TITLE: S-Trap™ column digestion protocol (Protifi) of proteins for LC-MS / proteomics
AUTHORS: ronan.ocualain, Jamesallsey, Davidknight, Staceywarwood, Emmakeevill

[DESCRIPTION]
This protocol details the in-house BioMS procedure of S-Trap™ protein clean-up and subsequent column digestion/conversion of protein to peptide using trypsin.

It is adapted from the long protocol from Protifi (as on August 2022) - https://files.protifi.com/protocols/s-trap-micro-long-4-7.pdf

[BEFORE_START]
Locate the Eppendorf Thermomixer

Use the 1.5 mL adaptor for the Eppendorf Thermomixer, and set the thermomixer to 47 °C, 60 min, and a speed of 0 rpm (i.e. no shaking).


[GUIDELINES]

You may purchase S-trap columns and other consumables directly from us at the BioMS core facility on PPMS - https://corefacilities.manchester.ac.uk/?BioMS).

Allow 150 min for this process 

This protocol is to be used for protein loads columns are best for loadings less than 100 µg of starting material (protein). 1

You have cell or tissue lysates in 25 µL or 50 µL of S-Trap lysis buffer (5% SDS with 50 mM TEAB pH 7.5). If your lysate volume is lower than these two volumes, make up to either with 5% S-Trap lysis buffer (working concentration). 2

SDS is essential for the S-trapping process and subsequent clean-up, it is important that samples contain close to 5 % (v/v) SDS. This can be achieved by dilution of the sample using the 10 % (v/v)stock of S-trap lysis buffer.

Protein lysates have been sheared of nucleic acid (e.g. from BioMS’s Covaris LE220+ AFA sonication system). Alternatively, a nuclease such as Benzonase may be used.3 

Protein lysates have been reduced and alkylated 4  and clarified by centrifuging at 14000 x g for 10 min (see reduction and alkylation protocol in this series). 

The protein concentration of a pool of the protein lysates has been quantified and samples prepared to be less than 4 µg/µL of protein (Use the Millipore Direct Detect with the 5% SDS calibration file to check).

Please take care not to touch the S-trap matrix in the column with pipette tips during the process.

Notes:
If you have more than 100 µg of protein - use the S-trap plate method "S-Trap™ 96-well plate digestion protocol" in this collection. 
Alternatively, if your lysate volume is larger than 50uL, simply remove a 25 µL sample aliquot for processing. 
This step is essential as omitting it may clog the S-Trap column. If you do not have access to ultrasonication, see protocol from EMDmillipore for details.
If it is thought that the insoluble pellet contains proteins of interest, then proceed with homogenising (with a pipette and tip) the insoluble pellet as much as possible with fresh 5% S-trap lysis buffer. A suspension of insoluble particles may be formed this way. Proceed with a separate S-trap column to process this additional sample. 


[STEPS]
SECTION: Sample preparation
1. To the reduced and alkylated sample of volume either of 25 µL or 50 µL, add a volume of  2.5 µL or 5.0 µL  respectively of  12 % (v/v) aqueous phosphoric acid at a ratio of 1:10 for a final concentration of 1.2 % (v/v) phosphoric acid and vortex mix.
 


 



SECTION: Sample preparation
2. Add 165 µL or 330 µL of S-Trap binding buffer to the 27.5 µL or 55 µL volumes of acidified protein lysates respectively and mix.
 

SECTION: Sample Trapping
4. Add enough of the acidified methanolic lysate to the S-Trap column.
 

SECTION: Sample Trapping
5. Centrifuge the column/tube combination at 4000 rcf for 2 min in the Eppendorf 5430R centrifuge.
 

SECTION: Sample Trapping
6. Repeat the previous two steps if there is additional sample to be processed.

 

SECTION: Sample Washing
7. Wash captured protein with one wash of 150 µL of MTBE solution, simply add 150 µL of the MTBE solution to the column, and spin at 4000 x g for 2 min. This will remove methanol-insoluble biomolecules from the quartz filter.
 

SECTION: Sample Washing
8. Following this, perform four washes of 150 µL of S-Trap binding buffer, again, add 150 µL of the S-trap binding buffer, and centrifuge at 4000 x g for 2 min.
 

 

SECTION: Sample Trapping
3. Place the S-Trap column on top of a 2 mL Eppendorf tube. This will collect the flow-through.
SECTION: Digestion
9. Move S-Trap column to a clean digestion 1.5 mL Eppendorf tube.
SECTION: Digestion
10. Locate the trypsin aliquots. They are in the top shelf of freezer 3.
 

SECTION: Digestion
11. The frozen aliquots are at a volume of 10 µL containing 20 µg of trypsin (concentration of 2 µg/µL). 
20 µL of digestion buffer is needed for each column.
If working from 50 µg of protein starting material, then add 75 µL of digestion buffer to the trypsin aliquot.  This gives a total volume of 85 µL, enough for 4 S-Trap digestions.
 


SECTION: Digestion
12. Add 20 µL of digestion buffer containing protease at 1:10 wt:wt into the top of the micro column. Use a gel loading tip (blue box) for this step.
 

SECTION: Digestion
13. Cap the S-Trap column loosely to limit evaporative loss. A suggested way to do this is to close the cap until you feel resistance, then loosen one half-turn.
SECTION: Digestion
14. Incubate in the Eppendorf thermomixer for 60 min at 47 °C for trypsin.
 


SECTION: Elution of peptides
16. Add 65 µL of digestion buffer to the S-Trap column. Centrifuge the column / tube at 4000 x g for 2 min, and collect.
SECTION: Elution of peptides
17. Add 65 µL of 0.1% aqueous formic acid (FA) to the S-Trap column.
SECTION: Elution of peptides
17.1. Centrifuge the column / tube at 4000 x g for 2 min, and collect, this is now combined with the first elution through the centrifugation process.
SECTION: Elution of peptides
18. Add 30 µL of 30% aqueous acetonitrile containing 0.1% formic acid. Centrifuge the column / tube at 4000 x g for 2 min, and collect.
SECTION: Elution of peptides
19. This elution assists in recovery of hydrophobic peptides. This is now combined with the the first and second elutions. The total volume will be approximately 200 µL.
 

SECTION: Elution of peptides
20. Proceed to R3 desalting or store in a refrigerator.

 

SECTION: Digestion
15. If you wish, you may also set up this digestion step overnight, with no impact on the S-trap process. To do this, set the Thermomixer to 37 °C  and incubate overnight, again with no shaking.