12. Place gently the plate in a sonicator bath and make sure that the
bottom of the 96-well plate is in contact with water, sonicate
for 10 min
13. Carefully, transfer the liquid containing the extracted peptide
to a clean 0.5-mL microfuge tube
14. Add in each well 100 μL of extraction buffer.
15. Sonicate for 10 min and then add the liquid to the previous
tube to pool the extracted peptides. Repeat once.
16. Dry the extracted peptide using a speed vacuum concentrator.
3.3.3 Mass Spectrometry
Analysis
Mass spectrometry analysis is carried out by LC-MSMS using a Q
Exactive Plus Hybrid Quadrupole-Orbitrap online with a nanoLC
Ultimate 3000 chromatography system (Thermo Fisher Scientific™, San Jose, CA) (see Note 24).
1. For each biological sample, inject in triplicate 5 μL
corresponding to 25 % of digested sample on the system.
2. After pre-concentration and washing of the sample on a
Acclaim PepMap 100 column (C18, 2 cm  100 μm i.d. 100
Å pore size, 5 μm particle size), peptides are separated on a LC
EASY-Spray column (C18, 50 cm  75 μm i.d., 100 Å, 2 μm,
100 Å particle size) at a flow rate of 300 nL/min with a
two-step linear gradient (2–22% acetonitrile–H 2 O; 0.1% formic acid for 100 min and 22–32% acetonitrile–H 2 O; 0.1%
formic acid for 20 min).
3. For peptides ionization in the EASYSpray source, set spray
voltage at 1.9 kV and the capillary temperature at 250
C. All
samples are measured in a data-dependent acquisition mode.
4. Each run is preceded by a blank MS run in order to monitor
system background.
5. The peptide masses are measured in a survey full scan (scan
range 375–1500 m/z, with 70 K FWHM resolution at m/
z¼400, target AGC value of 3 Â 10
6 and maximum injection
time of 100 ms).
6. Following the high-resolution full scan in the Orbitrap, the
10 most intense data-dependent precursor ions are successively
fragmented in HCD cell and measured in Orbitrap (normalized collision energy of 25%, activation time of 10 ms, target
AGC value of 1 Â 10
3 , intensity threshold 1 Â 10
4 maximum
injection time 100 ms, isolation window 2 m/z, 17.5 K FWHM
resolution, scan range 200–2000 m/z).
7. Dynamic exclusion is implemented with a repeat count of 1 and
exclusion duration of 20 s.
Identification of Associated PDZ Proteins
31
bottom of the 96-well plate is in contact with water, sonicate
for 10 min
13. Carefully, transfer the liquid containing the extracted peptide
to a clean 0.5-mL microfuge tube
14. Add in each well 100 μL of extraction buffer.
15. Sonicate for 10 min and then add the liquid to the previous
tube to pool the extracted peptides. Repeat once.
16. Dry the extracted peptide using a speed vacuum concentrator.
3.3.3 Mass Spectrometry
Analysis
Mass spectrometry analysis is carried out by LC-MSMS using a Q
Exactive Plus Hybrid Quadrupole-Orbitrap online with a nanoLC
Ultimate 3000 chromatography system (Thermo Fisher Scientific™, San Jose, CA) (see Note 24).
1. For each biological sample, inject in triplicate 5 μL
corresponding to 25 % of digested sample on the system.
2. After pre-concentration and washing of the sample on a
Acclaim PepMap 100 column (C18, 2 cm  100 μm i.d. 100
Å pore size, 5 μm particle size), peptides are separated on a LC
EASY-Spray column (C18, 50 cm  75 μm i.d., 100 Å, 2 μm,
100 Å particle size) at a flow rate of 300 nL/min with a
two-step linear gradient (2–22% acetonitrile–H 2 O; 0.1% formic acid for 100 min and 22–32% acetonitrile–H 2 O; 0.1%
formic acid for 20 min).
3. For peptides ionization in the EASYSpray source, set spray
voltage at 1.9 kV and the capillary temperature at 250
C. All
samples are measured in a data-dependent acquisition mode.
4. Each run is preceded by a blank MS run in order to monitor
system background.
5. The peptide masses are measured in a survey full scan (scan
range 375–1500 m/z, with 70 K FWHM resolution at m/
z¼400, target AGC value of 3 Â 10
6 and maximum injection
time of 100 ms).
6. Following the high-resolution full scan in the Orbitrap, the
10 most intense data-dependent precursor ions are successively
fragmented in HCD cell and measured in Orbitrap (normalized collision energy of 25%, activation time of 10 ms, target
AGC value of 1 Â 10
3 , intensity threshold 1 Â 10
4 maximum
injection time 100 ms, isolation window 2 m/z, 17.5 K FWHM
resolution, scan range 200–2000 m/z).
7. Dynamic exclusion is implemented with a repeat count of 1 and
exclusion duration of 20 s.
Identification of Associated PDZ Proteins
31