Oct 21, 2023
Protein Digestion with S-trap Spin Columns
- J P Rose1,
- M Watson1,
- B Schilling1,
- Joanna Bons1
- 1Buck Institute for Research on Aging
Protocol Citation: J P Rose, M Watson, B Schilling, Joanna Bons 2023. Protein Digestion with S-trap Spin Columns. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x7zdlqe/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: October 10, 2023
Last Modified: October 21, 2023
Protocol Integer ID: 89096
Keywords: Trypsin Digestion, S-trap spin columns, Proteomics, Mass Spectrometry, trap spin columns trypsin digestion, protein digestion, downstream proteomic profiling, trap spin columns in preparation, isolated protein, trap spin column, protein
Abstract
Trypsin digestion of isolated proteins using S-trap Spin columns in preparation for downstream proteomic profiling.
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
Troubleshooting
In a 2-mL microcentrifuge tube add up to 300 μg of the protein lysate, 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 for ≤ 100 μg of protein or S-trap mini spin columns for 100-300 µg of protein. Ensure that the S-Trap spin column is in a 2.0-mL flow-through catch tube.
Add 100 μL (S-trap micro spin column) or 200 μL (S-trap mini spin column) 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 (S-trap micro spin column) or 400 μL (S-trap mini spin column) 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 (S-trap micro spin column) or 400 μL (S-trap mini spin column) of S-Trap buffer and set aside.
Prepare a 1:25 (wt/wt) trypsin/protein ratio 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 125 μL of the sequencing-grade trypsin solution to the column at a 1:25 (wt/wt) trypsin/protein ratio. 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 125 μL 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.