TITLE: S-Trap™ plate digestion protocol (Protifi) of proteins for LC-MS / proteomics
AUTHORS: ronan o'cualain, David Knight, Stacey Warwood, James Allsey, Emmakeevill

[DESCRIPTION]
This protocol details the in-house BioMS procedure of S-Trap™ 96-well plate protein clean-up and digestion.

It is adapted from the long protocol from Protifi (as on August 2022) - https://files.protifi.com/protocols/s-trap-96-well-plate-long-1-4.pdf


[BEFORE_START]
Prepare your protein samples using the other protocols in this collection.

The following steps are optimised for volumes of 50 µL and 100 µL of protein.

For other volumes and amounts of protein, adjust accordingly, by dilution into S-trap lysis buffer. It is recommended that  the final concentration of SDS be at least greater than 3 % (v/v), and up to 15 % (v/v) , and a protein load between 50 µg to 250 µg for the process to work successfully.

Use the 1.5 mL adaptor for the Eppendorf Thermomixer, and set the thermomixer to 47 °C, 75 min, and a speed of 0 rpm (i.e. no shaking).

[GUIDELINES]
You may purchase S-trap plate positions (or full plates) 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 plate based protocol is to be used for samples with a protein load between 50 µg  to  250 µg of starting material.1

You have cell or tissue lysates in 50 µL of S-Trap lysis buffer (5 % (v/v) SDS with 50 millimolar (mM) TEAB pH 7.5).2

Protein lysates have been sheared of nucleic acid (e.g. from BioMS’s LE220+ AFA sonication system).3

Protein lysates have been reduced and alkylated and clarified by centrifuging at 14000 x g for 10 min (see the protocol "Reduction and alkylation of protein lysates for LC-MS (proteomics) using dithiothreitol (DTT) and iodoacetamide (IAM)" in this collection).4

The protein concentration of a pool of the protein lysates has been quantified and samples prepared to be 100 ug of protein in 50ul of 5% SDS (see notes).

Notes:
If you have less than 50 µg of protein - use the "S-Trap™ column digestion protocol (Protifi) of proteins for LC-MS / proteomics" in this collection. Speak with a member of BioMS if you are unsure which option to use. 
Alternatively, if your lysate volume is larger than 50 µL 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 50 µLvolume of sample in S-trap lysis buffer, add 5 µL of 12 % (v/v) aqueous phosphoric acid at 1:10 for a final concentration of 1.2 % (v/v) phosphoric acid and vortex mix.

 

 

SECTION: Sample preparation
2. Add 350 µL of S-Trap binding buffer to the acidified lysis buffer and mix.
SECTION: Sample preparation
3. Put the S-Trap plate on top of a clean 96 well plate, add the acidified methanolic SDS lysate into the plate.
 

SECTION: Sample Trapping
4. Locate an S-trap balance plate, with a receiver 96 well plate beneath. Centrifuge the plate at 1000 x g for 2 min in the Thermo megafuge 16 centrifuge.

 

SECTION: Sample Trapping
5. Repeat the previous two steps until there all sample has been applied to the S-Trap plate.
 

SECTION: Sample Washing
6. Wash captured protein with one wash of 200 µL of MTBE solution, simply add 200 µL of the MTBE solution to the column, and spin at 1000 x g for 2 min.
 

SECTION: Sample Washing
7. Following this, perform three washes of 200 µL of S-Trap binding buffer, again, add 200 µL of the S-trap binding buffer, and centrifuge at 1000 x g for 2 min.
 

 

SECTION: Digest proteins
8. Move S-Trap digestion plate on top of a clean receiver plate.
SECTION: Digest proteins
9. Locate the trypsin aliquots. They are in the top shelf of freezer 3.
 

SECTION: Digest proteins
10. 
The frozen aliquots are at a volume of 10 µL containing 20 µg of trypsin (concentration of 2 µg/µL1).

SECTION: Digest proteins
11. Add 250 µL of digestion buffer to the aliquot. This gives a total volume of 260 µL, enough for 2 S-Trap digestions.
SECTION: Digest proteins
12. Add 125 µL of digestion buffer containing protease into the top of the wells.
SECTION: Digest proteins
13. Place cover over the stacked plates.
SECTION: Digest proteins
14. Incubate in the thermomixer for 75 min at 47 °C for trypsin.
 

 

SECTION: Elute peptides:
16. Add 80 µL of digestion buffer to all wells of the S-Trap digestion plate.
SECTION: Elute peptides:
17. Centrifuge the plate at 1000 x g for 2 min or until all solution has passed through.
 

SECTION: Elute peptides:
18. Add 80 µL of 0.1% aqueous formic acid to all wells of the S-Trap digestion plate and spin through at 1000 x g for 2 min.
SECTION: Elute peptides:
19. Further elute peptides with 55 µL of 30% aqueous acetonitrile containing 0.1% formic acid and spin through at 1000 x g for 2 min.
SECTION: Elute peptides:
20. This elution assists in recovery of hydrophobic peptides.
 

SECTION: Elute peptides:
22. Proceed to R3 plate desalting or store in a refrigerator 2 min.

 



SECTION: Digest proteins
15. OPTIONALSTEP: 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.

SECTION: Elute peptides:
21. Add 125 µL of 0.1% aqueous formic acid to all wells of the sample/collection plate. This is to account for any evaporation that may have taken place during digestion, particularly if left overnight.