Mr. An,

There is unable without MS/MS to do this.

Hi there

How did you enrich the phosphopeptides? was it with TiO2?

Have you done any previous protein purification?

If so you can analyse your sample for total phosphate.

There are many ways to do it, but some of the buffers (for the enrichment) may interfere with the phosphate analysis, ICP-OES is an option

If you haven't purified the protein, a lot of phosphate may come from the cell lysate or growing media

Hope this helps

Dear Ran An,

I am sorry, but as Bodjidarka mentioned, the proof of the pudding will be by eating it. I have been using phosphoprotein enrichment technologies, both Zr and Ti based, but I have to admit that for me the differences are less related to the material, but rather seem to be protocol related. It is an area in which one could easily achieve a breakthrough. I, for one, have not succeeded. Kind regards, Harrie Verhoeven.

Thank all of you! I am using TiO2 enrichment kit.

If I cannot know the quality of my phosphopeptides, may I know approximate amount of phosphopeptides or peptides in my sample? E.g. nanoDrop or UV method?

Thank you!

Hi Ran,

i am facing somewhat similar kind of problem. Have you figured it out yet?

Take an aliquote before drying the sample and try the "Dot blot method" using antibody against the most commonly occurring phosphopeptide. Since the same amount of protein is taken compare between before and after enrichment samples. Better is the blot in the enriched sample.

Hi everyone,

How did you enrich the phosphopeptides with TiO2?

What is buffer you use for phosphopeptide enrichment?

And Is there much difference in the number of phosphopeptides before and after enrichment?

I have a problem is the number of phosphopeptide before and after enrichment is same