3. Equilibrating of the resin was performed by adding 200 μl of
80% ACN and 0.1% FA and then centrifuged at 100 Â g for
2 min. Repeat 1 more time discarding the eluate. Equilibrate
the resin again by adding 200 μl of 2% ACN and 0.1% FA and
then centrifuge at 100 Â g for 2 min. Repeat 1 more time
discarding the eluate.
4. After revise the sample is at the correct pH 2, 200 μl of sample
is loaded into the column and then centrifuge the column at
100 Â g for 2 min (see Note 14).
5. Wash the column with 200 μl of 2% ACN and 0.1% FA and
then centrifuge at 100 Â g for 2 min. Repeat 2 more times
discarding the eluate.
6. Continuedly, change the Eppendorf and eluate the purified
peptides by adding 200 μl of 50% ACN and 0.1% FA and
then centrifuge at 100 Â g for 2 min. Repeat this last step
1 more time collecting the eluate.
7. Peptide sample is dried under vacuum in a Speedvac (see Note
15).
8. Freeze (À80
C) until LC–MS analysis.
3.6 Shotgun LC–MS/
MS Analysis
1. Resuspend the peptide sample in 100 μl of 2% ACN and
0.2% TFA.
2. Sonicate for 2 min.
3. Take a 5 μl aliquot and dilute it to a final protein-equivalent
peptide concentration of 0.25 μg/μl.
4. Vortex the sample for 10 s and transfer it to a low-volume
HPLC vial.
5. Inject and analyze 2 μl of the sample by LC–MS with the
following analytical method: trap-elute configuration, with
5 μl/min of 2% ACN and 0.1% TFA for 5 min; LC gradient
for the analytical column: 5% to 35% B in 120 min, with A 0.1%
FA in water and B 0.1% FA in ACN, flow rate 300 nl/min; MS
in positive mode using a data-dependent acquisition method,
repeating cycles of one survey TOF MS scan (250 ms acquisition time, 350–1250 m/z range) followed by 65 MS/MS
(60 ms acquisition time, 230–1700 m/z range, rolling collision
energy) of the highest intensity precursors found in the survey
MS scan, using a mass tolerance window of 50 mDa and
excluding former target ions for 15 s and ion with a charge
state of 1 and above 5; spray voltage is 2600 V, interface heater
temperature is 100
C (see Note 16).
3.7 LC–MS/MS Mass
Spectrometry Data
Processing
1. Search each individual LC–MS run against the bacteria (see
Note 17) protein UniProt/TrEMBL database using the search
engine Sciex Protein Pilot v5.0.1. Use these parameters: sample
210
Ana G. Abril et al.
80% ACN and 0.1% FA and then centrifuged at 100 Â g for
2 min. Repeat 1 more time discarding the eluate. Equilibrate
the resin again by adding 200 μl of 2% ACN and 0.1% FA and
then centrifuge at 100 Â g for 2 min. Repeat 1 more time
discarding the eluate.
4. After revise the sample is at the correct pH 2, 200 μl of sample
is loaded into the column and then centrifuge the column at
100 Â g for 2 min (see Note 14).
5. Wash the column with 200 μl of 2% ACN and 0.1% FA and
then centrifuge at 100 Â g for 2 min. Repeat 2 more times
discarding the eluate.
6. Continuedly, change the Eppendorf and eluate the purified
peptides by adding 200 μl of 50% ACN and 0.1% FA and
then centrifuge at 100 Â g for 2 min. Repeat this last step
1 more time collecting the eluate.
7. Peptide sample is dried under vacuum in a Speedvac (see Note
15).
8. Freeze (À80
C) until LC–MS analysis.
3.6 Shotgun LC–MS/
MS Analysis
1. Resuspend the peptide sample in 100 μl of 2% ACN and
0.2% TFA.
2. Sonicate for 2 min.
3. Take a 5 μl aliquot and dilute it to a final protein-equivalent
peptide concentration of 0.25 μg/μl.
4. Vortex the sample for 10 s and transfer it to a low-volume
HPLC vial.
5. Inject and analyze 2 μl of the sample by LC–MS with the
following analytical method: trap-elute configuration, with
5 μl/min of 2% ACN and 0.1% TFA for 5 min; LC gradient
for the analytical column: 5% to 35% B in 120 min, with A 0.1%
FA in water and B 0.1% FA in ACN, flow rate 300 nl/min; MS
in positive mode using a data-dependent acquisition method,
repeating cycles of one survey TOF MS scan (250 ms acquisition time, 350–1250 m/z range) followed by 65 MS/MS
(60 ms acquisition time, 230–1700 m/z range, rolling collision
energy) of the highest intensity precursors found in the survey
MS scan, using a mass tolerance window of 50 mDa and
excluding former target ions for 15 s and ion with a charge
state of 1 and above 5; spray voltage is 2600 V, interface heater
temperature is 100
C (see Note 16).
3.7 LC–MS/MS Mass
Spectrometry Data
Processing
1. Search each individual LC–MS run against the bacteria (see
Note 17) protein UniProt/TrEMBL database using the search
engine Sciex Protein Pilot v5.0.1. Use these parameters: sample
210
Ana G. Abril et al.