In boh water and urea you might have some problem : in water, the solubility may be poor. You need to add some salt as tris and NaCl to obtain a complete dissolution of the casein. In urea, you may have some problem of stability of the casein denaturation (urea is a chaotropic agent) and you could add a detergent.

Just a remark, i think that trypsin works better in in-gel digestion than in pure solution. It is a reason of the SDS-PAGE before LC-MS.

I would not go for either. 6 M Urea is way too much for a tryptic digest. That's why you would need to dilute it to at least 4 M better less.

There are plenty of "in-solution" protocols out there.

If you really struggle with solubility you can go with SDS buffer but you need to get rid of that prior LC-MS otherwise your column will hate you.

But there is also a solution to that.

"Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat Methods 6, 359-U360, doi:Doi 10.1038/Nmeth.1322 (2009)."

I don't agree with Stéphane that an SDS-PAGE is reasonable for that.

I use BSA as a "standard" and I just dissolved that in bicarbonate buffer which is one trypsine loves :) and then simply follow the standard procedure of in-solution digest.

You anyway do an purification step of the peptides afterwards... so "quick and dirty" will do here, I guess.

Dissolve it in 6M Urea but digest with Lys-C first, then dilute urea down with ammonium bicarbonate and digest the rest with Trypsin.