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.

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).

Add 250 mM IAA for a final concentration of 40 mM and incubate for 30min at RT in the dark to alkylate the proteins.

Acidify the sample with 12% phosphoric acid for a final concentration of 1.2%.

Add 7 volumes of S-Trap buffer to the acidified lysate and mix immediately by inversion. Formation of protein colloid may be observed.

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.

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.

Repeat step 7 until the entire acidified lysate and S-Trap buffer mix has passed through the column.

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.

Add 200 μL of S-Trap buffer and set aside.

Prepare the solution of sequencing-grade trypsin.

Centrifuge the S-trap spin column at 4,000 x g for 10 seconds or until the column is dry.

Place the S-Trap spin column into a clean 2.0-mL elution tube.

Add 2 μg of the sequencing-grade trypsin solution to the column. Be careful not to pierce the trap material.

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.

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.

After overnight incubation take the samples out of the incubator and elute sequentially in the same 2.0-mL elution tube as follows:

a. Add 80 μL of elution buffer 1 (50 mM TEAB, pH 8) and centrifuge at 1,000 x g for 1 minute.

b. Add 80 μL of elution buffer 2 (0.5% FA) and centrifuge at 1,000 x g for 1 minute.

c. Add 80 μL of elution buffer 3 (50% ACN, 0.5% FA) and centrifuge at 4,000 x g for 1 minute.

Vacuum dry the eluted peptide solution in a centrifugal vacuum concentrator.

Reconstitute the dried peptides in 0.2% FA and thoroughly mix the solution before desalting.