How do I prepare and submit a sample for protein molecular weight determination?
Sample recommendations:
We recommend submitting 20 ug of protein in a volume of 20 uL at a concentration of 1 ug/ul. The sample should be pure or the deconvolution may not work.
Detergents are not compatible with mass spec and must be removed from your buffer prior to analysis. Remove all polymers or detergents such as TritonX, NP-40, SDS, or similar polymers in the buffer. They have strong ions that swamp the protein MS signal, and also will gum up the protein trap and contaminate our lines.
Remove low viscosity buffer compoments such as glycerol or PEG as they can clog the tubing in the system.
A 10 mM ammonium acetate buffer is ideal for analysis. Buffers such as Tris or phosphate can be used, however we do not recommend this as the salt in these buffers will reduce signal in the mass spec, resulting in poor deconvolution and mass determination.
Cleaning up the sample:
FPLC/HPLC purification with water/acetonitrile buffer, acetone or TCA precipitation with extensive washing are two common options for sample clean up. MW filtering with centrifugation and dialysis will work on some molecules, but some detergents form micelles and behave like large MW species, so they cannot be removed by MW filtering or dialysis. Resins to remove detergents can be used.
If you are exchanging the buffer, we recommend exchanging into 0.1% formic acid and water. If you need neutral pH, you can use ammonium acetate buffer.
Submitting the sample:
Fill out the MW sample submission form in FBS https://fbs.research.utexas.edu/Anon/Logon.aspx and bring your samples by the lab at MBB 1.420. We require information such as: buffer components, protein concentration, expected molecular weight, and a valid account number. If you need help, have questions, or need to consult specifically about the analysis, please come by our lab at MBB 1.420 and we will assist you.
Additional information:
A good resource on protein MW determination from NCSU is here: