Tag Archives: IP sample

Detergent removal 2: Affinity resin

To continue our detergent theme, here’s an affinity-based detergent removal method that is faster than the gel-assisted proteolysis but removes only the detergents, leaving the salts and chaotropic agents for you to deal with later. Unlike the gel-assisted method, it works for both proteins and peptides. The gel-assisted method is best suited for proteins, because the small, more soluble peptides are likely to elute out of the gel matrix during the washing.

I use Pierce detergent removal spin columns (0.125 mL format) in my lab, but there are other options available such as Bio-Beads or HyperD, each with its own pros and cons in the business of detergent removal. Pierce also sells a so-called HiPPR detergent removal resin (high protein and peptide recovery) for low-protein-concentration samples. The initial % detergent in such samples must also be low (ca. 1%).

The Pierce resin removes common ionic, nonionic, and zwitterionic detergents from protein and peptide solutions. This oligosaccharide-based affinity resin has a small hydrophobic cavity which creates a microenvironment for a detergent’s nonpolar moiety to enter and form an inclusion complex.

The workflow is simple: (1) centrifuge the column to remove storage buffer, (2) wash 3 times with your favorite buffer (pH 5-10), discarding the buffer each time, (3) add protein or peptide solution and let the resin do its magic for 2-5 min at room temperature, (4) centrifuge to collect your >95% detergent-free sample, i.e. don’t discard the flow-through this time!

Antharavally and co-workers from Thermo Scientific Pierce Protein Research published a study examining the detergent removal efficiency and protein recovery using their resin under several conditions (doi:10.1016/j.ab.2011.05.013). A table from this reference gives you some idea of the detergent concentrations removable with the Pierce resin.

Detergent removal efficiency (640x243)

Samples (0.1 ml containing 0.100 mg BSA + detergent at maximum concentration) were processed through 0.5 ml of Pierce detergent removal resin, and the residual detergent was measured as described in Materials and Methods. Protein concentration was determined by BCA protein assay (Pierce).

Detergent removal 1: Gel-assisted proteolysis

As promised, here’s a straightforward way to remove detergents, urea, and other LC-MS incompatible nasties from small-volume samples. The literature calls it ‘gel-assisted’ proteolysis. The idea is to entrap the protein solution in a polyacrylamide gel matrix, wash out detergents, salts, and chaotropic agents, and perform in-gel digestion. This technique works great for membrane proteins which are notoriously difficult to dissolve, and it is quite useful for any protein sample clean-up.


For my little demo, I used a 1 mg/mL BSA solution in 2% SDS. The disulfides were reduced with TCEP and alkylated with IAA, after which the protein solution was very thoroughly mixed with a 30% T acrylamide monomer solution. I quickly added 10% APS and TEMED and immediately vortexed and centrifuged this mixture so that the liquid is collected at the bottom of the tube. The polymerization time is very short, a minute or two! I left it to completely polymerize for another 20 min.


Using scalpel I removed the gel plug from the tube and diced it into small pieces. After 6 washes with 8 mg/mL ammonium bicarbonate in 50% acetonitrile, I dried the gel pieces in neat acetonitrile, removed the acetonitrile and added trypsin (see the in-gel digestion tutorial for details).



Fast forward to the MS analysis: Since the original BSA solution was very concentrated, I dissolved the peptides in 540 uL of mobile phase and injected 1 uL of this solution (55 ng total protein on column or approximately 1 pmol).








Second example shows an ion chromatogram from a 10-uL IP eluent containing 2% detergent which I cleaned up and digested using this technique.





















As always, let me know if you have questions!


doi: 10.1074/mcp.M500138-MCP200

doi: 10.1074/mcp.M800068-MCP200