2. Separate a total of 1 μg of peptides on an RP column
(75 μm  10 cm) packed in-house with C 18 resin (Magic C 18
AQ 3 μm) using 0.1% FA in Milli-Q water and 0.1% FA in 98%
ACN as mobile phases A and B, respectively. Use a 60-min
linear gradient from 5% to 35% B at a flow rate of 300 nL/min.
3. For ionization, follow the next parameters: 1.95 kV spray
voltage and 230
C capillary temperature. Peptides are analyzed in positive-ion mode, and complete high-resolution full
scans are obtained from 400 to 1600 amu (1 μscan) in the
Orbitrap analyzer, followed by four data-dependent collisioninduced dissociation (CID) MS/MS scans (1 μscans) using an
isolation width of 3 amu and a normalized collision energy
of 35%.
4. Fragmented masses are assigned during dynamic exclusion for
30 s after the second fragmentation event, and unassigned
charged ions are excluded from the MS/MS analysis.
3.6 MS Data
Processing
1. MS/MS spectra are analyzed using BYONIC™ and
SEQUEST-HT™ (Proteome Discoverer 2.2 package) against
the human UniProt/SwissProt protein database [10, 11] (see
Note 4).
2. Use the following constraints for the searches: semi-tryptic
cleavage with up to two missed cleavage sites and tolerances
of 25 ppm for precursor ions and 0.5 Da for MS/MS fragment
ions. The variable modifications allowed are methionine oxidation (+15.99492 Da) and phosphorylation (+79.966331 Da)
of serine, threonine, and tyrosine. Carbamidomethylation
(+57.021465 Da) on cysteine is selected as a static
modification.
3. Finally, the results are subjected to statistical analysis with the
Percolator algorithm to ensure that the false discovery rate
(FDR) was less than 1% (see Notes 5 and 6).
4 Notes
1. Different protein samples and different lysis buffer need to be
optimized.
2. DTT (DL-dithiothreitol) is used to stabilize proteins, to reduce
disulfide bonds, and to maintain monothiols in a reduced state.
This can be substituted by TCEP (Tris(2-carboxyethyl)
phosphine hydrochloride). IAA (iodoacetamide) is a
sulfhydryl-reactive alkylating reagent used to bind covalently
with the thiol group of cysteines in order to avoid disulfide
bond formation for protein characterization and peptide
mapping.
264
Mo ´ nica Carrera et al.
(75 μm  10 cm) packed in-house with C 18 resin (Magic C 18
AQ 3 μm) using 0.1% FA in Milli-Q water and 0.1% FA in 98%
ACN as mobile phases A and B, respectively. Use a 60-min
linear gradient from 5% to 35% B at a flow rate of 300 nL/min.
3. For ionization, follow the next parameters: 1.95 kV spray
voltage and 230
C capillary temperature. Peptides are analyzed in positive-ion mode, and complete high-resolution full
scans are obtained from 400 to 1600 amu (1 μscan) in the
Orbitrap analyzer, followed by four data-dependent collisioninduced dissociation (CID) MS/MS scans (1 μscans) using an
isolation width of 3 amu and a normalized collision energy
of 35%.
4. Fragmented masses are assigned during dynamic exclusion for
30 s after the second fragmentation event, and unassigned
charged ions are excluded from the MS/MS analysis.
3.6 MS Data
Processing
1. MS/MS spectra are analyzed using BYONIC™ and
SEQUEST-HT™ (Proteome Discoverer 2.2 package) against
the human UniProt/SwissProt protein database [10, 11] (see
Note 4).
2. Use the following constraints for the searches: semi-tryptic
cleavage with up to two missed cleavage sites and tolerances
of 25 ppm for precursor ions and 0.5 Da for MS/MS fragment
ions. The variable modifications allowed are methionine oxidation (+15.99492 Da) and phosphorylation (+79.966331 Da)
of serine, threonine, and tyrosine. Carbamidomethylation
(+57.021465 Da) on cysteine is selected as a static
modification.
3. Finally, the results are subjected to statistical analysis with the
Percolator algorithm to ensure that the false discovery rate
(FDR) was less than 1% (see Notes 5 and 6).
4 Notes
1. Different protein samples and different lysis buffer need to be
optimized.
2. DTT (DL-dithiothreitol) is used to stabilize proteins, to reduce
disulfide bonds, and to maintain monothiols in a reduced state.
This can be substituted by TCEP (Tris(2-carboxyethyl)
phosphine hydrochloride). IAA (iodoacetamide) is a
sulfhydryl-reactive alkylating reagent used to bind covalently
with the thiol group of cysteines in order to avoid disulfide
bond formation for protein characterization and peptide
mapping.
264
Mo ´ nica Carrera et al.