Utilization of lysozyme charge ladders to examine the effects of protein surface charge distribution on binding affinity in ion exchange systems.
A lysozyme library was employed to study the effects of protein surface modification on protein retention and to elucidate preferred protein binding orientations for cation exchange chromatography. Acetic anhydride was used as an acetylating agent to modify protein surface lysine residues. Partial acetylation of lysozyme resulted in the formation of a homologous set of modified proteins with varying charge densities and distribution. The resulting protein charge ladder was separated on a cation exchange column, and eluent fractions were subsequently analyzed using capillary zone electrophoresis and direct infusion electrospray ionization mass spectrometry. The ion exchange separation showed a significant degree of variation in the retention time of the different variants. Several fractions contained coelution of variants, some with differing net charge. In addition, several cases were observed where variants with more positive surface charge eluted from the column prior to variants with less positive charge. Enzymatic digest followed by mass spectrometry was performed to determine the sites of acetylation on the surface of the variants eluting in various fractions. Electrostatic potential maps of these variants were then generated to provide further insight into the elution order of the variants.
Chung
Wai Keen
WK
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Evans
Steven T
ST
Freed
Alexander S
AS
Keba
James J
JJ
Baer
Zachary C
ZC
Rege
Kaushal
K
Cramer
Steven M
SM
D000089:Acetic Anhydrides / Q000737:chemistry; D000107:Acetylation; D002852:Chromatography, Ion Exchange; D007474:Ion Exchange; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008962:Models, Theoretical; D009113:Muramidase / Q000737:chemistry*; D011485:Protein Binding
D000089:Acetic Anhydrides; C031800:acetic anhydride; D009113:Muramidase; D008239:Lysine