hello,
we conducted an ICP-MS test to verify our enzyme is indeed a metalloenzyme containing 2 zinc ions. the expected ratio between zinc atoms to protein molecules was 2:1.
the test showed a close ratio of 2.65:1.
I wonder what can be the cause, whether it's the buffer itself or maybe the test tubes.
anyway, I'm looking for an article that might relate to that so I can add a reference.
Thanks,
Sapir
How confident are you in the accuracy of your protein concentration measurement?
Zinc is a common contaminant in laboratories and just about everywhere else. Assuming your ICPMS was not contaminated with Zn from previous samples and you ran a blank before the protein-Zn complex sample, I can think of a number of issues right away before considering your stochiometry:
Did you acid wash your test tubes for the ICPMS, any glassware used in the protein extraction and so on? Was your 18Mohm.cm water purification system working properly? What was in your buffer solution? Was that free of Zn?
Did you use powder free nitrile gloves? Did you wash your hands after putting them on? Gloves are a huge source of Zinc contamination. There are numerous examples if you google them:
Friel et al 1996Article Laboratory gloves as a source of trace element contamination
,Garçon et al. (2017)
Article Nitrile, Latex, Neoprene and Vinyl Gloves: A Primary Source ...
also a poster I found online: https://www.covance.com/content/dam/covance/assetLibrary/posters/ZywickiAOAC14.pdf
Garcon et al showed even holding pipette tips can transfer Zn from gloves, so just work through every step in your process to be sure you haven't inadvertently added Zn somewhere.
Zinc is also one of the most abundant metal ions in the cell. How careful were you in your protein purification to remove cellular, not enzyme-bound zinc?
Thank you for all of the answers.
First of all, this result is preliminary. I just need to add this result to my progress report for my master's thesis, but to relate to the inaccuracy.
I'm not sure about the accuracy and all of the other questions because I was responsible for only some of the steps (digestion, dilution and ICP-MS test itself were done by the help of someone else), therefore I will consult them about the possible explanations everyone suggested.
Anyway, the experiment should be repeated and I will pay close attention to all of the possibilities you suggested.
Thank you very much, you've helped me a lot.
Sapir
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