4. Add 200 μL of Activation Solution and centrifuge 1500 Â g for
1 min. Discard the flow-through and repeat step.
5. Add 200 μL of Equilibration Solution and centrifuge 1500 Â g
for 1 min. Discard the flow-through and repeat step.
6. Load sample on top of the resin bed in the spin column and
place the column into a receiver tube. Centrifuge 1500 Â g for
1 min. Recover flow-through and reload on the resin bed to
ensure complete sample binding.
7. Place column into a receiver tube and add 200 μL of Wash
Solution and centrifuge at 1500 Â g for 1 min. Discard flowthrough and repeat step.
8. Place column into a clean receiver tube and add 20 μL of
Elution Buffer on top of the resin bed. Centrifuge at
1500 Â g for 1 min and repeat step using the same
receiver tube.
9. Dry sample in a vacuum concentrator and suspend sample in
0.1% formic acid in water prior LC-MS/MS analysis.
3.2.3 Tandem Mass
Spectrometry (LC-MS/MS)
1. Perform the protein identification using 1200 Series Nanoflow
LC (Agilent, Santa Clara, CA, USA) connected to AccurateMass Q-TOF 6550 iFunnel with nano-electrospray ionization
source.
2. Reconstitute the dried/lyophilized samples (digested peptides)
in 10 μL of 0.1% formic acid in ddH 2 O (Buffer A). Load the
analytes to Large Capacity Chip LC Column, 300 A ˚ , C18,
160 nL enrichment column with 75 μm  150 mm analytical
column (P/N: G4240–62010).
3. Set injection volume to 1 μL per sample and adjust the flow rate
to 2 μL/min for capillary pump and 0.4 μL/min, with a linear
gradient of 5–70% buffer B for 40 min.
4. Set mass spectrometer run parameters to 5.0 L/min at 325
C
for the drying gas, fragmentor voltage at 360 V and capillary
voltage at 1800 V. Acquire the spectrum using Mass Hunter
acquisition software (Agilent, CA, USA) in auto MS/MS mode
with a mass range (m/z) of 110–3000 for MS and 50–3000 for
MS/MS. Set the acquisition rate (spectra/s) for MS and MS/
MS at 2 and 4, respectively.
3.3 Protein Data
Analysis
This section will describe data mining using PEAKS Studio (Peptide de-novo sequencing, identification, and quantification software) (see Note 7).
1. Acquire the raw data from the mass spectrometer (.d format for
Agilent instrument) and process with PEAKS Studio (Bioinformatic Solution Inc., Waterloo, Canada) version 7.5 against
merged database (SwissProt and trEMBL) from Uniprot.org
on the sample species origin.
Proteomics and Mass Spectrometry
237
1 min. Discard the flow-through and repeat step.
5. Add 200 μL of Equilibration Solution and centrifuge 1500 Â g
for 1 min. Discard the flow-through and repeat step.
6. Load sample on top of the resin bed in the spin column and
place the column into a receiver tube. Centrifuge 1500 Â g for
1 min. Recover flow-through and reload on the resin bed to
ensure complete sample binding.
7. Place column into a receiver tube and add 200 μL of Wash
Solution and centrifuge at 1500 Â g for 1 min. Discard flowthrough and repeat step.
8. Place column into a clean receiver tube and add 20 μL of
Elution Buffer on top of the resin bed. Centrifuge at
1500 Â g for 1 min and repeat step using the same
receiver tube.
9. Dry sample in a vacuum concentrator and suspend sample in
0.1% formic acid in water prior LC-MS/MS analysis.
3.2.3 Tandem Mass
Spectrometry (LC-MS/MS)
1. Perform the protein identification using 1200 Series Nanoflow
LC (Agilent, Santa Clara, CA, USA) connected to AccurateMass Q-TOF 6550 iFunnel with nano-electrospray ionization
source.
2. Reconstitute the dried/lyophilized samples (digested peptides)
in 10 μL of 0.1% formic acid in ddH 2 O (Buffer A). Load the
analytes to Large Capacity Chip LC Column, 300 A ˚ , C18,
160 nL enrichment column with 75 μm  150 mm analytical
column (P/N: G4240–62010).
3. Set injection volume to 1 μL per sample and adjust the flow rate
to 2 μL/min for capillary pump and 0.4 μL/min, with a linear
gradient of 5–70% buffer B for 40 min.
4. Set mass spectrometer run parameters to 5.0 L/min at 325
C
for the drying gas, fragmentor voltage at 360 V and capillary
voltage at 1800 V. Acquire the spectrum using Mass Hunter
acquisition software (Agilent, CA, USA) in auto MS/MS mode
with a mass range (m/z) of 110–3000 for MS and 50–3000 for
MS/MS. Set the acquisition rate (spectra/s) for MS and MS/
MS at 2 and 4, respectively.
3.3 Protein Data
Analysis
This section will describe data mining using PEAKS Studio (Peptide de-novo sequencing, identification, and quantification software) (see Note 7).
1. Acquire the raw data from the mass spectrometer (.d format for
Agilent instrument) and process with PEAKS Studio (Bioinformatic Solution Inc., Waterloo, Canada) version 7.5 against
merged database (SwissProt and trEMBL) from Uniprot.org
on the sample species origin.
Proteomics and Mass Spectrometry
237