Protein Stability and Structure in HIC: Hydrogen Exchange Experiments and COREX Calculations

Abstract

Hydrogen exchange mass spectrometry (HXMS) coupled to proteolytic digestion has been used to probe the conformation of bovine β-lactoglobulin (BLG), bovine α-lactalbumin (BLA), and human serum albumin (HSA) in solution and while adsorbed to the hydrophobic interaction chromatography media Phenyl Sepharose 6FF. All three proteins show evidence of EX1 exchange kinetics, indicating a loss of stability on the surface. HX protection patterns for all three proteins also indicate that the unfolded form is only partially solvent exposed. The hydrogen-deuterium exchange patterns of BLG and BLA on the surface suggest a structure that resembles each protein's respective solution phase molten globule state. The low stability of Domain II of HSA observed on Phenyl Sepharose 6FF also suggests a link to solution stability because Domain II is frequently cited as the least stable domain in solution unfolding pathways. COREX, an algorithm used to compute protein folding stabilities, correctly predicts solution hydrogen-deuterium exchange patterns for BLG and offers insight into its adsorbed phase stabilities but is unreliable for BLA predictions. The results of this work demonstrate a link between solution-phase local stability patterns and the nature of partially unfolded states that proteins can adopt on HIC surfaces.

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

“…Partial unfolding in solution has been observed from increased temperature, from adding denaturants such as urea or GdnHCl, and at extremes in pH [29,30] and on HIC surfaces [31][32][33]. Chemical denaturation and the mechanism of surface unfolding are not as well understood as are thermal and pH induced denaturation in solution.…”

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry

“…This requires an in‐depth characterization of chromatographic media under operating conditions . Various methods such as pulse response experiments, attenuated total reflectance (ATR) FTIR spectroscopy , hydrogen–deuterium exchange as analyzed by MS , circular dichroism and Raman spectroscopy as well as isothermal titration calorimetry (ITC) have been used to investigate conformational changes. Kinetics for this so called “protein spreading” has been determined for the adsorption of BSA and β‐lactoglobulin to Toyopearl Butyl‐650 M .…”

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

“…The four mAb samples outlined in Section 3.2.2.2 were prepared for HX-MS analysis according to the following protocol adapted from Gospodarek et al [57]. For this purpose, a 20-µL volume of each mAb sample was mixed with 180 µL of D 2 O containing 40 mM NaCH 3 COO at pH 5.0 (the pH value for D 2 O buffer was directly from pH meter reading without correction for the deuterium isotope effect).…”

“…Stability of other proteins, including &-chymotrypsinogen A, &-lactoglobulin B, holo-&-lactalbumin, bovine !-lactoglobulin, and human serum albumin was also studied using HX-MS on a variety of HIC stationary phases by Deitcher et al [56] and Gospodarek et al [57].…”