5. C18 stage tips are washed by adding 100 μL of 5% ACN and
0.1%TFA. This step is repeated one time.
6. Peptides are eluted by adding 20 μL of Solution B (80% ACN
and 0.1%TFA) into the C18 stage tip and centrifuged at 1500 g
at room temperature for 5 min. The flow-through is collected
into a new 1.5 mL Eppendorf tube. This step is repeated one
time. The two elutions are combined together.
7. Desalted eluates are dried in a speed vacuum concentrator at
30
C.
3.5 LC-MS/MS
Analysis and Peptide
Identification
(Timing ~ 2.5 h Per
Sample)
Dried peptides are resuspended into 5 μL solution A at room
temperature. The enriched peptide mixture, consisting of phosphopeptides and nonphosphopeptides, are analyzed via LC-MS/MS
using standard settings (see Note 14). The parameters used for the
LC-MS/MS analysis are list in Table 1.
Table 1
LC-MS/MS parameters for the analysis of phosphopeptids [28]
Instrument
Parameter
Setting
LC system
Sample loading
At maximum pressure 800 bar
Gradient length (min)
135
Gradient flow rate (nL/min)
250
Linear gradient (percentage of LC
solvent B in LC solvent A)
5–35% for 115 min
35–45% for 15 min
45–90% for 5 min
Ion source
Column heater temperature (
C)
35
Spray voltage (kV)
2.1
Capillary temperature (
C)
250
Mass spectrometer: full MS
Resolution (at m/z 400)
70,000
Automatic gain control target (ions)
1,000,000
Maximum injection time (ms)
120
Scan range (m/z)
300–1600
Mass spectrometer: dd-MS2
Resolution (at m/z 400)
35,000
Automatic gain control target (ions)
500,000
Maximum injection time (ms)
120
Loop count
12
Isolation window (m/z)
1.2
TopN
12
Scan range (m/z)
200–2000
Normalized collision energy
25
Mass spectrometer: general
Polarity
Positive
Intensity threshold
100,000
Charge exclusion
Unassigned, 1, 8, >8
Peptide match
Preferred
Dynamic exclusion (s)
20
Plant Membranes Phosphoproteome
447
0.1%TFA. This step is repeated one time.
6. Peptides are eluted by adding 20 μL of Solution B (80% ACN
and 0.1%TFA) into the C18 stage tip and centrifuged at 1500 g
at room temperature for 5 min. The flow-through is collected
into a new 1.5 mL Eppendorf tube. This step is repeated one
time. The two elutions are combined together.
7. Desalted eluates are dried in a speed vacuum concentrator at
30
C.
3.5 LC-MS/MS
Analysis and Peptide
Identification
(Timing ~ 2.5 h Per
Sample)
Dried peptides are resuspended into 5 μL solution A at room
temperature. The enriched peptide mixture, consisting of phosphopeptides and nonphosphopeptides, are analyzed via LC-MS/MS
using standard settings (see Note 14). The parameters used for the
LC-MS/MS analysis are list in Table 1.
Table 1
LC-MS/MS parameters for the analysis of phosphopeptids [28]
Instrument
Parameter
Setting
LC system
Sample loading
At maximum pressure 800 bar
Gradient length (min)
135
Gradient flow rate (nL/min)
250
Linear gradient (percentage of LC
solvent B in LC solvent A)
5–35% for 115 min
35–45% for 15 min
45–90% for 5 min
Ion source
Column heater temperature (
C)
35
Spray voltage (kV)
2.1
Capillary temperature (
C)
250
Mass spectrometer: full MS
Resolution (at m/z 400)
70,000
Automatic gain control target (ions)
1,000,000
Maximum injection time (ms)
120
Scan range (m/z)
300–1600
Mass spectrometer: dd-MS2
Resolution (at m/z 400)
35,000
Automatic gain control target (ions)
500,000
Maximum injection time (ms)
120
Loop count
12
Isolation window (m/z)
1.2
TopN
12
Scan range (m/z)
200–2000
Normalized collision energy
25
Mass spectrometer: general
Polarity
Positive
Intensity threshold
100,000
Charge exclusion
Unassigned, 1, 8, >8
Peptide match
Preferred
Dynamic exclusion (s)
20
Plant Membranes Phosphoproteome
447