PTMScan^® HS SUMO Immunoenrichment Companion Reagents Kit #98883

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.