The patterning of protein features on poly[(tert-butylmethacrylate)-co-(methyl methacrylate)] via e-beam lithography was achieved using two mechanisms which control the radiation-assisted, spatially addressable, selective attachment of the protein on the polymer surface. The hydrophobicity-controlled adsorption on the unexposed hydrophobic polymeric surface produces negative tone, high-contrast, high-resolution images. The chemical linkage of the protein amino groups to the carboxylic groups generated by the e-beam radiolysis produces images with a contrast tunable by the exposure energy. The performance measures of protein patterning, that is, the contrast, resolution, and level of defects, were discussed in the context of the respective mechanisms. As PtBuMA is the radiation sensitive component of a class of deep-UV resists, the study applies to the protein patterning via deep-UV lithography, with potential impact on the fabrication of biodevices and combinatorial chemistry.