I am working on 260kDa iron-sulfur cluster containing protein using FTICR (ESI source) MS. We found the tetramer formation, but there is a strong adduct formation, not clear what kind of adduct it is, it is very broad peak. We were trying to alter Mass parameters, but still no further improvement. I have desalted the protein 3 times using PD-10 column also. Could anyone suggest any ideas. related to parameters? Any ideas are really appreciated. Thanks!
NP: Attached file is the peak shape.
Senthil: Your protein behaves as a typical glycoprotein with complex carbohydrates containing negatively charged sialic acids, which yield a broad "peak". One way to get a sharp peak is to heat denature your protein followed by treatment with N-glycosidase F (NGF) to remove the complex carbohydrates. This approach can also be coupled with adding a reducing agent such as beta-mecaptoethanol (bME) to observe its monomer(s). In terms of what kind of metal it carries, you can use EDTA to treat your protein followed by PD-10 to see its apo-protein form, which should be smaller and the size difference is/are the metal it normally binds. Good luck.
@Ke-wei Zhao, Thanks a lot for the comment. I am working with Iron-sulfur enzymes. We would like to see the oligomeric state of the enzyme with metal cluster. I could see monomer in denatured condition. But I want it in native form.
Hello Senthil,
If you would like to observe the protein in its native form (oligomeric with metal cluster), you should avoid desalting as this may be altering the complex inadvertently by removing stabilising salts. Using high quality water and glassware (preferably acid-washed) may reduce the adducts. In addition, a slight decrease in pH (perhaps try a titration series of small pH decreases?) may help in removing the adducts as they are displaced with a higher proton (H+) concentration (potential competitive binding).
Finally, it is also important to remember if you working with a multimeric protein complex, those additional adducts are stabilising the desolvated gas-phase complex from your sample solution through the optics and into the FT detector. Once there, maybe trap the complex for longer and apply some additional energy/heat to see if that gently "nudges" the broad peak / adduct off.
Who knows, maybe the broad peak is something of interest!?
Kind regards,
Peter
Hello Peter,
Thanks a lot for the valuable suggestion. We have tried already with different pH, the complex is very sensitive in near acidic. So we are maintaining in basic medium. But I must take into account, to try without desalting and also i will modify the trapping condition.