Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered‐charge peptide adsorbents

Abstract

Ion-exchange chromatography (IEX) relies on electrostatic interactions between the adsorbent and the adsorbate, and is used extensively in protein purification. Conventional IEX utilizes ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered-charge adsorbent surfaces using Fluorescence Correlation Spectroscopy. We can resolve protein free diffusion, hindered diffusion and association with bare glass, agarose-coated, and agarose-clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered charge adsorbent and other surface interactions at the molecular scale.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

“…This control demonstrates that there are no long-lived (>3 ms) (SI Appendix, Fig. S2) interactions between the glass substrate and the α-lactalbumin, consistent with previous observations (58,60).…”

“…Steric screening of peptides impairs protein interaction with the charged ligand, reducing interaction energy, leading to shorter desorption times and longer adsorption times, and the inverse relationship between k a and k d observed. The importance of agarose sterics in α-lactalbumin separations is consistent with our previously reported findings (58) and is one reason why affinity chromatography supports often use a "spacer arm" between ligand and matrix (67). Future studies using the single-molecule technology developed in this work are needed to investigate the use of spacer arms with clustered-charge peptides to control steric effects.…”

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

“…40 Finally, for the diffusion properties, correlation analysis finds an average of log(D/nm 2 ·s -1 ) = 4.8 ± 0.8, in agreement with expectations based on FCS ( Figure S11b). 41 SPT also accurately finds log(D/nm 2 ·s -1 ) = 5.3 ± 0.3. However, fcsSOFI analysis super-resolves the spatial heterogeneity of diffusion coefficients (arrows, Figure 2e).…”

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

“…Super-resolution imaging allows for long-sought direct observations at the nanometer scale of the interfacial interactions that control chromatography. Using improved experimental and analysis methods, we showed the importance of charge-clustering of ligands and the kinetic heterogeneity even of nominally chemically-identical adsorption sites [12,21,26,38]. These results demonstrate the potential of single molecule techniques to advance a mechanistic understanding of chromatography difficult to obtain with ensemble techniques.…”

“…Foundational single molecule work resolved variability present at heterogeneous ion-exchange and reverse-phase stationary phase interfaces using confocal microscopy combined with fluorescence correlation spectroscopy and blip analysis [26-32] and total internal reflection microscopy [33-37]. We have recently extended single molecule techniques to the super-resolution level (< 250 nm, below the diffraction limit of light) to investigate adsorptive separations at the single ligand-single protein level [21].…”