Mar 28, 2024

Public workspaceProtein Digestion with S-trap Spin Columns using Conditioned Concentrated Media

 Forked from Protein Digestion with S-trap Spin Columns
DOI
dx.doi.org/10.17504/protocols.io.x54v928eml3e/v1
  • 1Buck Institute for Research on Aging
M A Watson
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v928eml3e/v1
Protocol Citation: Joanna Bons, J P Rose, M A Watson, B Schilling 2024. Protein Digestion with S-trap Spin Columns using Conditioned Concentrated Media. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v928eml3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 25, 2024
Last Modified: March 28, 2024
Protocol Integer ID: 97346
Keywords: Trypsin Digestion, S-trap spin columns, Proteomics, Mass Spectrometry, Conditioned Media, SASP, Secretome
Abstract
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.
Materials
  • S-trap spin columns (Protifi)
  • Centrifuge
  • Centrifugal vacuum concentrator
  • Small hot box/incubator
  • HPLC-grade water
  • 10% SDS solution
  • 1 M triethylamonium bicarbonate (TEAB) solution, pH 8
  • 100 mM triethylamonium bicarbonate (TEAB) solution, pH 8 in water
  • Dithiothreitol (DTT; 250 mM in 100 mM TEAB, pH 8)
  • Iodoacetamide (IAA; 250 mM in 100 mM TEAB, pH 8)
  • S-trap buffer (90% methanol in 100 mM TEAB)
  • Sequencing-grade trypsin
  • 12% phosphoric acid in water
  • Digestion buffer/Elution buffer 1: 50 mM triethylamonium bicarbonate (TEAB) solution, pH 8 in water
  • Elution buffer 2: 0.5% formic acid (FA) in water
  • Elution buffer 3: 50% acetonitrile (ACN), 0.5% formic acid (FA) in water
  • 0.2% formic acid (FA) in water
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.