overlap for replicate analysis of phosphopeptides (~50%), suggesting that TMT labeling allowed for the identification of phosphopeptides that otherwise would have been overlooked with
traditional analytical strategies. Indeed, evaluation of the changes
in peptide retention time before and after TMT0 labeling showed
that TMT0 labeling led to greatly increased peptide retention time
(Fig. 2c) and thus allowed the identification of phosphopeptides
with lower hydrophobicity (Fig. 2d). We further used the
TMT-based method to identify a previously unreported phosphopeptide located on the C-terminus of the GPCR CXCR3,
QPpSSSR, which is thought to contribute to the regulation of its
signaling mechanism. This simple method is expected to be broadly
used not only for profiling hydrophilic phosphoproteomes but also
for effective RPLC-MS analysis of other highly hydrophilic
analytes.
2 Materials
All buffers are made with sequencing-grade chemicals and ultrapure water (Milli-Q).
2.1 Cell Lysis
Experiments were performed using MCF7 and HEK 293 cell lines
in ATCC-formulated Eagle’s minimum essential medium supplemented with 0.01 mg/mL human recombinant insulin and a final
concentration of 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific). Use appropriate media for your
cell line of choice.
1. ATCC MCF7 and HEK 293 cell lines.
2. Sterile petri dishes (150 mm).
3. Phosphatase inhibitor cocktail.
4. Lysis buffer (MCF7): 100 mM NH 4 HCO 3 , pH 8.0, 8 M urea,
and a 1% phosphatase inhibitor.
5. Lysis buffer (HEK 293): 50 mM Tris–HCl, 150 mM NaCl, 1%
NP-40 at pH 7.5 containing a phosphatase inhibitor.
6. Phosphate-buffered saline (PBS).
7. Rubber policeman cell scraper.
8. Sonicator UTR200 for cell lysis.
9. Eppendorf centrifuge 5810R for centrifugation.
10. BCA-based protein concentration quantification assay.
2.2 Protein Digestion
1. 200 mM dithiothreitol (DTT).
2. 300 mM fresh iodoacetamide (IAA).
3. 1 M trimethyl ammonium bicarbonate (TMAB).
250
Chia-Feng Tsai et al.
traditional analytical strategies. Indeed, evaluation of the changes
in peptide retention time before and after TMT0 labeling showed
that TMT0 labeling led to greatly increased peptide retention time
(Fig. 2c) and thus allowed the identification of phosphopeptides
with lower hydrophobicity (Fig. 2d). We further used the
TMT-based method to identify a previously unreported phosphopeptide located on the C-terminus of the GPCR CXCR3,
QPpSSSR, which is thought to contribute to the regulation of its
signaling mechanism. This simple method is expected to be broadly
used not only for profiling hydrophilic phosphoproteomes but also
for effective RPLC-MS analysis of other highly hydrophilic
analytes.
2 Materials
All buffers are made with sequencing-grade chemicals and ultrapure water (Milli-Q).
2.1 Cell Lysis
Experiments were performed using MCF7 and HEK 293 cell lines
in ATCC-formulated Eagle’s minimum essential medium supplemented with 0.01 mg/mL human recombinant insulin and a final
concentration of 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific). Use appropriate media for your
cell line of choice.
1. ATCC MCF7 and HEK 293 cell lines.
2. Sterile petri dishes (150 mm).
3. Phosphatase inhibitor cocktail.
4. Lysis buffer (MCF7): 100 mM NH 4 HCO 3 , pH 8.0, 8 M urea,
and a 1% phosphatase inhibitor.
5. Lysis buffer (HEK 293): 50 mM Tris–HCl, 150 mM NaCl, 1%
NP-40 at pH 7.5 containing a phosphatase inhibitor.
6. Phosphate-buffered saline (PBS).
7. Rubber policeman cell scraper.
8. Sonicator UTR200 for cell lysis.
9. Eppendorf centrifuge 5810R for centrifugation.
10. BCA-based protein concentration quantification assay.
2.2 Protein Digestion
1. 200 mM dithiothreitol (DTT).
2. 300 mM fresh iodoacetamide (IAA).
3. 1 M trimethyl ammonium bicarbonate (TMAB).
250
Chia-Feng Tsai et al.