While myelin is commonly assessed in mice as a model for humans, it remained unclear to which extent their myelin protein composition is similar. We analyzed the proteome of myelin biochemically purified from human white matter by two steps of discontinuous sucrose density gradient centrifugation intermitted by osmotic shocks. We utilized a data-independent acquisition (DIA) workflow with alternating low and elevated energy (MSE) and an ion mobility-enhanced version thereof (referred to as UDMSE) to achieve both, a correct quantification of exceptionally abundant myelin proteins and a comprehensive coverage of the myelin proteome. Label-free protein quantification revealed that the relative abundance of the structural myelin proteins PLP, MBP, CNP and SEPTIN8 correlates well with that in c57Bl/6N-mice. Conversely, multiple other proteins were identified exclusively or predominantly in human or mouse myelin. Species-dependent diversity of myelin protein composition can be instructive when translating from mouse models to humans.