8. KCl.
9. Formic acid (FA).
10. C18 MicroSpin™ column.
2.7 LC-MS/MS
Analysis
1. Proxeon EASY-nLC II LC system (Thermo Fisher Scientific)
coupled to an LTQ-Orbitrap XL (Thermo Fisher Scientific).
2. Reverse-phase (RP) column (75 μm  10 cm) packed in-house
with C 18 resin (Magic C 18 AQ 3 μm; Michrom BioResources,
Auburn, CA, USA).
3. Mobile phase A: 0.1% FA in Milli-Q water.
4. Mobile phase B: 0.1% FA in 98% acetonitrile (ACN).
2.8 MS Data
Processing
1. Proteome Discoverer 2.2 package (Thermo Fisher Scientific)
implemented with BYONIC™ and SEQUEST-HT licenses.
3 Methods
3.1 Cell Culture
and Cell Lysis
1. Grow human Jurkat T-cells (E6.1) in RPMI 1640 medium
supplemented with FBS and antibiotics during several weeks
in a humidified 37
C incubator with 5% CO 2 (see Note 1).
2. Harvest the T-cells by centrifugation at 2000 Â g for 5 min at
4
C and wash them with 5 mL of ice-cold PBS.
3. Aspirate PBS and lyse a total of 10 Â 10
8 of Jurkat T-cells by
mechanical disruption for 5 min at 4
C using a glass potter
homogenizer in 1.5 mL of lysis buffer previously chilled at
4
C.
4. Centrifuge protein extracts at 500 Â g for 15 min at 4
C and
purify the supernatants into new tubes.
5. Measure protein concentration in the extracts using Lowry,
Bradford, or the bicinchoninic acid (BCA) method. The concentration is expected to be approximately 1–3 mg/mL. It is
recommended to continue with Subheading 3.2 immediately
to minimize protein degradation. If lysates need to be stored at
this point, store them at À80
C until further use.
3.2 Trypsin Protein
Digestion Using HIFU
1. Dissolve a total of 5 mg of protein in 8 M urea and 25 mM of
ammonium bicarbonate, pH 8.0. Add DTT to a final concentration of 5 mM and iodoacetamide to a final concentration of
25 mM. Incubate for 10 min at 56
C in the dark. Dilute 4Â
the sample with 25 mM of ammonium bicarbonate, pH 8.0 (see
Note 2).
2. Digest a total of 5 mg of protein with 100 μg of trypsin in
25 mM ammonium bicarbonate, pH 8, with simultaneous
application of HIFU [6, 7].
262
Mo ´ nica Carrera et al.
9. Formic acid (FA).
10. C18 MicroSpin™ column.
2.7 LC-MS/MS
Analysis
1. Proxeon EASY-nLC II LC system (Thermo Fisher Scientific)
coupled to an LTQ-Orbitrap XL (Thermo Fisher Scientific).
2. Reverse-phase (RP) column (75 μm  10 cm) packed in-house
with C 18 resin (Magic C 18 AQ 3 μm; Michrom BioResources,
Auburn, CA, USA).
3. Mobile phase A: 0.1% FA in Milli-Q water.
4. Mobile phase B: 0.1% FA in 98% acetonitrile (ACN).
2.8 MS Data
Processing
1. Proteome Discoverer 2.2 package (Thermo Fisher Scientific)
implemented with BYONIC™ and SEQUEST-HT licenses.
3 Methods
3.1 Cell Culture
and Cell Lysis
1. Grow human Jurkat T-cells (E6.1) in RPMI 1640 medium
supplemented with FBS and antibiotics during several weeks
in a humidified 37
C incubator with 5% CO 2 (see Note 1).
2. Harvest the T-cells by centrifugation at 2000 Â g for 5 min at
4
C and wash them with 5 mL of ice-cold PBS.
3. Aspirate PBS and lyse a total of 10 Â 10
8 of Jurkat T-cells by
mechanical disruption for 5 min at 4
C using a glass potter
homogenizer in 1.5 mL of lysis buffer previously chilled at
4
C.
4. Centrifuge protein extracts at 500 Â g for 15 min at 4
C and
purify the supernatants into new tubes.
5. Measure protein concentration in the extracts using Lowry,
Bradford, or the bicinchoninic acid (BCA) method. The concentration is expected to be approximately 1–3 mg/mL. It is
recommended to continue with Subheading 3.2 immediately
to minimize protein degradation. If lysates need to be stored at
this point, store them at À80
C until further use.
3.2 Trypsin Protein
Digestion Using HIFU
1. Dissolve a total of 5 mg of protein in 8 M urea and 25 mM of
ammonium bicarbonate, pH 8.0. Add DTT to a final concentration of 5 mM and iodoacetamide to a final concentration of
25 mM. Incubate for 10 min at 56
C in the dark. Dilute 4Â
the sample with 25 mM of ammonium bicarbonate, pH 8.0 (see
Note 2).
2. Digest a total of 5 mg of protein with 100 μg of trypsin in
25 mM ammonium bicarbonate, pH 8, with simultaneous
application of HIFU [6, 7].
262
Mo ´ nica Carrera et al.