2. Run the Lumos Tribrid mass spectrometer in the datadependent mode at a top 12 method. The following parameters are used: resolution for full scans 60,000, the AGC
value of 4 Â 10
5 , maximum injection time of 50 ms, and scan
range from 300–1500 m/z; and resolution for HCD MS/MS
scan 50,000, the AGC value of 1 Â 10
5 , maximum injection
time of 300 ms, a normalized collision energy of 30%, a cycle
time of 2 s, and a dynamic exclusion time of 45 s.
3.9 Data Analysis
1. Analyze the raw files using the freely available MaxQuant
against human UniProt database (version May 20, 2015). Variable modifications: the acetylation (protein N-term), oxidation (M), and phospho (STY); the fixed modification: the
carbamidomethyl (C). 1% false discovery rate (FDR) is used
for the levels of protein, peptide, and modification.
2. Results can be read out from the modificationSpecificPeptides
and Phospho (STY) Sites tab-delimited txt output files generated by MaxQuant.
4 Notes
1. QPSSSR and its phosphorylated forms from CXCR3 are too
hydrophilic to detect using standard protocols, as without
TMT0 labeling they cannot bind to a C18 column or cartridge.
2. The TMT labeling efficiency is sensitive to the buffer pH. Prior
to the TMT0 labeling, we test the pH value to ensure pH ! 8
using pH paper. This is a key step for high-efficiency TMT
labeling.
3. We employ the recently optimized TMT-to-peptide ratio of 1:1
(w/w) to have both robust and cost-efficient TMT0
labeling [18].
4. After TMT0 labeling, the ACN concentration needs to be
reduced below 5%. The remaining high concentration of
ACN will affect the recovery of TMT0-labeled peptides for
C18 desalting to remove unreacted TMT0 reagent.
5. For preparation of IMAC tip, the frits are soft and seal very
easily at the end of tip. Applying a large pressure results in more
tightly packing, and the back pressure will be too high to be
difficult for buffer passing through the IMAC tip.
6. 2 mg of Ni-NTA silica resin is used for 100 μg of tryptic
peptides. Adjustment is required for small amounts of tryptic
peptides.
7. Ni-NTA silica resin settles very quickly and must be mixed
thoroughly immediately before pipetting to ensure that the
resin is evenly distributed.
Analysis of Hydrophilic Phosphopeptides
255
value of 4 Â 10
5 , maximum injection time of 50 ms, and scan
range from 300–1500 m/z; and resolution for HCD MS/MS
scan 50,000, the AGC value of 1 Â 10
5 , maximum injection
time of 300 ms, a normalized collision energy of 30%, a cycle
time of 2 s, and a dynamic exclusion time of 45 s.
3.9 Data Analysis
1. Analyze the raw files using the freely available MaxQuant
against human UniProt database (version May 20, 2015). Variable modifications: the acetylation (protein N-term), oxidation (M), and phospho (STY); the fixed modification: the
carbamidomethyl (C). 1% false discovery rate (FDR) is used
for the levels of protein, peptide, and modification.
2. Results can be read out from the modificationSpecificPeptides
and Phospho (STY) Sites tab-delimited txt output files generated by MaxQuant.
4 Notes
1. QPSSSR and its phosphorylated forms from CXCR3 are too
hydrophilic to detect using standard protocols, as without
TMT0 labeling they cannot bind to a C18 column or cartridge.
2. The TMT labeling efficiency is sensitive to the buffer pH. Prior
to the TMT0 labeling, we test the pH value to ensure pH ! 8
using pH paper. This is a key step for high-efficiency TMT
labeling.
3. We employ the recently optimized TMT-to-peptide ratio of 1:1
(w/w) to have both robust and cost-efficient TMT0
labeling [18].
4. After TMT0 labeling, the ACN concentration needs to be
reduced below 5%. The remaining high concentration of
ACN will affect the recovery of TMT0-labeled peptides for
C18 desalting to remove unreacted TMT0 reagent.
5. For preparation of IMAC tip, the frits are soft and seal very
easily at the end of tip. Applying a large pressure results in more
tightly packing, and the back pressure will be too high to be
difficult for buffer passing through the IMAC tip.
6. 2 mg of Ni-NTA silica resin is used for 100 μg of tryptic
peptides. Adjustment is required for small amounts of tryptic
peptides.
7. Ni-NTA silica resin settles very quickly and must be mixed
thoroughly immediately before pipetting to ensure that the
resin is evenly distributed.
Analysis of Hydrophilic Phosphopeptides
255