8. Centrifuge 5000 Â g for 2 min at 15
C.
9. Discard the liquid and repeat twice.
10. Add 300 μL of sample (~100 μg) in 0.1% TFA. Centrifuge at
3000 Â g for 2 min at 15
C.
11. Pass the sample back through the column and centrifuge again
at 3000 Â g for 2 min at 15
C.
12. Pick up in the collector and save them again (see Note 7).
13. Add to the column the tubes (H 2 O-8) already prepared in step
2 from six sample fractionation steps.
14. Centrifuge at 3000 Â g for 2 min at 15
C.
15. Each eluate (~300 μL) is collected in tubes. Aliquot for peptide
quantitation and LC-MS/MS analysis.
16. Dry in speed-vac and proceed to the next step or store at
À80
C.
3.7 LC-MS/MS
Analysis and Data
Processing
Analyze by liquid chromatography-tandem mass spectrometry in a
nano Easy-nLC 1000 coupled to a high-resolution mass spectrometer Q-Exactive HF.
1. The peptides were concentrated “online” by reverse-phase
chromatography (RP) using an Acclaim PepMap
100 pre-column (20 mm  75 μm ID, C18 of 3 μm particle
diameter and 100 A ˚ pore size).
2. Peptides were then separated into a C18 Picofrit reverse-phase
analytical column (500 mm  75 μm ID, 2 μm particle diameter, 100 A ˚ pore size) with an integrated spray tip, thermostated,
operating at a flow of 250 nL/min.
3. The peptides were eluted using a gradient from 2% to 35% of
buffer B in 150 min and up to 40% in 10 min. 0.1% FA in water
as buffer B and 0.1% formic acid in ACN as buffer A were used.
4. The nano-HPLC is coupled online to the nanoelectric source
of the Q-exactive HF mass spectrometer with which the peptides were analyzed. Peptide entry was performed by electrospray ionization using the integrated tip in the analytical
column.
5. Data acquisition was carried out with a voltage of 1.8 kV for the
electrospray, and the ion transfer tube that guides the ions from
the spray to the interior of the mass spectrometer had a temperature of 270
C.
6. The peptides were detected with a resolution of 120,000 in
full-scan MS mode in an m/z mass range of 340–1600 Da.
7. The MS/MS data were acquired in the data-dependent acquisition (DDA) mode of the MS. Thus, in each microscan up to
15 precursors with a load of 2+ to 4+ were selected, depending
10
Miriam Morente-Lo ´ pez et al.
C.
9. Discard the liquid and repeat twice.
10. Add 300 μL of sample (~100 μg) in 0.1% TFA. Centrifuge at
3000 Â g for 2 min at 15
C.
11. Pass the sample back through the column and centrifuge again
at 3000 Â g for 2 min at 15
C.
12. Pick up in the collector and save them again (see Note 7).
13. Add to the column the tubes (H 2 O-8) already prepared in step
2 from six sample fractionation steps.
14. Centrifuge at 3000 Â g for 2 min at 15
C.
15. Each eluate (~300 μL) is collected in tubes. Aliquot for peptide
quantitation and LC-MS/MS analysis.
16. Dry in speed-vac and proceed to the next step or store at
À80
C.
3.7 LC-MS/MS
Analysis and Data
Processing
Analyze by liquid chromatography-tandem mass spectrometry in a
nano Easy-nLC 1000 coupled to a high-resolution mass spectrometer Q-Exactive HF.
1. The peptides were concentrated “online” by reverse-phase
chromatography (RP) using an Acclaim PepMap
100 pre-column (20 mm  75 μm ID, C18 of 3 μm particle
diameter and 100 A ˚ pore size).
2. Peptides were then separated into a C18 Picofrit reverse-phase
analytical column (500 mm  75 μm ID, 2 μm particle diameter, 100 A ˚ pore size) with an integrated spray tip, thermostated,
operating at a flow of 250 nL/min.
3. The peptides were eluted using a gradient from 2% to 35% of
buffer B in 150 min and up to 40% in 10 min. 0.1% FA in water
as buffer B and 0.1% formic acid in ACN as buffer A were used.
4. The nano-HPLC is coupled online to the nanoelectric source
of the Q-exactive HF mass spectrometer with which the peptides were analyzed. Peptide entry was performed by electrospray ionization using the integrated tip in the analytical
column.
5. Data acquisition was carried out with a voltage of 1.8 kV for the
electrospray, and the ion transfer tube that guides the ions from
the spray to the interior of the mass spectrometer had a temperature of 270
C.
6. The peptides were detected with a resolution of 120,000 in
full-scan MS mode in an m/z mass range of 340–1600 Da.
7. The MS/MS data were acquired in the data-dependent acquisition (DDA) mode of the MS. Thus, in each microscan up to
15 precursors with a load of 2+ to 4+ were selected, depending
10
Miriam Morente-Lo ´ pez et al.