Monthly Archives: January 2014

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




January 11, 2014

I just stopped by to check on Orbi today. It is running samples for Joana (almost done), Sarah and Allen (in the queue), and Matt (next). I am also running one of my demo samples for gel-assisted proteolysis – a very simple, fast, inexpensive, and effective technique for cleaning up and digesting protein samples, which I will share with you next week. Enjoy your weekend!


January 9, 2014

It is still very hot in the lab, 81 F (27 C)… OPP is doing their best to keep up with our requests, fighting the aging equipment. I will be moving my office today and tomorrow to provide access to the HVAC equipment for the OPP staff. I am currently working on preparing the samples submitted by Sarah, Joana, Matt, and Allen. If I can cool the LC, I will start running them tonight.

2 PM ETA: Thanks to Neal, Ron, Bob, and Steve from OPP area 4 the temperature in the lab is coming down! Looks like we are back to running samples!

LC-MS compatible detergents

They do exist! They are compatible with the reverse-phase chromatography! They solubilize stubborn proteins and improve proteolysis! Here are their names:

Acid-labile surfactants are hydrolyzed at low pH, and the hydrolysis products are compatible with reversed-phase separations and MS. These include RapiGest SF and PPS Silent Surfactant.

Invitrosol is a homogeneous surfactant whose elution profile does not overlap with the proteolytic peptides elution profiles.

ProteaseMAX is a surfactant that degrades during proteolysis, and its degradation products do not interfere with LC-MS.

All other detergents are not compatible with LC-MS and must be removed from the samples prior to analysis. These detergents include SDS (sodium dodecyl sulfate) and LDS (Lithium dodecyl sulfate), NP-40, Triton, Octyl glucoside and octyl thioglucoside, CHAPS, sodium deoxycholate, lauryl maltoside, Brij-35, etc. There are several ways to remove detergents, which is a topic for another blog post!


January 7, 2014

Orbi is still recovering from vacation: can’t get stable temperature, so no samples will be run today. On the bright side, the weather is lovely! Stay warm, watch out for those white spots on your face!


January 6, 2014

Happy New Year everyone! We are back from a nice 2-week vacation, re-energized and ready to run your samples! Orbi has finally established communication with all its parts and now is baking out. LCT is down: it lost one of its pumps, and we are waiting for the pump to be repaired and sent back to us. The open access instrument will be up and running tomorrow as well as the triple quad.