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.

Slide1

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.

Slide2

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).

Slide3

 

 

 

 

 

 

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

Slide4

 

Slide5

 

 

Slide6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

As always, let me know if you have questions!

References:

doi: 10.1074/mcp.M500138-MCP200

doi: 10.1074/mcp.M800068-MCP200

 

 

1 thought on “Detergent removal 1: Gel-assisted proteolysis

  1. Pingback: Detergent removal 2: Affinity resin | Proteomics and Mass Spectrometry Core Facility Blog

Comments are closed.