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98883
PTMScan® HS SUMO Immunoenrichment Companion Reagents Kit
Proteomic Analysis Products
PTMScan

PTMScan® HS SUMO Immunoenrichment Companion Reagents Kit #98883

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PTMScan® HS SUMO Immunoenrichment Companion Reagents Kit: Image 1
PTMScan® HS SUMO Immunoenrichment Companion Reagents Kit: Image 2
Before performing a PTMScan® HS immunoenrichment, use S-Trap columns to prepare samples from cell or tissue lysates all the way to purified peptides.
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Product Includes Volume (with Count)
S-Trap Midi Spin Columns 1 x 10 Pack
S-TrapTM SDS Solubilization Buffer 1 x 5 ml
S-TrapTM Phosphoric Acid 1 x 1 ml
S-TrapTM Binding/Wash Buffer 6 x 25 ml
S-TrapTM Elution Buffer #1 (TEAB) 1 x 8.5 ml
S-TrapTM Elution Buffer #2 (TFA) 1 x 5 ml
S-TrapTM Elution Buffer #3 (ACN/TFA) 1 x 5 ml
Water, LC-MS Grade (Burdick and Jackson) 27732 1 x 21 ml
Phosphatase Inhibitor Cocktail (100X) 5870 1 x 200 µl
Iodoacetamide, PTMScan® Qualified 88931 5 x 3.7 mg
DTT (Dithiothreitol) 7016 1 x 192.8 mg
PTMScan® Wild Type Alpha-Lytic Protease (WaLP) 33036 1 x 100 µg

Storage

All components in this kit are stable for at least 12 months when stored at the recommended temperature.

Protocol

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S-Trap Sample Preparation Protocol (WaLP)

Day 1: Digest Proteins to Peptides

Reagents not supplied and equipment required:

  1. Phosphate Buffered Saline (PBS-20X) (#9808)
  2. Protein Quantitation Colorimetric Assay Kit, such as BCA Protein Assay Kit (#7780)
  3. Centrifuges capable of handling 15mL tubes and 1.5mL tubes
  4. Vacuum concentrator

A. Solutions and Reagents

    NOTE: The S-Trap Midi Spin Columns can support the digestion of 0.3 – 5 mg soluble protein. Approximately 1 mg is recommended for a PTMScan® HS experiment.

  1. 1X Lysis Buffer: Mix 5 mL of S-Trap SDS Solubilization Buffer (2X) with 5 mL of DI water to make Lysis Buffer 1X. Store at room temperature; the SDS will precipitate if stored cold. Prior to each experiment, for each sample, take out 1 ml of 1X S-Trap SDS Solubilization Buffer and mix with 20 uL of Phosphatase Inhibitor Cocktail (100X), which should be used at 1/50 for PTMScan® experiments.
  2. Dithiothreitol (DTT): Make 1.25 M stock: Resuspend one tube containing 192.8 mg with 1 mL DI water. Divide into 25 uL aliquots. Store at -20°C for up to three months. Thaw one aliquot for each experiment.
  3. Iodoacetamide solution: Dissolve one vial in 200 uL of DI water immediately before use. Each vial is sufficient to alkylate four samples or 2mL of lysate. Keep the solution protected from light.

B. Preparation of Cell Lysate, Cultured Cells

  1. Grow approximately 1-4 x 107 cells for each experimental condition (enough cells to produce approximately 1-2 mg of soluble protein). The cell number corresponds to approximately one 150 mm culture dish (depending on the cell type).
  2. Wash adherent cells in cold PBS, then harvest by scraping in 500 uL of 1X S-Trap SDS Solubilization Buffer per plate. For suspension cells, collect the cells by centrifugation and wash the pellet with cold 1X PBS. Harvest the pellet with 500 uL of 1X S-Trap SDS Solubilization Buffer. Do not cool lysate on ice as this may cause precipitation of the SDS. The harvested cells can be frozen and stored at -80°C for several weeks or proceed to sonication. (SAFE STOP)
  3. Using a sonicator with a microtip, sonicate lysate at 5 W output with 3 bursts of 15 sec each. Cool on ice for 1 min between each burst. Clear the lysate by centrifugation at 10,000 x g for 15 min at room temperature and transfer the protein extract (supernatant) into a new tube. Store the cleared lysate at -80°C at this point or continue directly through the Protein Quant and Digestion steps. (SAFE STOP)

    NOTE: Lysate sonication fragments DNA and reduces sample viscosity. Ensure that the sonicator tip is submerged in the lysate. If the tip is not submerged properly, it may induce foaming and degradation of your sample.

    NOTE: Centrifugation is performed at room temperature to prevent SDS from precipitating out of solution.

C. Preparation of Tissue Lysates

  1. Select a fresh-frozen piece of tissue and place it in a 50 mL conical tube. Add approximately 2 mL of 1X S-Trap SDS Solubilization Buffer per 100 mg of wet tissue, or enough to submerge it completely.
  2. Lyse the tissue using a homogenizer. Wash the tool with DI water in between each sample. Keep the moving parts submerged in buffer to avoid excessive foaming.
  3. Using a sonicator with a microtip, sonicate lysate at 5 W output with 3 bursts of 15 sec each. Cool on ice for 1 min between each burst. Clear the lysate by centrifugation at 10,000 x g for 15 min at room temperature and transfer the protein extract (supernatant) into a new tube. Store the cleared lysate at -80°C at this point or continue directly through the Protein Quant and Digestion steps. (SAFE STOP)

    NOTE: Lysate sonication fragments DNA and reduces sample viscosity. Ensure that the sonicator tip is submerged in the lysate. If the tip is not submerged properly, it may induce foaming and degradation of your sample.

    NOTE: Centrifugation is performed at room temperature to prevent SDS from precipitating out of solution.

D. Protein Quantitation

  1. Measure soluble protein concentration using a colorimetric assay kit that is compatible with the SDS lysis buffer. The bicinchoninic acid (BCA) assay is recommended.
  2. Normalize all samples so that equal amounts of protein are prepared for each condition and replicate. Alternatively, design the experiment so that equal numbers of cells are prepared for each sample.

E. Reduction and Alkylation of Proteins

  1. Add 1/278 volume of 1.25 M DTT to the cleared cell supernatant to reach 4.5mM DTT final concentration (e.g. 1.8 µl of 1.25 M DTT for 500 uL of protein extract). Mix well, and place the tube into a 55°C incubator for 30 min.
  2. Cool the solution on ice briefly until it has reached room temperature.
  3. Add 1/10 sample volume of iodoacetamide solution to the cleared cell supernatant, mix well, and incubate for 15 min at room temperature in the dark.

F. S-Trap Column Cleanup and Protease Digestion

  1. Add 1/10 sample volume of S-Trap Phosphoric Acid to the sample and mix well.
  2. Transfer the sample to a 15 mL tube and add 6.6x the sample volume (eg. 3.3 mL) of S-Trap Binding/Wash Buffer. Mix well. A cloudy protein colloid should form immediately. 
  3. IMPORTANT: The protein in the sample is insoluble in the Loading Buffer. DO NOT CENTRIFUGE the sample or allow it to settle before loading onto the S-Trap column in the next step.

  4. Load the full volume of sample onto the S-Trap column, ensuring all the cloudy precipitate is transferred. Centrifuge 2 min at 4000 x g at room temperature until all the solution has passed through. Discard the flow-through.
  5. NOTE: All centrifugation steps for S-Traps columns should be performed at room temperature and 4000 x g.

  6. Wash the S-Trap column with 3 mL of S-Trap Binding/Wash Buffer and centrifuge 2 min. Discard the flow-through. Repeat 3 times for 4 washes total.
  7. Transfer S-Traps columns to clean collection tubes prior to digestion.
  8. Prepare 350 uL of WaLP working solution for each sample. Use WaLP at 1:100 (w/w) enzyme : substrate (eg. 10 ug WaLP for 1 mg sample). Dilute the WaLP stock (0.4 mg/mL) up to 350uL total with S-Trap Elution Buffer #1 (TEAB) and verify that the pH is ~8.
  9. Add the 350 uL WaLP solution to each cartridge and cap the tubes LOOSELY so that the digestion solution can absorb into the cartridge without creating negative air pressure. The cartridges may be centrifuged briefly but transfer any liquid that passes through back to the top of the cartridge. Place them in an incubator or water bath overnight at 37°C.
  10. IMPORTANT: Do not shake the tubes. Set the tubes in a rack so they remain secure and straight upright and the enzyme solution will absorb evenly into the cartridge bed.

    IMPORTANT: Complete digestion is critical for maximum peptide recovery in the following elution steps. Cutting incubation time short or using less than the recommended amount of enzyme will reduce performance.

  11. The next day, add 500 uL of S-Trap Elution Buffer #1 (TEAB) to the S-Traps columns and centrifuge 1 min or until all solution passes through at 4000 x g. Keep the cartridge in the same collection tube for all three elution steps (see steps 9 & 10).
  12. Add 500 uL of S-Trap Elution Buffer #2 (TFA) to the S-Traps columns, centrifuge 1 min or until all the solution passes through, at 4000 x g.
  13. Add 500 uL of S-Trap Elution Buffer #3 (ACN/TFA) to the S-Traps columns, centrifuge 1 min.
  14. Verify that the combined elutions have a pH<3. Transfer the eluate (1.85 mL altogether) to a 2 mL microcentrifuge tube.
  15. NOTE: At this stage, the peptides are clean for LCMS if analysis of the unmodified proteome is desired. 1% of the total volume can be set aside for this purpose.

  16. Dry the peptide solution in a vacuum concentrator set to ambient temperature overnight or until completely dry. The pellet should be visible at the end.
  17. NOTE: Peptide solutions may be frozen at -80°C for 1 hr or longer before placing in the vacuum concentrator; this will prevent full tubes from spilling when placed at an angle to dry. (SAFE STOP)

    NOTE: A standard lyophilization apparatus is also acceptable in place of a vacuum concentrator.

    NOTE: Dry, digested peptides are stable at -80°C for several months (seal the closed tube with parafilm for storage). (SAFE STOP)

posted October 2020

Protocol Id: 2324

Product Description

The PTMScan® HS SUMO Immunoenrichment Companion Reagents Kit includes ten S-Trap™ columns and all the necessary reagents to prepare purified peptides ready for SUMO remnant (K-ε-GG) enrichment. Cells or tissues from any species are supported, as long as the SUMO linkage sequence [T/S]GG-[substrate] is conserved in the species of interest. The kit includes supplies to prepare ten wild type alpha-lytic protease (WaLP) digestions of 1 mg each, which is sufficient for ten immunoenrichments with the PTMScan® HS Ubiquitin/SUMO Remnant Motif (K-ε-GG) Kit #59322. The soluble protein content of cells and tissues vary, but 1 x107 cells or 15-30 mg of wet tissue from mammalian sources should be sufficient. Clean peptides are ready for PTM enrichment with only one day of hands-on work and one day of hands-off drying time.
CAS 13721-39-6, 7681-49-4, 144-48-9, 3483-12-3, 76-05-1, 75-05-8, 15715-58-9, 67-56-1, 151-21-3, 7664-38-2, 7732-18-5

Background

Proteomics analyses of posttranslationally modified peptides rely on both high-quality enrichments and pre-enrichment sample preparation. Suspension Trap (S-Trap™) columns are convenient tools to combine protein digestion and peptide cleanup into a single procedure. The S-Trap™ column is a spin filter made out of quartz that can trap large particles, such as precipitated proteins, but has little to no affinity for smaller particles, such as peptides (1). Cells and tissues can be prepared in any lysis buffer of choice, but buffers containing SDS are reported to extract more protein per cell than other buffers and help to maximize S-Trap™ column performance (2). Acidifying the cell lysate and mixing it with methanol causes the proteins to precipitate, so that they can be captured on the S-Trap™ column with a brief centrifugation step. Detergents, along with lipids and any other contaminants, are washed away with additional methanol. The captured proteins can then be digested with trypsin or any other appropriate enzyme. Digesting on a solid substrate such as the S-Trap™ column enhances the enzymatic efficiency, reducing missed cleavages in comparison to in-solution methods (3). The peptides elute easily into mass spectrometry-friendly solutions. Once dried, these purified peptides are ready to be enriched for any PTM of interest using techniques such as IMAC #20432 or PTMScan®. If desired, the peptides are also ready for direct mass spectrometry analysis without further cleanup.

The S-Trap™ workflow is much faster and less laborious than in-solution digestion because it avoids a separate peptide purification procedure. Once protein extracts are collected, reduction and alkylation takes 45 minutes, loading and washing the S-Trap™ columns takes 15 minutes, and then the samples are allowed to digest overnight. Clean peptides are collected the next morning in less than 15 minutes. Drying in a vacuum concentrator (or lyophilizer) can be completed over the next night, so that enrichment can be completed on the third day of sample preparation.
  1. Zougman, A. et al. (2014) Proteomics 14, 1006-0.
  2. Elinger, D. et al. (2019) J Proteome Res 18, 1441-5.
  3. Ludwig, K.R. et al. (2018) J Proteome Res 17, 2480-90.

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
ProtiFi and S-Trap are trademarks of ProtiFi, LLC.
PTMScan is a trademark of Cell Signaling Technology, Inc.
Burdick and Jackson is a trademark of Honeywell International, Inc.