Disulfide Analysis with Mass Spectrometry: Toward Biotherapeutic Characterization

Proteins continue to increase in use as biotherapeutic agents but need to be analyzed to verify efficacy and safety. For instance, disulfide bonds are a protein posttranslational modification (PTM) that are critically important to protein stability and function; therefore, they also impact safety and efficacy. Proteins can assume non-native bonds during recombinant expression, sample handling, or sample purification, leading to undesirable products. Currently, mapping of disulfide bonds is problematic due to sample requirements and data analysis difficulties. In this dissertation, many aspects of disulfide bonding analysis are improved. First, the fragmentation products of disulfide bonded peptides are analyzed to assist efforts in the automated analyses of these species when the analytical workflow involves LC-MS and collision-induced dissociation. Additionally, a new analysis method is introduced to more easily assign disulfide bonding partner peptides. This method utilizes a recent innovation in fragmentation, electron transfer dissociation. The method can also be used to easily detect alternate disulfide bonding patterns and confirmed the findings that there are multiple disulfide structures in a recombinant HIV glycoprotein, gp120. The method is then expanded to incorporate reporter peptides to ensure the sample preparation is not causing these alternate structures. Finally, a software tool capable of automated assignments is also outlined to expedite the data analysis. In sum, this dissertation advances the field of disulfide analysis of proteins, by introducing new methods to expedite the work and new techniques to ensure the reliability of the analyses

Collision Induced Dissociation Products of Disulfide-bonded Peptides: Ions Result from the Cleavage of More than One Bond

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1007/s13361-010-0064-x.Disulfide bonds are a posttranslational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is difficult due, to various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during Collision Induced Dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a QTOF instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. This data shows that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not

Characterizing O-linked glycopeptides by electron transfer dissociation: fragmentation rules and applications in data analysis

Studying protein O-glycosylation remains an analytical challenge. Different from N-linked glycans, the O-glycosylation site is not within a known consensus sequence. Additionally, O-glycans are heterogeneous with numerous potential modification sites. Electron transfer dissociation (ETD) is the method of choice in analyzing these glycopeptides since the glycan side chain is intact in ETD, and the glycosylation site can be localized on the basis of the c and z fragment ions. Nonetheless, new software is necessary for interpreting O-glycopeptide ETD spectra in order to expedite the analysis workflow. To address the urgent need, we studied the fragmentation of O-glycopeptides in ETD and found useful rules that facilitate their identification. By implementing the rules into an algorithm to score potential assignments against ETD-MS/MS data, we applied the method to glycopeptides generated from various O-glycosylated proteins including mucin, erythropoietin, fetuin and an HIV envelope protein, 1086.C gp120. The site-specific O-glycopeptide composition was correctly assigned in every case, proving the merits of our method in analyzing glycopeptide ETD data. The algorithm described herein can be easily incorporated into other automated glycomics tools

Characterizing the Impact of Primer-Template Mismatches on Recombinase Polymerase Amplification

Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3'-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping

The Community Land Model version 5 : description of new features, benchmarking, and impact of forcing uncertainty

The Community Land Model (CLM) is the land component of the Community Earth System Model (CESM) and is used in several global and regional modeling systems. In this paper, we introduce model developments included in CLM version 5 (CLM5), which is the default land component for CESM2. We assess an ensemble of simulations, including prescribed and prognostic vegetation state, multiple forcing data sets, and CLM4, CLM4.5, and CLM5, against a range of metrics including from the International Land Model Benchmarking (ILAMBv2) package. CLM5 includes new and updated processes and parameterizations: (1) dynamic land units, (2) updated parameterizations and structure for hydrology and snow (spatially explicit soil depth, dry surface layer, revised groundwater scheme, revised canopy interception and canopy snow processes, updated fresh snow density, simple firn model, and Model for Scale Adaptive River Transport), (3) plant hydraulics and hydraulic redistribution, (4) revised nitrogen cycling (flexible leaf stoichiometry, leaf N optimization for photosynthesis, and carbon costs for plant nitrogen uptake), (5) global crop model with six crop types and time‐evolving irrigated areas and fertilization rates, (6) updated urban building energy, (7) carbon isotopes, and (8) updated stomatal physiology. New optional features include demographically structured dynamic vegetation model (Functionally Assembled Terrestrial Ecosystem Simulator), ozone damage to plants, and fire trace gas emissions coupling to the atmosphere. Conclusive establishment of improvement or degradation of individual variables or metrics is challenged by forcing uncertainty, parametric uncertainty, and model structural complexity, but the multivariate metrics presented here suggest a general broad improvement from CLM4 to CLM5

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb29