Leveraging complementary ion activation methods with proton transfer charge reduction reactions for comprehensive characterization of monoclonal antibody heavy chain subunits v2
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: Jennifer S. Brodbelt
PROVIDER: PXD064275 | JPOST Repository | Sun May 25 00:00:00 GMT+01:00 2025
REPOSITORIES: jPOST
Dataset's files
Items per page: 5 1 - 5 of 20 |
Similar Datasets
Project description:The use of LC-MS, 193 nm UVPD, EThcD, gas-phase fractionation, and PTCR to characterize mAbs and ADCs.
2025-03-13 | PXD060606 | JPOST Repository
Project description:An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.
| S-EPMC4966340 | biostudies-literature
Project description:Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation. ᅟ.
| S-EPMC6669196 | biostudies-literature
Project description:The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.
| S-EPMC4661386 | biostudies-literature
Project description:The reaction kinetics of many metal redox couples on electrode surfaces are enhanced in the presence of halides (i.e., Cl-, Br-, I-). Using first-principles metadynamics simulations, we show a correlation between calculated desorption barriers of V3+-anion complexes bound to graphite via an inner-sphere anion bridge and experimental V2+/V3+ kinetic measurements on edge plane pyrolytic graphite in H2SO4, HCl, and HI. We extend this analysis to V2+/V3+, Cr2+/Cr3+, and Cd0/Cd2+ reactions on a mercury electrode and demonstrate that reported kinetics in acidic electrolytes for these redox couples also correlate with the predicted desorption barriers of metal-anion complexes. Therefore, the desorption barrier of the metal-anion surface intermediate is a descriptor of kinetics for many metal redox couple/electrode combinations in the presence of halides. Knowledge of the metal-anion surface intermediates can guide the design of electrolytes and electrocatalysts with faster kinetics for redox reactions of relevance to energy and environmental applications.
| S-EPMC8494035 | biostudies-literature
Project description:Charge inversion ion/ion reactions can provide a significant reduction in chemical noise associated with mass spectra derived from complex mixtures for species composed of both acidic and basic sites, provided the ions derived from the matrix largely undergo neutralization. Amino acids constitute an important class of amphoteric compounds that undergo relatively efficient charge inversion. Precipitated plasma constitutes a relatively complex biological matrix that yields detectable signals at essentially every mass-to-charge value over a wide range. This chemical noise can be dramatically reduced using multiply charged reagent ions that can invert the charge of species amenable to the transfer of multiple charges upon a single interaction and by detecting product ions of opposite polarity. The principle is illustrated here with amino acids present in precipitated plasma subjected to ionization in the positive mode, reaction with anions derived from negative nanoelectrospray ionization of poly (amido amine) dendrimer generation 3.5, and mass analysis in the negative ion mode.
| S-EPMC3084898 | biostudies-literature
Project description:Macromolecular radicals are receiving growing interest as functional materials in energy storage devices and in electronics. With the need for enhanced conductivity, researchers have turned to macromolecular radicals bearing conjugated backbones, but results thus far have yielded conjugated radical polymers that are inferior in comparison to their non-conjugated partners. The emerging explanation is that the radical unit and the conjugated backbone (both being redox active) transfer electrons between each other, essentially "quenching" conductivity or capacity. Here, the internal charge transfer process is quantified using a polythiophene loaded with 0, 25, or 100% nitroxide radicals (2,2,6,6-tetramethyl-1-piperidinyloxy [TEMPO]). Importantly, deconvolution of the cyclic voltammograms shows mixed faradaic and non-faradaic contributions that contribute to the internal charge transfer process. Further, mixed ion-electron transfer is determined for the 100% TEMPO-loaded conjugated radical polymer, from which it is estimated that one triflate anion and one propylene carbone molecule are exchanged for every electron. Although these findings indicate the reason behind their poor conductivity and capacity, they point to how these materials might be used as voltage regulators in the future.
| S-EPMC8162116 | biostudies-literature
Project description:The isolation of human monoclonal antibodies with broadly neutralizing breadth can provide a promising countermeasure for influenza A viruses infection. Most broadly neutralizing antibodies against influenza A viruses bind to the conserved stem region or the receptor-binding cavity of hemagglutinin and the interaction is dominated by the heavy chain. The light chain, however, contributes few or no direct contacts to the antigen. Here we report an H3-clade neutralizing human monoclonal antibody, AF4H1K1, which recognizes the hemagglutinin glycoproteins of all group 2 influenza A viruses. This human monoclonal antibody has been obtained through the screening by pairing different heavy and light chains from an H7N9-infected patient based on the next-generation sequencing technology. Further structural studies revealed that light chains modulate the neutralizing spectrum by affecting the local conformation of heavy chains, instead of direct interaction with the antigen. These findings provide important clues to understand the molecular basis of light chains in antigen recognition and to explore the strategies in particular of the use of light chain modification to develop broadly protective monoclonal antibodies against influenza A viruses and other emerging viruses.
| S-EPMC6465249 | biostudies-literature
Project description:AIMS:To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS:Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS:Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION:The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.
| S-EPMC1770088 | biostudies-literature
Project description:In the present work, we report an update and extension of the previous ion-pair formation study of Hubers, M.M.; Los, J. Chem. Phys.1975, 10, 235-259, noting new fragment anions from time-of-flight mass spectrometry. The branching ratios obtained from the negative ions formed in K + SF6 collisions, in a wide energy range from 10.7 up to 213.1 eV in the centre-of-mass frame, show that the main anion is assigned to SF5- and contributing to more than 70% of the total ion yield, followed by the non-dissociated parent anion SF6- and F-. Other less intense anions amounting to <20% are assigned to SF3- and F2-, while a trace contribution at 32u is tentatively assigned to S- formation, although the rather complex intramolecular energy redistribution within the temporary negative ion is formed during the collision. An energy loss spectrum of potassium cation post-collision is recorded showing features that have been assigned with the help of theoretical calculations. Quantum chemical calculations for the lowest-lying unoccupied molecular orbitals in the presence of a potassium atom are performed to support the experimental findings. Apart from the role of the different resonances participating in the formation of different anions, the role of higher-lying electronic-excited states of Rydberg character are noted.
| S-EPMC11397648 | biostudies-literature