In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after
the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions.
Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back
exchange averages roughly 30% because of the long exposure to H2O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during
quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show
that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced
retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated.
The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample
throughput, and high reproducibility and reliability. 相似文献
When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein–protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, “traditional” continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry.
Management of the enormous amount of data produced during solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has stimulated software analysis development. The proteolysis step of the experiment generates multiple peptide fragments, most of which overlap. Prior automated data reduction algorithms extract the deuteration level for individual peptides, but do not exploit the additional information arising from fragment overlap. Here, we describe an algorithm that determines discrete rate constant values to each of the amide hydrogens in overlapped fragments. By considering all of the overlapped peptide segments simultaneously, sequence resolution can be improved significantly, sometimes to the individual amino acid level. We have validated the method with simulated deuterium uptake data for seven overlapped fragments of a poly-Ala nonapeptide, and then applied it to extract rate constant values for the first 29?N-terminal amino acids of C22A FK506-binding protein. 相似文献
The adsorption isotherms at 25, 45, and 65 degrees C of molybdenum solutions of concentration ranges between 10(-3) and 3x10(-2) M(Mo) (pH 4-5) on different alumina samples are investigated. The analysis is conducted using a modified Frumkin isotherm which takes a more realistic account of the lateral interaction between adsorbed species and considers that the adsorption takes place on the most basic OH groups on the surface of alumina. The results are discussed in view of the difference in solutions speciation, and the changes in the pH of the remaining supernatant solutions. The solution temperature, PZC of the used aluminas, the configuration of the basic OH groups on their surface, and the pore structure have been shown to intervene effectively. Copyright 2000 Academic Press. 相似文献
Introduction of a chemical change to one or more amino acids in a protein’s polypeptide chain can result in various effects on its higher-order structure (HOS) and biophysical behavior (or properties). These effects range from no detectable change to significant structural or conformational alteration that can greatly affect the protein’s biophysical properties and its resulting biological function. The ability to reliably detect the absence or presence of such changes is essential to understanding the structure–function relationship in a protein and in the successful commercial development of protein-based drugs (biopharmaceuticals). In this paper, we focus our attention on the latter by specifically elucidating the impact of oxidation on the HOS, structural dynamics, and biophysical properties of interferon beta-1a (IFNβ-1a). Oxidation is a common biochemical modification that occurs in many biopharmaceuticals, specifically in two naturally-occurring sulfur-containing amino acids, methionine and cysteine. To carry out this work, we used combinations of hydrogen peroxide and pH to differentially oxidize IFNβ-1a (to focus on only methionine oxidation versus methionine and cysteine oxidation). We then employed several analytical and biophysical techniques to acquire information about the differential impact of these two oxidation scenarios on IFNβ-1a. In particular, the use of MS-based techniques, especially HDX-MS, play a dominant role in revealing the differential effects.
