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I would like to know how phosphorylated sites in proteins are detected in practice. I read some papers where the authors were talking about mass spectrometry techniques.

But my question is that why can't X-ray crystallography or NMR techniques capture phosphorylated sites?

(Note : My background is electronics engineering, so I am not an expert in this area.)

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In principle X-ray crystallography or NMR could detect phosphorylation sites but they are much more complex and expensive techniques than mass spec. So for simply figuring out phosphorylation patterns in a protein is much easier using mass spec.

Detailed reasons:

For X-ray you need to crystallize the protein which is often very difficult/impossible and requires quite a lot of sample material. Afterwards you need a good X-ray source (ideally a synchrotron beamline) and record the scattering pattern over an extended range of angles (at least 180 degrees) before your crystal evaporates in the intense X-ray beam. Last but not least, you need additional chemical tricks, elaborate mathematical models and a large amount of computing power to compensate for the fact that in X-Ray crystallography you lose the phase information (because there are no suitable lenses for X-rays).

For NMR, you need large, very strong superconducting magnets, sophisticated radio transmitters and receivers, complicated math and computing power. However even if you have all that, you are still limited to very small proteins because otherwise the NMR spectrum is simply too complicated to be solved.

In contrast, for mass spec, you need a (highly sophisticated) spray gun, a moderate magnetic field and an electron multiplying detector. This is still a very sophisticated machine but much simpler compared to the above. In addition, data analysis is simpler than in the above cases.

Basically if you don't need to know the structural changes induced by phosphorylation (for which you would need to compare the structures of two crystals), you would stick to mass spec.

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X-ray crystallography has been used to detect phosphorylated sites.

The RCSB protein database currently contains 856 structures that have both a resolution below 3 angstroms and the keyword "phosphorylated" in their listing.

It also appears to be possible to use NMR to study phosphorylated proteins.

The situations where NMR/x-ray crystallography or mass spectrometry are used differ. These differences may explain why a paper discussing the detection of phosphorylation using mass spectrometry would make no mention of NMR or x-ray crystallography.

NMR/x-ray crystallography provide structural data and almost always utilise known protein sequences.

Mass spectrometry has two major strengths for phosphorylation site detection and these highlight the reasons for using it. The first strength is the ability to gain information about many phosphorylation events, including unknown ones, from a single liquid chromatography MS/MS run. The second strength is the potential to quantify multiple phosphorylation sites from a minimum of 3 runs of a single sample.

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