Hello Maafi Islam

You could use Mass spectrometry (MS) technique for the detection of phosphorylation. You may use several enrichment strategies for phospho-protein analysis by MS such as immobilized metal affinity chromatography (IMAC), phosphospecific antibody enrichment, chemical-modification-based methods such as beta-elimination of phospho-serine and -threonine, and replacement of the phosphate group with biotinylated moieties, because sometimes it becomes difficult to observe the signals from low-abundance phospho-proteins of interest in the high-background of abundant non-phosphorylated proteins.

You could also use collision-induced dissociation (CID) and electron transfer dissociation (ETD) which provide comprehensive parallel analysis of peptide sequences and post-translational modifications like phosphorylation.

Best.

Thank you Sebastian Schmitt for your answer.

Yes, I tried doing IP didn't work but I didn't get to do western using phosphorylation-specific antibody because since my IP didn't work I was not able to purify my protein and look at the phosphorylation sites which is my aim.

I was thinking to do the reverse as you mentioned but I was just worried whether it will be acceptable since I couldn't find any papers doing the reverse way.

I am not familiar with the titanium dioxide method could you please help me with that. Thanks again.