The electrospray ionization method we use requires that the sample be free from salts and from substances such as detergents that suppress ionization. Many affinity tag procedures and kits have detergents in their buffers! If you can’t avoid detergents in your elution buffers, they must be removed prior to enzymatic digestion of the sample. High CMC detergents may be removed by dialysis, but commonly used detergents like triton X-100 can only be removed by special techniques. Remember, the ideal sample contains nothing but peptides!
You may want to try substituting an acid hydrolzable detergent in your protocol. There are also commercial detergent removal kits that we have seen success with. Ask us for details.
The protocol below is generally useful for removing a variety of small molecule contaminants, although not many detergents.
TCA precipitation of proteins
Protocol prepared by Lori Kohlstaedt.
1. Add 100% (w/v) TCA (trichloro acetic acid, see preparation method below) to the
sample to bring the TCA concentration to 20%.
2. Incubate on ice for at least 1 hr. Dilute samples may be left overnight.
3. Spin at maximum speed at 4 degrees in a microcentrifuge for 10 min.
4. Wash the pellet 3X with a solution of ice cold 0.01 M HCl / 90% acetone.
5. Allow the pellet to air dry.
The pellet can then be resuspended directly in 100 mM tris, pH 8.5, 8 M urea for
enzymatic digestion for mass spectrometry
Preparation of 100% TCA:
(Don’t try to weigh out TCA; it’s too hygroscopic)
1. Obtain a fresh bottle of crystalline TCA.
2. Read the weight in the container from the label.
3. Add distilled water to give a 100 g / 100 ml solution at final volume.
Store the solution in an acid compatible container.
Another common problem is the introduction into the sample of contaminants that leach out of plastics. Pay close attention to solvent compatibilities of tubes and pipets you use during sample preparation. For example, do not pipet concentrated acid with plastic pipet tips.
Sample submission form here.