A cytochrome C homologous library with varying charge distribution was employed to study the effects of protein surface modification on protein retention in cation exchange chromatography. Varying quantities of succinic anhydride were added to horse cytochrome c to control the degree of succinylation of protein surface lysine residues. Cation exchange chromatography was then carried out using a succinylated protein mixture containing primarily single lysine modifications and the collected fractions were evaluated using direct infusion and tryptic digest-mass spectrometry to determine the degree of succinylation and the sites of modification on the protein surface. Electrostatic potential (EP) maps of the native protein and the variants were generated to provide insight into the elution order of the variants and the results indicated that there were two major binding regions on the protein surface. Variants showing the largest change in retention time as compared to the native protein had modified residues within the binding regions. On the other hand, variants which showed a smaller change in protein retention time had succinylated residues at locations which were not part of the two distinct binding regions. This study demonstrates that the use of controlled protein surface modification offers a convenient means of studying the relationship between protein surface properties and chromatographic binding affinity.