2. Homogenize samples by ultrasonication (2–3 short pulses,
15μm of amplitude) to break down chromosomal DNA and
bacterial cell walls.
3. Centrifuge samples at 14,000 Â g for 5 min to pellet any debris.
3.3 Filter-Aided
Sample Preparation
(FASP)
1. Mix up to 60μl of the sample with 200μl of UA in the filter unit
and centrifuge at 7000 Â g for 5 min.
2. Add 200μl of UA to the filter unit and centrifuge at 7000 Â g
for 5 min.
3. If the sample volume is larger, repeat steps 1–2 until all the
sample is loaded.
4. Discard the flow-through from the collection tube.
5. Add 100μl of IAA solution and mix at 600 rpm in a thermomixer for 1 min and incubate without mixing for 20 min in
the dark.
6. Centrifuge the filter units at 7000 Â g for 5 min.
7. Add 100μl of UB to the filter unit and centrifuge at 7000 Â g
for 10 min. Repeat this step twice.
8. Add 35μl of UB with 5μl of Lys-C (0.5μg) and mix at 600 rpm
in thermomixer for 1 min.
9. Incubate the units at room temperature for 4–5 h.
10. Transfer the filter units to new collection tubes (see Fig. 1c).
11. Activate trypsin by heating at 30
C for 15 min before use (see
Note 5).
12. Dilute 2.5μl of activated trypsin (0.25μg) in 120μl of ABC and
add to the filter unit, and mix at 600 rpm in thermomixer for
1 min.
13. Incubate the units at room temperature for 4–18 h.
14. Centrifuge the filter units at 10,000 Â g for 20 min.
15. Add 50μl of ABC and centrifuge the filter units at 10,000 Â g
for 20 min.
16. Check the peptide concentration by measuring the absorbance
at 280 nm using a microvolume spectrophotometer.
17. Acidify with TFA to a final concentration of 0.5% and store at
À20
C or proceed with Subheading 3.4.
3.4 C18-StageTip
Cleanup
1. Make a hole in the lid of a 2-ml Eppendorf tube cap and insert
C18-StageTip (see Fig. 1d).
2. Add 50μl of MetOH to the top of the filter and centrifuge at
400 Â g for 3–4 min.
3. Add 50μl of buffer B, and centrifuge at 400 Â g for 3–4 min.
172
Sjoerd van der Post and Liisa Arike
15μm of amplitude) to break down chromosomal DNA and
bacterial cell walls.
3. Centrifuge samples at 14,000 Â g for 5 min to pellet any debris.
3.3 Filter-Aided
Sample Preparation
(FASP)
1. Mix up to 60μl of the sample with 200μl of UA in the filter unit
and centrifuge at 7000 Â g for 5 min.
2. Add 200μl of UA to the filter unit and centrifuge at 7000 Â g
for 5 min.
3. If the sample volume is larger, repeat steps 1–2 until all the
sample is loaded.
4. Discard the flow-through from the collection tube.
5. Add 100μl of IAA solution and mix at 600 rpm in a thermomixer for 1 min and incubate without mixing for 20 min in
the dark.
6. Centrifuge the filter units at 7000 Â g for 5 min.
7. Add 100μl of UB to the filter unit and centrifuge at 7000 Â g
for 10 min. Repeat this step twice.
8. Add 35μl of UB with 5μl of Lys-C (0.5μg) and mix at 600 rpm
in thermomixer for 1 min.
9. Incubate the units at room temperature for 4–5 h.
10. Transfer the filter units to new collection tubes (see Fig. 1c).
11. Activate trypsin by heating at 30
C for 15 min before use (see
Note 5).
12. Dilute 2.5μl of activated trypsin (0.25μg) in 120μl of ABC and
add to the filter unit, and mix at 600 rpm in thermomixer for
1 min.
13. Incubate the units at room temperature for 4–18 h.
14. Centrifuge the filter units at 10,000 Â g for 20 min.
15. Add 50μl of ABC and centrifuge the filter units at 10,000 Â g
for 20 min.
16. Check the peptide concentration by measuring the absorbance
at 280 nm using a microvolume spectrophotometer.
17. Acidify with TFA to a final concentration of 0.5% and store at
À20
C or proceed with Subheading 3.4.
3.4 C18-StageTip
Cleanup
1. Make a hole in the lid of a 2-ml Eppendorf tube cap and insert
C18-StageTip (see Fig. 1d).
2. Add 50μl of MetOH to the top of the filter and centrifuge at
400 Â g for 3–4 min.
3. Add 50μl of buffer B, and centrifuge at 400 Â g for 3–4 min.
172
Sjoerd van der Post and Liisa Arike