TITLE: Brainless S-trap Proteomics Protocol Mini Columns or 96 well
AUTHORS: Edwin Yoo

[DESCRIPTION]
Brainless S-trap Proteomics Protocol Mini Columns or 96 well

[STEPS]
SECTION: Solubilize In SDS, reduce, alkylate
2. Add 50 μL 1X lysis buffer with reductant and alkylator (heated to 99C) to tissue or pelleted cells. Tissue/cells should be greater than 300 ug. 
 

SECTION: Solubilize In SDS, reduce, alkylate
6. Measure protein concentration with BCA. Make sure final sample has 1-300 ug protein in 50 ul of 1x lysis buffer. 
*100 ug protein is more than sufficient for general proteome. 300 ug required for phosphoproteomics.
SECTION: Solubilize In SDS, reduce, alkylate
3. Boil on heat block (99C) for 10 min.                                                                                                                                                                                                            

SECTION: Solubilize In SDS, reduce, alkylate
4. Sonicate with micro tip probe for 15s with pulses of 5 s on and 1 s off at an amplitude of 50 %. Alternatively use bead homogenizer if tissue is too big/tough for sonication. Sonication may be preferred for small tissue pieces.
*If the sample is viscous, sheer DNA thoroughly via sonication or treatment with Benzonase. This is essential as DNA can clog the S-Trap column.
SECTION: Solubilize In SDS, reduce, alkylate
7. Add 5 ul acidifier to 50 ul sample (final concentration ~1.1% phosphoric acid). Vortex.
SECTION: Trap Protein In Column
8. Add 350 ul binding/wash buffer to the sample and mix. 
SECTION: Trap Protein In Column
9. Place S-trap mini column into a 2 ml receiver tube for waste flow through. For S-trap 96 well plate, make sure there is waste plate underneath. Using pipette, transfer to S-trap column or 96 well plate. Do not centrifuge yet.
*For larger initial volumes, can load column multiple times with volumes < 600ul.
SECTION: Solubilize In SDS, reduce, alkylate
1. Make up solutions in the materials section ahead of time (at least day before). Trypsin, reducing agent, and alkylator should be made fresh before necessary step.
SECTION: Trap Protein In Column
10. Centrifuge S-trap column at 4000 g for 30s to trap proteins (vaccum manifold or 1500g for 2 min for 96 well plate).
*Can also use negative pressure via vacuum manifold or positive pressure from above.
*Clogged columns may be centrifuged as high as 15,000g.
SECTION: Clean Protein
11. Add 400 ul binding/wash buffer; centrifuge 4000g for 30s (vaccum manifold or 1500g for 2 min for 96 well plate). Repeat 3x and discard flow through as necessary.
*Can rotate columns 180 degrees between centrifugations to ensure homogenous flow.
SECTION: Clean Protein
12. Centrifuge S-trap column at 4000g for 1 min (vaccum manifold or 1500g for 2 min for 96 well plate) to fully remove binding/wash buffer. Failure to do so may result in dripping.
SECTION: Clean Protein
13. Transfer S-trap mini column to a clean 2 ml sample tube or new 96 well receiver plate for digestion.
SECTION: Incubate and digest protein
14. Add 125 ul digestion buffer containing trypsin at 1:10 wt:wt. It is okay for sample ranges from 1-300 ug to just apply 10 ug trypsin in 125 ul digestion buffer.
*Make sure there are no air bubbles on top of the trap because that prevents sample from entering trap and being properly digestion. Can use tricks to try and break up bubbles if any are present.
SECTION: Incubate and digest protein
15. Cap the s-trap loosely to limit evaporative loss. Cannot be air tight. Incubate for 1-2 hrs at 47C for trypsin or overnight at 37C. Water bath is best since the humid environment limits evaporative loss. If using dry incubator, put a beaker of water in there to humidify. Do not shake.
SECTION: Elution
16. Add 80 ul elution buffer 1 to S-trap and then centrifuge (4000 rcf for 1 min),  (vaccum manifold or 1500g for 2 min for 96 well plate).
*Do not centrifuge the digestion through before applying elution buffer 1. Apply elution buffer 1 directly into the trap containing the digestion buffer that was incubated.
SECTION: Elution
17. Add 80 ul of elution buffer 2 to S-trap then centrifuge (4000 rcf, 1 min),  (vaccum manifold or 1500g for 2 min for 96 well plate).
SECTION: Elution
18. Add 80 ul elution buffer 3 to S-trap then centrifuge (4000 rcf, 1 min),  (vaccum manifold or 1500g for 2 min for 96 well plate).
*This elution assists in recovery of hydrophobic peptides. Other organics may also be used as needed.
SECTION: Elution
19. Pool elutions. Measure peptide concentrations using pierce quantitative colorimetric peptide assay. Transfer to 96 well axygen PCR microplate if ready to inject in mass-spec. Dry plate in speed vac 2h-overnight. Can also dry down and freeze sample in plate or tube. Dry peptides are a stable stop point.
SECTION: Elution
20. If proceeding to phosphoenrichment, optionally save 50 ug peptides for bulk proteomics and proceed with phosphoenrichment with remaining 250 ug peptides.
SECTION: Solubilize In SDS, reduce, alkylate
5. Clarify sample of debris by centrifugation (5 min at 13,000 g to pellet debris). Transfer 50 ul supernatant to new tube or plate.