Suspension Trapping method (STrap)
Tryptic digest, maximum protein load of 50 μg
1. Materials
Solutions and Reagents
Milli-Q water (H2O)
AmBic (Ammonium Bicarbonate) solution: 50 mM NH4HCO3 in H2O
Lysis buffer: 5% (w/v) sodium dodecyl sulfate (SDS), 50mM Tris/HCl pH 7.6
STrapping buffer: 90% methanol, 100 mM Tris/HCl pH 7.1
Phosphoric acid stock solution: 12.15% in H2O
DTT stock solution: 1 M dithiothreitol in H2O, prepared on the day of experiment
IAA stock solution: 0.9 M iodoacetamide in H2O, prepared on the day of experiment
Trypsin solution: 0.033 μg/μl of trypsin (03708985001, Roche or V5111, Promega) in 50 mM
NH4HCO3, prepared prior to starting the STrap processing step and kept on ice
TFA solution 1: 0.5% trifluoroacetic acid in H2O
TFA solution 2: 10% trifluoroacetic acid in H2O
Elution solution: 70% acetonitrile, 0.5% formic acid in H2O
FA solution: 0.2% formic acid in H2O
Equipment
Bench-top centrifuge (for example MiniSpin, Eppendorf)
Probe Sonicator (for example Soniprep 150, MSE)
Heating block suitable for handling 1.5 ml microtubes (for example PHMT, Grant Bio)
Plastic Syringe, 20 ml (for example 301031, BD) with a custom adapter to fit into 200 μl pipette tips
Vacuum Concentrator (for example SpeedVac, Thermo)
STrap-tip (S-tip): The quartz fibre S-tip is made either from MK360 (Munktell ) or combination of
QM-A (Whatman) with MK360 membrane and Empore C18 (3M) membrane plugs stacked together
in a pipette tip (D200, Gilson) using the gauge 14 blunt end needle (Z261394, Sigma). Eleven MK360
quartz plugs or six QM-A and five MK360 quartz plugs, and three or four C18 plugs are forced into the
200 μl pipette tip end with the aid of a pusher – the piece of 1/16” OD PEEK tubing (1535, Upchurch
Scientific). The stack is further pressed down and compacted with a piece of the 1/16” OD PEEK
tubing several times.
O-tube: A 1.5 mL microcentrifuge tube (72.690.001, Sarstedt) with an opening punctured in the
tube’s lid (alternatively, a pipette tip lid-adapter for microcentrifuge tubes could be used).
The S-tip and O-tube comprise the Spin-unit.
Filter tips, 10 μl (TF-300-R-S, Axygen).
2. Methods
2.1 Cell lysis and reduction of cysteine residues
Cells are lysed in excess of the Lysis buffer (ca. 1:8-1:10 sample-to-Lysis buffer volume ratios) at
room temperature. To shear the DNA, the lysate is shortly sonicated using a probe sonicator. DTT
stock solution is added to a final concentration of 20 mM. The extract is heated up at 95oC for 5 min.
The extract is clarified by centrifugation at 12,100 x g for 10 min. Protein concentration could be
measured by tryptophan fluorescence (Wisniewski, J.R., Dus, K. & Mann, M. Proteomics Clin Appl
2013).
Notes:
Temperatures below 15°C cause SDS precipitation and thus must be avoided during the sample
processing steps. The lysate could be aliquoted and stored at -20oC and, when needed, processed
further after heating it up for 2 min at 95oC.
2.2 Alkylation of cysteine residues
IAA stock solution is added to a final concentration of 150 mM with the incubation step being at
least 15 min in a dark.
2.3 Preparation of the Trypsin solution
Trypsin solution (0.033 μg/μl in 50 mM NH4HCO3) is prepared prior to the step 2.4 and placed on ice.
2.4 Sample processing by STrap (tryptic digest and peptide desalting)
1. Pre-heat the heating block to 47oC.
2. Insert the S-tip into the O-tube.
3. (See Notes) Add 120 μl of the STrapping buffer into the S-tip onto the top of the quartz stack.
Wait for 1 min.
4. (See Notes) To 18 μl of the sample add 2 μl of the Phosphoric acid stock solution. Mix by pipetting
up and down.
5. Slowly add the acidified sample into the upper quarter of the STrapping buffer in the S-tip. Insert
the S-tip into the O-tube. Place the Spin-unit into the centrifuge and mark the S-tip part facing
outwards.
6. (See Notes) Centrifuge the Spin-unit at 2800 x g for 2 min.
7. Dispose of the tube with the flow-through.
8. Add 70 μl of the STrapping buffer into S-tip. Insert the S-tip into the fresh O-tube. Place the Spinunit into the centrifuge with the S-tip mark facing inwards. Centrifuge the Spin-unit for 45 sec at
2800 x g.
9. Add 30 μl of the AmBic solution into S-tip and centrifuge the Spin-unit for 30 sec at 2800 x g.
10. Add 22 μL of the Trypsin solution into the S-tip onto the top of the plugs stack. Push down the
solution using the syringe with a customized tip adapter till the solution meniscus is positioned ca. 3
mm above the top of the plugs stack.
11. Close the top of the S-tip with the 10 μl filter tip.
12. Insert the closed S-tip into the fresh O-tube, place the unit into the heating block at 47oC and
cover with the aluminium foil.
13. (See Notes) Incubate for 60 min.
14. Remove the Spin-unit from the heating device. Take out the filter tip. Add 50 μl of the AmBic
solution into the S-tip onto the top of the plugs stack.
15. Centrifuge the Spin-unit at 2300 x g for 60 sec.
16. (Optional) Remove the S-tip from the O-tube. Add 3 μl of the TFA solution 2 to the flow-through.
Load the acidified flow-through into the S-tip. Insert the S-tip back into the O-tube. Centrifuge the
Spin-unit at 2300 x g for 60 sec.
16. Add 100 μl of the TFA solution 1 into the S-tip. Centrifuge the Spin-unit at 2500 x g for 90 sec.
17. Place the S-tip into the fresh O-tube. Add 80 μl of the Elution solution into the S-tip, centrifuge
the Spin-unit for 5 sec at 2500 x g, wait 30 sec and then centrifuge the Spin-unit for 1.0 min at 2500 x
g.
18. (Optional) Add 50 μl of the Elution solution into the S-tip, centrifuge the Spin-unit for 60 sec at
2500 x g.
19. The eluate in the O-tube, containing desalted peptides, is concentrated in the SpeedVac to the
final volume of 5 - 12 μL. If needed, the concentrated peptide mixture could be diluted with the FA
solution up to the required volume.
Notes:
1. After each centrifugation step make sure that all added solution has gone through the S-tip.
2. A properly assembled S-tip can tolerate the centrifugal acceleration of at least 4000 x g.
3. The typical working ratio between the STrapping buffer in the S-tip (step 3) and the acidified
sample (step 4) is 6:1 (acceptable tested ranges 4.5:1 to 7:1), e.g. the STrapping buffer 120 μl
and the added acidified sample 20 μl.
4. In step 4, the sample could be diluted with the Lysis buffer up to the required volume before
the acidification.
5. The final concentration of the phosphoric acid in the sample (step 4) is 1.2% which is
achieved by addition of the Phosphoric acid stock solution to the sample at 1:10 ratio.
6. Alternatively to the high-temperature (47oC) digestion, digestion for 4 hours at 37oC in a
humidified chamber could be performed.
7. The use of lower concentrations of acetonitrile (e.g. down to 50%) in the Elution buffer is
also suitable.