Close up photo of vials in autosampler of gas chromatography with spectrometer in chemical laboratory

How it works

Sample preparation is a vital first step for mass spectrometry and gel-based protein analysis.

At Impact Proteomics, we provide sample preparation tools to ensure your workflow begins with the highest quality sample so you can get the highest quality data.

The key to our incredible sample preparation tools is our patent pending reversible click chemistry tag, called ProMTag. One half of the tag reacts reversibly with primary amines on the surface of proteins and the other half covalently reacts with ProMTag capture resin. This allows us to quickly tag proteins, immobilize them covalently on resin, extensively wash them, and then release them in their original unmodified state. The possibilities are endless for how this technology can help you accomplish your research goals.

All of our protein extraction and cleanup kits contain the same four basic steps:

  • 1) Label proteins with ProMTag, our reversible protein tag
  • 2) Covalently bind ProMTag labeled proteins to ProMTag capture resin
  • 3) Wash away contaminants and/or exchange buffers
  • 4) Release the clean, unmodified protein sample from the beads

 

For the generation of peptide samples for mass spectrometry, additional steps include:

  • 5) Reduction and alkylation
  • 6) Digestion with stabilized and tagged trypsin
  • 7) Removal of trypsin
  • 8) Elution of clean, digested peptides

 

For multi-omics applications to purify nucleic acids and proteins, additional steps include:

  • 3.1) Elute nucleic acids and split sample in half
  • 3.2) Digest one sample with DNase to acquire RNA
  • 3.3) Digest the other sample with RNase to acquire DNA

 

For immunoprecipitation and cleanup of antigens, steps include:

  • 1) Bind antibodies from either commercial product or patient serum to provided protein A beads
  • 2) Add ProMTag labeled antigens to protein A beads to bind potential antigens to antibodies on protein A beads
  • 3) Wash away unbound antigens
  • 4) Elute antibodies and antigens from protein A beads
  • 5) Bind ProMTag labeled antigens to ProMTag capture resin
  • 6) Wash away contaminating antibodies
  • 7) Reduce and alkylate antigens
  • 8) Release and digest antigens into peptides with MT-trypsin
  • 9) Elute antigen peptides ready for mass spectrometry

 

For enrichment of membrane proteins, additional steps include:

  • 1) Label proteins on the surface of the cell with ProMTag before cell lysis
  • 1.1) Wash cells extensively to remove excess ProMTag
  • 1.2) Lyse cells with provided cell lysis buffer
  • 1.3) Spin out cellular debris and transfer supernatant to ProMTag capture resin

 

All reagents and supplies for all steps are included in our kits for convenience and reproducibility

Whether you are working with whole proteins, whole proteomes, or peptides and more our protein sample preparation tools can enhance your workflows using our patent pending reversible tagging system. We have taken advantage of natural protein chemistry to create a way to capture, clean up, and release any protein-based sample.

Our ProMTag reversible protein tagging agent reacts with primary amines on the surface of every protein and/or peptide to covalently link the tag to the surface.  The ProMTag agent then reacts with the surface of the ProMTag Capture Resin to covalently attach the proteins to the surface of the resin, allowing for extensive washing and buffer exchange.  The covalent bond between the protein and the ProMTag is reversed by adding the mass spec compatible elution buffer.  This leaves the amines on the protein in their original, unmodified state ensuring no changes to the mass or isoelectric point of the protein that could interfere with analysis.  If the proteins are being digested into peptides we provide MT-trypsin which is stabilized to prevent autodigestion and labeled with an irreversible tag to prevent contamination of the final sample.  Additional steps can be added to allow for additional purification of nucleic acids, enrichment of membrane proteins, or capture and cleanup of immunoprecipitation samples. What can Impact Proteomics do for you?

Check out our resources page for more data describing how our technology helps answer new questions about biology.

How it works: