Use of small molecules for isolation of particular sub-proteomes is often complicated by the need for chemical modification of a parent compound for affinity sorbent preparation. Isatin (indoledione-2,3) is an endogenous indole that exhibits a wide spectrum of biological activities. Using 5-aminocaproylisatin for proteomic profiling of fractionated rodent brain homogenates, we previously identified more than sixty individual proteins. However, proteins tested in an optical biosensor study for validation of their isatin-binding capacity demonstrated different affinity for immobilized 5-aminocaproylisatin and 5-aminoisatin. In this study, we comparatively evaluated proteomic profiles of isatin-binding proteins separated using both isatin analogs as the affinity ligands. The total number of identified proteins was higher with the shorter isatin analog (88 versus 66), and only 22 proteins were identical in the two proteomic profiles. Thus, proteomic profiling of brain isatin-binding proteins is significantly influenced by the length of the spacer between the amino group used for affinity ligand coupling to Sepharose and the isatin moiety. This suggests that the actual number of brain proteins interacting with endogenous (unmodified) isatin still remains underestimated due to different affinity of proteins for the isatin analogs used for the affinity-based proteomic profiling.