3.4 TMTzero (TMT0)
Labeling
1. Reconstitute the tryptic peptides with 200 mM HEPES
(pH 8.5).
2. Check the pH value (see Note 2).
3. Mix the tryptic peptides with a TMT0 reagent using the
recently optimized TMT-to-peptide ratio of 1:1 (w/w) (see
Note 3).
4. Incubate for 1 h at room temperature and terminate the reaction by the addition of 5% hydroxylamine for 15 min.
5. Acidify the TMT0-labeled peptides using 10% TFA with the
final concentration of 0.5% TFA.
6. Dilute the TMT0-labeled peptides with buffer A (RPLC) (see
Note 4).
7. Desalt the TMT0-labeled peptides using a C18 SPE cartridge
(see Subheading 3.5) for large amounts of peptides or a C18
StageTip for small amounts of peptides ( 20 μg) (see Subheading 3.7).
3.5 Peptide
Desalting by C18
Cartridge for Large
Amounts of Samples
1. Activate a Waters Sep-Pak C18 cartridge with 1 mL of MeOH
and then buffer B.
2. Equilibrate the C18 cartridge with 1 mL of buffer A.
3. Load the tryptic peptides onto the C18 cartridge.
4. Wash the C18 cartridge with 1 mL of buffer A twice.
5. Elute the peptides with 500 μL of buffer B.
6. Determine the amount of tryptic peptides by BCA and then
dry using a SpeedVac.
3.6 Phosphopeptide
Enrichment by IMAC
1. Prepare IMAC tip (20-μL pipette tip) by capping one end with
a 20-μm polypropylene frits (see Note 5).
2. Resuspend 20 mg of Ni-NTA silica resin from Ni-NTA spin
column with 200 μL of 1% acetic acid (see Note 6).
3. Transfer the resuspended Ni-NTA silica resin to the IMAC tip
and then pack the Ni-NTA beads by centrifugation at 1000 Â g
at 25
C for 1 min (see Note 7).
4. Remove Ni
2+ ions with 200 μL of 50 mM EDTA in 1 M NaCl.
5. Wash away the remining 50 mM EDTA with 200 μL of 1%
acetic acid (see Note 8).
6. Activate the IMAC tip with 200 μL of 100 mM FeCl 3 by
centrifugation at 300 Â g at 25
C for 2 min.
7. Equilibrate the IMAC tip with 200 μL of 1% acetic acid before
sample loading.
8. Reconstitute the desalted TMT0-lableled peptides with 100 μL
of 80% ACN and 0.1% TFA (see Note 9).
Analysis of Hydrophilic Phosphopeptides
253
Labeling
1. Reconstitute the tryptic peptides with 200 mM HEPES
(pH 8.5).
2. Check the pH value (see Note 2).
3. Mix the tryptic peptides with a TMT0 reagent using the
recently optimized TMT-to-peptide ratio of 1:1 (w/w) (see
Note 3).
4. Incubate for 1 h at room temperature and terminate the reaction by the addition of 5% hydroxylamine for 15 min.
5. Acidify the TMT0-labeled peptides using 10% TFA with the
final concentration of 0.5% TFA.
6. Dilute the TMT0-labeled peptides with buffer A (RPLC) (see
Note 4).
7. Desalt the TMT0-labeled peptides using a C18 SPE cartridge
(see Subheading 3.5) for large amounts of peptides or a C18
StageTip for small amounts of peptides ( 20 μg) (see Subheading 3.7).
3.5 Peptide
Desalting by C18
Cartridge for Large
Amounts of Samples
1. Activate a Waters Sep-Pak C18 cartridge with 1 mL of MeOH
and then buffer B.
2. Equilibrate the C18 cartridge with 1 mL of buffer A.
3. Load the tryptic peptides onto the C18 cartridge.
4. Wash the C18 cartridge with 1 mL of buffer A twice.
5. Elute the peptides with 500 μL of buffer B.
6. Determine the amount of tryptic peptides by BCA and then
dry using a SpeedVac.
3.6 Phosphopeptide
Enrichment by IMAC
1. Prepare IMAC tip (20-μL pipette tip) by capping one end with
a 20-μm polypropylene frits (see Note 5).
2. Resuspend 20 mg of Ni-NTA silica resin from Ni-NTA spin
column with 200 μL of 1% acetic acid (see Note 6).
3. Transfer the resuspended Ni-NTA silica resin to the IMAC tip
and then pack the Ni-NTA beads by centrifugation at 1000 Â g
at 25
C for 1 min (see Note 7).
4. Remove Ni
2+ ions with 200 μL of 50 mM EDTA in 1 M NaCl.
5. Wash away the remining 50 mM EDTA with 200 μL of 1%
acetic acid (see Note 8).
6. Activate the IMAC tip with 200 μL of 100 mM FeCl 3 by
centrifugation at 300 Â g at 25
C for 2 min.
7. Equilibrate the IMAC tip with 200 μL of 1% acetic acid before
sample loading.
8. Reconstitute the desalted TMT0-lableled peptides with 100 μL
of 80% ACN and 0.1% TFA (see Note 9).
Analysis of Hydrophilic Phosphopeptides
253