Hi Edna,

If you have tried long kinase reactions (as you did), I don't think adding more kinase will have a big impact on phosphorylation levels. Unless you have reason to believe that your kinase loses activity quickly, it should normally continue to phosphorylate substrate proteins during the time of the assay. If other substrates show much higher phosphorylation levels I doubt that this is the case, but you can always try a dose response just to be sure.

I have never tried a phospho-protein purification kit, but one way to potentially circumvent the problem with mass-spec sensitivity is to do mutational analysis instead: you mutate candidate phosphosites and express and purify all mutants in E. coli and compare their phosphorylation by your kinase. Whether this approach is feasible depends on your substrate and the number of putative phosphosites of course. The other downside is that it is of course a much more indirect way of verifying phosphosites.

I'd be curious to hear other people's ideas as well.

Best regards,

Liesbeth

Dear Edna,

I have some questions that might help. Why not pulling down the endogenous protein (the substrate) from human cells after enriching for the conditions that gets you maximal phosphorylation? You can overexpress a tagged protein and pull it down and send it to mass spec after. I don't know the pathway that this protein is part of, but it's possible that there is a phosphatase that reduces the signal. Using a phosphatase inhibitor in this case will enhance your signal. Let me know how do you see my suggestions.

Dear Edna,

how about the in vitro incubation conditions? perhaps your kinase needs a different cofactor. I work with plant MAPkinases and their specific activities vary depending on metal ions availability (Mg2+, Mn2+). 

Best

Romina

How about using an antibody against phosphotyrosine (if that is what you are interested in) attached to magnetic beads, so you can elute your enriched phosphorylated proteins easily?

There are some anti-phosphotyrosine antibodies that are very good for IP available commercially.

Hi Edna,

I agree with most of the suggestions above; perhaps you can optimize your in vitro reactions to enhance protein phosphorylation, but adding more kinase itself is probably not going to make a huge difference.  Perhaps adjusting pH, ionic strength, adding cofactors, etc. may help.  Also - is it possible that your kinase needs a regulatory subunit or some type of other protein-protein interaction (or lipid or small molecule interaction) to be activated?  Would it be possible to isolate the kinase from cells in which you know (or at least predict) it would be active?

For the second part of your question - the phosphopeptide enrichment step - this depends on which phosphoresidues you're looking for.  As mentioned in some of the other comments, if you're looking for a phosphotyrosine, antibody-mediated enrichment will be your best bet.  There are some great ones out on the market; I've had good success with 4G10 in the past.  If you're looking for phosphorylation on a serine or threonine, we've had good success in our lab using IMAC (immobilized metal affinity chromatography) prior to mass spectrometry. 

Good luck!

Natalie

Article A Quantitative Map of the Liver Mitochondrial Phosphoproteom...

Thank you very much all for your answers, they are very helpful!!!

Hello Edna, I think you need to address the issue of low 32P incorporation in your in vitro assay. Are you positive that the protein you are studying is actually a substrate for this kinase or, alternatively, the conditions you are using have activated the kinase?

you could co-express the kinase and the substrate in E. coli.

Hi..You could put an invitro kinase reaction with cold ATP(non radioactive) and run it on SDS-PAGE. The gel is then stained with a phosphospecific ProQ diamond Stain (Invitrogen) and then do sypro staining for total protein estimation