I am trying to identify potential binding partners of a nitroimidazole compound, which gets entrapped only in hypoxic cells. The drug has a biontin moiety to facilitate pull down of the drug-protein adducts using streptavidin mutein beads.
Trouble is, I suspect my drug binds to cytoskletal proteins, which is usually considered as background contamination in mass spec data. I use tubulin to check for background contamination in my elutions. I see very high levels of tubulin in my hypoxic drug treated elutions, and low to no signal in my controls. My mass spec data also has proteins like actin, HSP 70/90, GAPDH. Pyruvate kinase, Aldolase, EIF4, ELF, histons, 40s/60s ribosomal proteins etc. While some of these proteins are present in my controls too, hypoxic drug treated elutions have, in general, very high ion score and PSMs.
In such scenario, should I completely disregard these proteins just because they are known mass spec background hits? I want to narrow down some proteins so that I can move on with confirming the interaction in co-IP experiments. What would be a logical way to screen out background hits?
Hi Faisal, I love the word "crapome" do you mind if I steal it?
Anyway, I would recommend passing the sample first though plain Sepharose to get rid of non-specific Sepharose binding proteins before the streptavidin column.
Also does your drug have a long spacer arm with the biotin? It can affect the exposure of the drug when bound to the avidin.
A short biotin spacer arm can potentially sterically hinder your protein from interacting with the drug.
Thank you Steingrimur for your suggestions.
My drug do not have the biotin on it per se; after incubating cells with the drug, we lyse the cells, and add the biotin later chemically.
Also, Crapome is not my invention (I wish it was); it is a database that lists common non-specific hits in mass spec.
Hello Faisal,
i would never disregard possible interaction partners without doing a statistic to compare if the quantity of recovered protein is significantly more than in the BG-control (even if it is listed in the crapome). It could allways happen that a true interaction partner has also got a low affinity to the resin / antibody etc. and you will detect it in LC-MS analysis. this doesn´t mean that it is not a binding partner.
Best, Murat
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