3. Use a high-intensity ultrasonic probe with a 1-mm tip to
perform the pulsed ultrafast digestion in 3 min at an amplitude
of 50%.
4. Add an additional 100-μg aliquot of trypsin to the sample, and
repeat the HIFU digestion for an additional 3 min.
5. Acidify the peptide digest by addition of 5% TFA to reduce the
pH of the solution to ~2.7.
6. Clean the samples on a SepPak C18 cartridge and dry in a
SpeedVac.
3.3 Phosphopeptide
Enrichment Using TiO 2
1. Reconstitute 5 mg of dried peptides in 300 μL of the washing
solution A.
2. Place 3 mg of TiO 2 packing in a 1-mL Mobicol spin column
and subsequently wash with 300 μL of Milli-Q water and
300 μL of methanol and finally equilibrate with 300 μL of
washing solution A for at least 10 min (the liquid from each
step is removed by centrifugation at 500 Â g) [8].
3. After removing the washing solution A, add the peptide solution to the Mobicol spin column filled with the blocked TiO 2
resin and incubate for 60 min with end-over-end rotation.
4. After the incubation step, the peptide solution is removed by
centrifugation, and the resin is thoroughly washed two times
each with 300 μL of washing solution B (80% ACN and 0.1%
TFA solution), followed by 300 μL of washing solution C
(0.1% TFA).
5. The phosphopeptides are eluted from the TiO 2 resin using two
150-μL aliquots of 0.3 M NH 4 OH (pH 10.5).
6. After elution, the pH of the pooled eluents is rapidly adjusted
to pH 2.7 using 10% TFA.
7. Finally, the phosphopeptides are purified using a C18 SepPak
cartridge and stored at À80
C.
3.4 Phosphopeptide
Fractionation Using
SCX
1. Aliquots of peptide solutions (1 mL) are loaded onto a PolyS
ULFOETHYL-A column equilibrated with 30% ACN containing 5 mM KH 2 PO 4 , pH 2.7 [9].
2. The buffer used for SCX separation contains 0.05% FA, 5 mM
KH 2 PO 4 , and 30% ACN. Phosphopeptide separation was performed by stepwise elution using the same buffer with increasing KCl concentrations from 0 to 350 mM.
3. Twelve SCX fractions are collected, acidified with 10% FA and
cleaned on a C18 MicroSpin™ column prior to MS analysis.
3.5 LC-MS/MS
Analysis
1. Analyze each SCX fraction by LC-MS/MS in a Proxeon EASYnLC II LC system coupled to an LTQ-Orbitrap XL mass
spectrometer (see Note 3).
Rapid Shotgun Phosphoproteomics Analysis
263
perform the pulsed ultrafast digestion in 3 min at an amplitude
of 50%.
4. Add an additional 100-μg aliquot of trypsin to the sample, and
repeat the HIFU digestion for an additional 3 min.
5. Acidify the peptide digest by addition of 5% TFA to reduce the
pH of the solution to ~2.7.
6. Clean the samples on a SepPak C18 cartridge and dry in a
SpeedVac.
3.3 Phosphopeptide
Enrichment Using TiO 2
1. Reconstitute 5 mg of dried peptides in 300 μL of the washing
solution A.
2. Place 3 mg of TiO 2 packing in a 1-mL Mobicol spin column
and subsequently wash with 300 μL of Milli-Q water and
300 μL of methanol and finally equilibrate with 300 μL of
washing solution A for at least 10 min (the liquid from each
step is removed by centrifugation at 500 Â g) [8].
3. After removing the washing solution A, add the peptide solution to the Mobicol spin column filled with the blocked TiO 2
resin and incubate for 60 min with end-over-end rotation.
4. After the incubation step, the peptide solution is removed by
centrifugation, and the resin is thoroughly washed two times
each with 300 μL of washing solution B (80% ACN and 0.1%
TFA solution), followed by 300 μL of washing solution C
(0.1% TFA).
5. The phosphopeptides are eluted from the TiO 2 resin using two
150-μL aliquots of 0.3 M NH 4 OH (pH 10.5).
6. After elution, the pH of the pooled eluents is rapidly adjusted
to pH 2.7 using 10% TFA.
7. Finally, the phosphopeptides are purified using a C18 SepPak
cartridge and stored at À80
C.
3.4 Phosphopeptide
Fractionation Using
SCX
1. Aliquots of peptide solutions (1 mL) are loaded onto a PolyS
ULFOETHYL-A column equilibrated with 30% ACN containing 5 mM KH 2 PO 4 , pH 2.7 [9].
2. The buffer used for SCX separation contains 0.05% FA, 5 mM
KH 2 PO 4 , and 30% ACN. Phosphopeptide separation was performed by stepwise elution using the same buffer with increasing KCl concentrations from 0 to 350 mM.
3. Twelve SCX fractions are collected, acidified with 10% FA and
cleaned on a C18 MicroSpin™ column prior to MS analysis.
3.5 LC-MS/MS
Analysis
1. Analyze each SCX fraction by LC-MS/MS in a Proxeon EASYnLC II LC system coupled to an LTQ-Orbitrap XL mass
spectrometer (see Note 3).
Rapid Shotgun Phosphoproteomics Analysis
263