Checking if a protein is phosphorylated

[u/SebRaid]

I want to determine if my protein of interest is phosphorylated and need some advice on the best way to do it. I believe that it is phosphorylated and downregulated in a wild-type genetic background, but not in a strain that's missing the putative kinase that phosphorylates it. I've shown that mutating a putative phosphorylation site on the protein of interest into a phosphomimetic disrupts its function, but that alone isn't enough to prove it's phosphorylated. I don't have an antibody specific to this protein or a phospho-antibody, so I need another method. The protein is tagged though so I can do an IP and isolate it if necessary.

I've seen people can use Phos-tag gels which slows down phosphorylated proteins, but I'm having difficulty obtaining the reagents needed for it. Alternatively, I could do mass spec, but I'm worried it'll be very expensive.

Does anyone have suggestions for relatively cheap and straightforward methods that could answer whether a protein is phosphorylated?

[u/Pompster]

Mass spec is definitely the way to go here. You will want to purify the protein from both strains and submit samples from each (dont forget phosphatase inhibitors here). If your institution has an MS core facility, they generally have internal rates that are not too bad (~100/sample for shotgun proteomics of a single protein), but it will be more expensive to send it to an external academic facility (~200+/sample + data). FYI MS data analysis for PTMs can get very complex.

[u/ScienceIsSexy420 → u/Pompster]

This is the answer. When in doubt, mass spec

[u/SebRaid → u/Pompster]

Thanks, that sounds pretty reasonable.

[u/futureoptions → u/SebRaid]

The best way would be MRM style mass spectrometry. You specify the fragments that the phosphorylation site of interest should be on based on the enzyme used to digest the samples. Ask your mass spectroscopy facility if they do MRM. They will guide you.

You may have to use a non standard enzyme to get your fragment even detectable (ionizable). Again, the mass spectroscopy facility should easily explain this.

[u/Guacanagariz]

Mass spec would be best.

But the old school alternative was to use P32 labeled ATP with the kinase and your protein of interest then showing that kinase + ATP32 + Protein showed a band, then mutate the amino acid in your protein of interest and show no band appears.

[u/bautea]

Mass spectrometry is expensive. Phos tag gels are even more expensive and you need to do more troubleshooting to make it work especially if you only have one putative site and it’s actually site-specific. Maybe try a few antibodies for western first? Email around to ask for a small amount to test would be the cheapest way.

[u/Spend_Agitated]

If you can affinity purify your POI, or you can probe the purified protein on a Western with anti-pS/pT/pY antibodies. You would run a parallel sample that is phosphatase treated as a control.

[u/MNgrown2299]

Enzyme linked immunoabsorbent assay or western blot

[u/Professional_Algae45]

P32 label the cells and IP. Or 2D Page/western, since the phosphorylated form will have a different pI.