3 Methods
3.1 Human Plasma
or Serum Protein
Depletion
1. Prepare the buffer solutions A and B for the HPLC system (see
Note 2).
2. Dilute 40 μL of human plasma sample using 120 μL of buffer A
and centrifuge the sample for 15 min at 21,000 Â g.
3. Transfer the supernatants to microtubes and keep them on ice.
4. Connect buffer A and buffer B flasks to pump inlets and purge
the HPLC system at 1 mL/min for 5 min (without a column).
5. Set the depletion method provided for column’s manufacturer
using Empower Pro 2 Software (see Note 3).
6. Run the HPLC method once without a column.
7. Attach the Agilent MARS Hu14 column to the HPLC system.
8. Equilibrate the column with buffer A for 30 min (see Note 4).
9. Inject 120 μL of the sample into the HPLC loop and start the
LC method (see Note 5).
10. Run the chromatographic method for depletion and collect
eluted fractions using 1.5-mL tubes.
11. Store collected fractions at À80
C until further analyses.
3.2 Desalting
and Buffer Exchange
1. Wash sample concentration tubes with ultrapure water.
2. Add 3 mL of 50 mM ammonium bicarbonate and centrifuge at
6600 Â g for 15 min.
3. Add the low-abundance fraction samples to each concentration
tube (see Note 6).
4. Centrifuge at 6600 Â g for 17 min or until the remaining
volume in the upper chamber is around 300–600 μL.
5. Remove the assembly and discard the flow through.
6. Complete the sample volume to 4 mL using 50 mM ammonium bicarbonate solution.
7. Concentrate the sample by centrifuging at 6600 Â g for 30 min
or until the remaining volume is around 300–600 μL (see
Note 7).
8. Mix the concentrated sample with an up-and-down pipette
motion several times and transfer it to a 1.5-mL tube (see
Note 8).
3.3 Tryptic Digestion
Using RapiGest™ SF
Surfactant
1. Preheat a block heater or thermoshaker to 80
C.
2. Add 50 μg of the sample (1 μg/μL) to 1.5-mL microtubes (see
Note 7).
3. Add 10 μL of 50 mM ammonium bicarbonate (pH 8.5) and
mix well by inverting.
158
Licia C. Silva-Costa et al.
3.1 Human Plasma
or Serum Protein
Depletion
1. Prepare the buffer solutions A and B for the HPLC system (see
Note 2).
2. Dilute 40 μL of human plasma sample using 120 μL of buffer A
and centrifuge the sample for 15 min at 21,000 Â g.
3. Transfer the supernatants to microtubes and keep them on ice.
4. Connect buffer A and buffer B flasks to pump inlets and purge
the HPLC system at 1 mL/min for 5 min (without a column).
5. Set the depletion method provided for column’s manufacturer
using Empower Pro 2 Software (see Note 3).
6. Run the HPLC method once without a column.
7. Attach the Agilent MARS Hu14 column to the HPLC system.
8. Equilibrate the column with buffer A for 30 min (see Note 4).
9. Inject 120 μL of the sample into the HPLC loop and start the
LC method (see Note 5).
10. Run the chromatographic method for depletion and collect
eluted fractions using 1.5-mL tubes.
11. Store collected fractions at À80
C until further analyses.
3.2 Desalting
and Buffer Exchange
1. Wash sample concentration tubes with ultrapure water.
2. Add 3 mL of 50 mM ammonium bicarbonate and centrifuge at
6600 Â g for 15 min.
3. Add the low-abundance fraction samples to each concentration
tube (see Note 6).
4. Centrifuge at 6600 Â g for 17 min or until the remaining
volume in the upper chamber is around 300–600 μL.
5. Remove the assembly and discard the flow through.
6. Complete the sample volume to 4 mL using 50 mM ammonium bicarbonate solution.
7. Concentrate the sample by centrifuging at 6600 Â g for 30 min
or until the remaining volume is around 300–600 μL (see
Note 7).
8. Mix the concentrated sample with an up-and-down pipette
motion several times and transfer it to a 1.5-mL tube (see
Note 8).
3.3 Tryptic Digestion
Using RapiGest™ SF
Surfactant
1. Preheat a block heater or thermoshaker to 80
C.
2. Add 50 μg of the sample (1 μg/μL) to 1.5-mL microtubes (see
Note 7).
3. Add 10 μL of 50 mM ammonium bicarbonate (pH 8.5) and
mix well by inverting.
158
Licia C. Silva-Costa et al.