room temperature under constant
gentle agitation for 90 min.
3.4.2 Receptor Capture
(All Steps Performed at
4
C)
1. Collect 2 Â 10
7 cells with gentle scraping (for adherent cells) or
centrifugation (for suspension cells) (see Notes 19 and 20).
2. Wash cells once with 50 mL PBS pH 6.5 in a 50 mL
centrifuge tube.
3. Centrifuge the cells at 300 Â g for 5 min at 4
C, discard the
supernatant, and resuspend the cells in the sodium (meta)
periodate buffer.
4. Incubate cells at 4
C in the dark for 15 min, under constant
gentle agitation.
5. Centrifuge the cells at 300 Â g for 5 min at 4
C, discard the
supernatant, and wash cells with 50 mL of PBS pH 7.4.
6. Centrifuge cells at 300 Â g for 5 min at 4
C and resuspend cells
in 20 mL 5-methoxyanthranilic acid buffer.
7. Split the cell suspension into two 15 mL centrifuge tubes.
8. Add the TRICEPS-coupled ligand of interest to one of the
tubes and the coupled control ligand to the other.
9. Incubate ligands with cells for 90 min at 4
C, under constant
gentle agitation.
10. Centrifuge the cells at 300 Â g for 5 min at 4
C and wash cells
with 10 mL PBS pH 7.4.
11. Centrifuge the cells at 300 Â g for 5 min at 4
C, remove the
supernatant, and freeze the cell pellets.
3.4.3 Cell Lysis and
Protein Enrichment
1. Resuspend the cell pellets in 800 μL of lysis buffer and transfer
the lysates to two Eppendorf tubes.
2. Sonicate the lysates using three 30 s sonication pulses and
remove debris by centrifugation at 16,000 Â g for 10 min.
3. Wash 200 μL alkyne agarose (per replicate) with 1.8 mL deionized water and add to the lysates.
4. Add 1 mL of 2Â click chemistry mastermix and incubate the
samples for 18 h (Æ2 h) at room temperature under gentle
agitation.
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