3.5 Filter-Aided
Sample Preparation
Protocol of SEC
Fraction for MS
Analysis (FASP
Protocol)
SEC fractions can be processed with standard protein digestion
protocols used for MS-based proteomic analysis. However, we
recommend the protocol described below. Key advantages of the
in-filter plate format workflow that we describe here are the high
level of recovery and reproducibility which arises from avoiding
both protein precipitation and peptide cleanup.
1. Centrifuge to remove SEC buffer at 1800 Â g for 30 min at
RT. Increase centrifugation time if needed until the filter membrane is dry (see Note 13).
2. Add 50 μL of buffer TUA (8 M Urea, 20 mM ammonium
bicarbonate, 5 mM TCEP) to each well of the filter plate and
incubate the filter plate at 1800 Â g at 37
C for 30 min in a
microplate shaker.
3. Add 10 μL of buffer IA (50 mM iodoacetamide, 20 mM
ammonium bicarbonate) to each well of the filter plate and
incubate the filter plate at 1800 Â g at RT up to 1 h in the dark.
4. Centrifuge at 1800 Â g for 30 min at RT to remove TUA and
IA buffer.
5. Add 100 μL of buffer ABC (20 mM ammonium bicarbonate)
to the filter plate.
6. Centrifuge at 1800 Â g for 20 min at RT.
7. Repeat one more time for a total of two washes (see Note 14).
8. Centrifuge at 1800 Â g for 60 min and make sure that all wells
are dry (<10 μL leftover volume) before proceeding to step 9
(see Note 15).
9. Transfer the filter plate to a new receiver plate to perform the
proteolysis. Add to each well 50 μL of buffer ABC, which
contains 0.3 μg of lysyl endopeptidase and 1 μg of trypsin.
10. Incubate overnight at 37
C at 300 rpm shaker speed.
11. Collect peptides by centrifuging the 96 well plate at 1800 Â g
for 30 min.
12. Add 100 μL of HPLC grade water to the filter plate and
centrifuge filter plate at 1800 Â g for 60 min.
13. Transfer each well to a low-binding Eppendorf tube.
14. Add 100 μL of a 1:1 acetonitrile/H 2 O mixture to the receiver
plate to increase the recovery of hydrophobic peptides and pool
the corresponding well in a low-binding Eppendorf tube.
15. Speedvac to complete dryness.
16. Resuspend samples in 20 μL of MS buffer A with iRT peptides
diluted in a 1:100 ratio.
280
Andrea Fossati et al.
Sample Preparation
Protocol of SEC
Fraction for MS
Analysis (FASP
Protocol)
SEC fractions can be processed with standard protein digestion
protocols used for MS-based proteomic analysis. However, we
recommend the protocol described below. Key advantages of the
in-filter plate format workflow that we describe here are the high
level of recovery and reproducibility which arises from avoiding
both protein precipitation and peptide cleanup.
1. Centrifuge to remove SEC buffer at 1800 Â g for 30 min at
RT. Increase centrifugation time if needed until the filter membrane is dry (see Note 13).
2. Add 50 μL of buffer TUA (8 M Urea, 20 mM ammonium
bicarbonate, 5 mM TCEP) to each well of the filter plate and
incubate the filter plate at 1800 Â g at 37
C for 30 min in a
microplate shaker.
3. Add 10 μL of buffer IA (50 mM iodoacetamide, 20 mM
ammonium bicarbonate) to each well of the filter plate and
incubate the filter plate at 1800 Â g at RT up to 1 h in the dark.
4. Centrifuge at 1800 Â g for 30 min at RT to remove TUA and
IA buffer.
5. Add 100 μL of buffer ABC (20 mM ammonium bicarbonate)
to the filter plate.
6. Centrifuge at 1800 Â g for 20 min at RT.
7. Repeat one more time for a total of two washes (see Note 14).
8. Centrifuge at 1800 Â g for 60 min and make sure that all wells
are dry (<10 μL leftover volume) before proceeding to step 9
(see Note 15).
9. Transfer the filter plate to a new receiver plate to perform the
proteolysis. Add to each well 50 μL of buffer ABC, which
contains 0.3 μg of lysyl endopeptidase and 1 μg of trypsin.
10. Incubate overnight at 37
C at 300 rpm shaker speed.
11. Collect peptides by centrifuging the 96 well plate at 1800 Â g
for 30 min.
12. Add 100 μL of HPLC grade water to the filter plate and
centrifuge filter plate at 1800 Â g for 60 min.
13. Transfer each well to a low-binding Eppendorf tube.
14. Add 100 μL of a 1:1 acetonitrile/H 2 O mixture to the receiver
plate to increase the recovery of hydrophobic peptides and pool
the corresponding well in a low-binding Eppendorf tube.
15. Speedvac to complete dryness.
16. Resuspend samples in 20 μL of MS buffer A with iRT peptides
diluted in a 1:100 ratio.
280
Andrea Fossati et al.