Hi all,
My name is Seoyeon. I am preparing metal binding experiment to a protein of interest. It was previously shown that zinc binds to the protein. For some reason I can't tell that the metal is bound (no m/z peak corresponding to protein+zinc).
There could be many things that went wrong.. but I am wondering if my sample prep was not proper.
Zinc stock prep:
2mM ZnCl2 in 150mM NH4OAc 10mM NH4HCO3 at pH 7.5 (buffer)
diluted to 100uM ZnCl2 and aliquoted in various volumes to meet certain final concentrations.
Buffer + ZnCl2 solutions were prepared in advance on the day of analysis..
Then I did buffer exchange with the protein. Finally, the protein was added and I waited for 5 minutes to let the binding to occur.
Is there any issue with my sample prep? Is my waiting time too short? About the waiting time, they were really sprayed at least few more minutes ~ few hours (for later samples..).
Thanks!
Could the problem be the insoluble Zinc carbonate that forms when you mix 2mM ZnCl2 with 10mM NH4HCO3? The solubility of Zinc carbonate in water at pH 7 is on the order of 25uM, see link. Try a different buffer; why not just use 150mM NH4OAc at pH 7.5?
http://www.nist.gov/srd/upload/jpcrd444.pdf
what are your MS conditions and what instrument are you using - unless th conditions inside the instrumenty are very carefully controlled the protein Zn complex will dissociate before it hits the detector.
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