I hope that this is can help you:

Article R2-P2 rapid-robotic phosphoproteomics enables multidimension...

To enrich their phosphorylated proteins the used Fe-NTA MagBeads.

Mr. Barzaghi,

In fact, you can carry out even exact quantification in vivo by mass spectrometry, despite the chemical reactions in the living systems.

It can be done by means of our more recently developed quantification approach and model equations. Please, consider detail in the following discussions and the reference sections, therein.

1. https://www.researchgate.net/post/any_expert_in_endotoxin_quantification_by_fluorescent_assay

2. https://www.researchgate.net/post/How_to_quantitate_endogenous_metabolites_in_plasma

In addition, you can consider some detail on the theory at reference [3], as well.

3. Article Electrospray ionization mass spectrometric solvate cluster a...

If you have any questions and comments on the computations/calculations accordint to our new theory in ordet to apply our method to your system adequately, do not hesitate to perform further discussion herein. Moreover, meanwhile we have developed even more simplistic quantitative MS approach using the same theoretical background as it is given in reference [1]-[3], which most probably should be also applicable to complex biological systems.

Phosphatases may quickly convert phosphorylated proteins into their non-phosphorylated forms during the isolation process. It is recommended therefore to include inhibitors for the several types of phosphataes (tyrosine, serine and threonine) to maintain the actual phosphorylation status. Vanadate and deltamethrin can be used for example.

Peter

Thanks for your answers. I actually already have a protocol for mass-spec and enrichment in phospho-peptides. My concern is more about the upstream steps, such as the use of the correct lysis buffer or some implementation of the IP steps.