TITLE: Protein Digestion with S-trap Spin Columns using Conditioned Concentrated Media
AUTHORS: Joanna Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Trypsin digestion of isolated proteins using S-trap Spin columns in preparation for downstream proteomic profiling. 

For trypsin digestion of proteins from conditioned media Step 1 is modified as we add a volume of media rather than a concentration of protein homogenate. Step 6 is modified because we use S-trap micro spin columns as conditioned concentrated media samples contain ≤ 100 μg of protein. In step 14 and 16 we use 2 μg sequencing-grade trypsin instead of the usual 1:25 (wt/wt) trypsin/protein ratio.

[STEPS]
1. In a 2-mL microcentrifuge tube add an appropriate proportion of volume from the concentrated condition media, 10% SDS for a final concentration of 4% SDS, 1M TEAB pH 8 solution for a final concentration of 50 mM TEAB, and HPLC-grade water if necessary to bring the volume up to a minimum of 50 µL. 
2. Add 250 mM DTT for a final concentration of 20 mM and incubate for 10min at 50°C to reduce the proteins. Then immediately leave for 10 min on the bench at room temperature (RT).
3. Add 250 mM IAA for a final concentration of 40 mM and incubate for 30min at RT in the dark to alkylate the proteins.
4. Acidify the sample with 12% phosphoric acid for a final concentration of 1.2%. 
5. Add 7 volumes of S-Trap buffer to the acidified lysate and mix immediately by inversion. Formation of protein colloid may be observed.
6. Use S-trap micro spin columns as conditioned concentrated media samples contain ≤ 100 μg of protein. Ensure that the S-Trap spin column is in a 2.0-mL flow-through catch tube.
7. Add 100 μL of the acidified lysate/S-Trap buffer mix into the S-Trap spin column. Centrifuge at 4,000 x g for 10 seconds or until all the solution has passed through the column. Discard the flow-though.
8. Repeat step 7 until the entire acidified lysate and S-Trap buffer mix has passed through the column.
9. Add 200 μL of S-Trap buffer to wash the column. Centrifuge at 4,000 x g for 10 seconds or until all solution has passed through the column. Discard the wash solution.
10. Add 200 μL of S-Trap buffer and set aside. 
12. Centrifuge the S-trap spin column at 4,000 x g for 10 seconds or until the column is dry.
13. Place the S-Trap spin column into a clean 2.0-mL elution tube. 
15. Loosely cap the S-trap micro spin column or close the S-trap mini spin column and incubate for 1 hour at 47°C with no agitation. 
16. After 1 hour, add another 2 μg of the sequencing-grade trypsin solution to the column and incubate overnight at 37°C with no agitation.
17.1. a.     Add 80 μL of elution buffer 1 (50 mM TEAB, pH 8) and centrifuge at 1,000 x g for 1 minute.
17. After overnight incubation take the samples out of the incubator and elute sequentially in the same 2.0-mL elution tube as follows:
17.2. b.     Add 80 μL of elution buffer 2 (0.5% FA) and centrifuge at 1,000 x g for 1 minute.
17.3. c.     Add 80 μL of elution buffer 3 (50% ACN, 0.5% FA) and centrifuge at 4,000 x g for 1 minute. 
18. Vacuum dry the eluted peptide solution in a centrifugal vacuum concentrator.
19. Reconstitute the dried peptides in 0.2% FA and thoroughly mix the solution before desalting.
14. Add 2 μg of the sequencing-grade trypsin solution to the column. Be careful not to pierce the trap material.
11. Prepare the solution of sequencing-grade trypsin.