4. Sequencing-grade modified trypsin (Promega).
5. Trifluoroacetic acid (TFA).
6. SpeedVac concentrator.
2.3 TMT Labeling
1. Reconstitution buffer: 100% ACN.
2. Reaction buffer: 200 mM HEPES (pH 8.5).
3. TMT0 reagent.
4. Stopping reagent: 5% hydroxylamine.
5. Acidifying reagent: 10% TFA.
2.4 Peptide
Desalting
1. Reversed-phase Waters C18 Sep-Pak cartridge.
2. 3 M Empore C18 membrane disks.
3. 16G and 20G flat-bottom needle and plunger.
4. 200-μL tip for low peptide input.
5. Wash buffer: 100% methanol (MeOH).
6. Buffer A: 0.1% TFA.
7. Buffer B: 80% ACN in 0.1% TFA.
8. SpeedVac concentrator.
2.5 Phosphopeptide
Enrichment
1. Ni-NTA silica resin.
2. Self-pack IMAC tip.
3. 20-μm polypropylene frit disk.
4. 18G flat-bottom needle and plunger.
5. 50 mM EDTA in 1 M NaCl.
6. Equilibration buffer (IMAC): 1% acetic acid.
7. Activation buffer: 100 mM FeCl 3 in equilibration buffer.
8. Loading buffer (IMAC): 80%ACN in 0.1%TFA.
9. Wash buffer (IMAC): 80% ACN in 1% TFA.
10. Elution buffer (IMAC): 200 mM phosphate salt.
11. 3 M Empore C18 membrane disks.
12. C18 StageTip.
13. Wash buffer (C18): 0.1% FA.
14. Elution buffer (C18): 80% ACN/0.1% TFA.
15. SpeedVac concentrator.
2.6 Liquid
Chromatography and
Electrospray Tandem
Mass Spectrometry
1. Phosphopeptide reconstitution buffer: 0.1% FA with 2% ACN.
2. A nanoACQUITY UPLC system.
3. An in-house packed analytical column (75 μm i.d. Â 20 cm)
containing 1.9-μm ReproSil C18 resin with a column heater set
at 50
C.
Analysis of Hydrophilic Phosphopeptides
251