Hi Sepideh, I don't have a specific reference, but many serine proteinases like trypsin and thrombin still show activity at -20oC.

If you look at a slow freezing process, free water freezes out first and concentrates the proteins in a small volume before they get frozen.

It is the same process that is used to make "ice beer". You slow freeze regular beer. Water freezes first, excluding compounds that don't easily fit into the water ice lattice, like ethanol and other complex molecules.

Anyway, for proteomic analysis, it is probably advisable to flash freeze the samples and thaw them quickly just before the analysis. Protein-protein interaction will probably be preserved if they are still intact after the freeze/thaw.

Thanks!