4. Buffer A (RPLC): 0.1% FA with 3% ACN.
5. Buffer B (RPLC): 0.1% FA in 90% ACN.
6. Orbitrap Fusion Lumos Tribrid mass spectrometer.
7. Data analysis software using MaxQuant software (www.
maxquant.org).
3 Methods
3.1 Cell Lysis
1. Gently aspirate cell culture media and wash cells twice with
ice-cold PBS.
2. Add lysis buffer to the MCF7 and HEK 293 cells.
3. Heat solution for 5 min at 95
C to facilitate lysis and to
inactivate endogenous proteases and phosphatases.
4. Homogenize the lysate with sonication at 4
C.
5. Perform a BCA assay to determine the protein concentration.
6. Dilute all samples to equal protein concentrations based on
BCA assay results to achieve the desired starting concentration
(e.g., 1–10 μg/μL).
3.2 Protein Digestion
1. Reduce disulfide bonds using 5 mM DTT for 1 h at 37
C.
2. Alkylate cysteine residues using 20 mM iodoacetamide in the
dark for 1 h at room temperature.
3. Digest with trypsin at an enzyme-to-substrate ratio of 1:50
(w/w) and incubate overnight at 37
C with gentle shaking.
4. Acidify the tryptic peptides with TFA at a final concentration of
0.5% (v/v).
3.3 Digestion of
Immunoprecipitated
Proteins
(Phosphorylated
CXCR3 in This
Example)
1. Treat HEK293 cells with and without the CXCR3 ligand
CXCL10 at a final concentration of 100 nM for 5 min.
2. Lyse cells as described in Subheading 3.1.
3. Incubate cell lysates with anti-FLAG magnetic beads for at least
4 h at 4
C.
4. Elute FLAG-CXCR3 with 0.1 M glycine HCl buffer (pH 3.0)
followed by neutralization with 0.5 M Tris–HCl, pH 7.4,
1.5 M NaCl.
5. Precipitate proteins with 100% acetone and then air-dry.
6. Reconstitute the immunoprecipitated CXCR3 with 200 mM
HEPES (pH ¼ 8.5) and directly digest with sequencing-grade
modified trypsin overnight at 37
C.
7. Label the digested peptides directly with TMT0 reagents (see
Note 1).
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5. Buffer B (RPLC): 0.1% FA in 90% ACN.
6. Orbitrap Fusion Lumos Tribrid mass spectrometer.
7. Data analysis software using MaxQuant software (www.
maxquant.org).
3 Methods
3.1 Cell Lysis
1. Gently aspirate cell culture media and wash cells twice with
ice-cold PBS.
2. Add lysis buffer to the MCF7 and HEK 293 cells.
3. Heat solution for 5 min at 95
C to facilitate lysis and to
inactivate endogenous proteases and phosphatases.
4. Homogenize the lysate with sonication at 4
C.
5. Perform a BCA assay to determine the protein concentration.
6. Dilute all samples to equal protein concentrations based on
BCA assay results to achieve the desired starting concentration
(e.g., 1–10 μg/μL).
3.2 Protein Digestion
1. Reduce disulfide bonds using 5 mM DTT for 1 h at 37
C.
2. Alkylate cysteine residues using 20 mM iodoacetamide in the
dark for 1 h at room temperature.
3. Digest with trypsin at an enzyme-to-substrate ratio of 1:50
(w/w) and incubate overnight at 37
C with gentle shaking.
4. Acidify the tryptic peptides with TFA at a final concentration of
0.5% (v/v).
3.3 Digestion of
Immunoprecipitated
Proteins
(Phosphorylated
CXCR3 in This
Example)
1. Treat HEK293 cells with and without the CXCR3 ligand
CXCL10 at a final concentration of 100 nM for 5 min.
2. Lyse cells as described in Subheading 3.1.
3. Incubate cell lysates with anti-FLAG magnetic beads for at least
4 h at 4
C.
4. Elute FLAG-CXCR3 with 0.1 M glycine HCl buffer (pH 3.0)
followed by neutralization with 0.5 M Tris–HCl, pH 7.4,
1.5 M NaCl.
5. Precipitate proteins with 100% acetone and then air-dry.
6. Reconstitute the immunoprecipitated CXCR3 with 200 mM
HEPES (pH ¼ 8.5) and directly digest with sequencing-grade
modified trypsin overnight at 37
C.
7. Label the digested peptides directly with TMT0 reagents (see
Note 1).
252
Chia-Feng Tsai et al.