We show that different ratios of bovine serum albumin (BSA) and lysozyme (LSZ) can be achieved in a mixed protein adsorption layer by tailoring the amounts of carboxyl (-COOH) and aluminum hydroxyl (Al-OH) groups on colloidal alumina particles (d50 = 180 nm). The particles are surface-functionalized with -COOH groups, and the resultant surface chemistry, including the remaining Al-OH groups, is characterized and quantified using elemental analysis, zeta potential measurements, acid-base titration, IR spectroscopy, electron microscopy, nitrogen adsorption, and dynamic light scattering. BSA and LSZ are subsequently added to the particle suspensions, and protein adsorption is monitored by in situ zeta potential measurements while quantified by UV spectroscopy and gel electrophoresis. A comparison of single-component and sequential protein adsorption reveals that BSA and LSZ have specific adsorption sites: BSA adsorbs primarily via Al-OH groups, whereas LSZ adsorbs only via -COOH groups (1-2 -COOH groups on the particle surface are enough to bind one LSZ molecule). Tailoring such groups on the particle surface allows to control the composition of a mixed BSA and LSZ adsorption layer. The results provide further insight into how particle surface chemistry affects the composition of protein adsorption layers on colloidal particles and is valuable for the design of such particles for biotechnological and biomedical applications.