Product Information
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.
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.
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.
IMPORTANT: The protein in the sample is insoluble in the S-Trap™ Binding/Wash Buffer. DO NOT CENTRIFUGE the sample or allow it to settle before loading onto the S-trap™ in the next step.
NOTE: All centrifugation steps for S-Traps™ should be performed at room temperature and 4000 x g.
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 tryptic digestion is critical for maximum peptide recovery in the following elution steps. Cutting incubation time short or using less than the recommended amount of trypsin will reduce performance.
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.
NOTE: Peptide solutions may be frozen at -80°C for 1 hr or longer before placing in the speedvac; 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 August 2020
revised October 2020
Protocol Id: 2184
Proteomics analyses of posttranslationally modified peptides rely on both high-quality enrichments and pre-enrichment sample preparation. Suspension Traps (S-Trap™ columns) are a convenient tool 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 the performance of S-Trap™ columns (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™ column 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.
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