Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting.

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.

Computational analysis of 140 years of US political speeches reveals more positive but increasingly polarized framing of immigration.

We classify and analyze 200,000 US congressional speeches and 5,000 presidential communications related to immigration from 1880 to the present. Despite the salience of antiimmigration rhetoric today, we find that political speech about immigration is now much more positive on average than in the past, with the shift largely taking place between World War II and the passage of the Immigration and Nationality Act in 1965. However, since the late 1970s, political parties have become increasingly polarized in their expressed attitudes toward immigration, such that Republican speeches today are as negative as the average congressional speech was in the 1920s, an era of strict immigration quotas. Using an approach based on contextual embeddings of text, we find that modern Republicans are significantly more likely to use language that is suggestive of metaphors long associated with immigration, such as "animals" and "cargo," and make greater use of frames like "crime" and "legality." The tone of speeches also differs strongly based on which nationalities are mentioned, with a striking similarity between how Mexican immigrants are framed today and how Chinese immigrants were framed during the era of Chinese exclusion in the late 19th century. Overall, despite more favorable attitudes toward immigrants and the formal elimination of race-based restrictions, nationality is still a major factor in how immigrants are spoken of in Congress.

Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry.

Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. difficile mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids, including Cys222, Lys224, His226, and Thr227, with levels ranging from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93, and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug-product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.

8OHdG is not a biomarker for Huntington disease state or progression.

OBJECTIVE: To evaluate plasma 8-hydroxy-deoxy-guanosine (8OHdG) levels as a potential biomarker of premanifest and early Huntington disease (HD). METHODS: Personnel from 2 independent laboratories quantified 8OHdG in blinded longitudinal plasma samples taken 24 months apart from 160 TRACK-HD participants, as well as samples containing control plasma with added ("spiked") 8OHdG. One laboratory used a liquid chromatography-electrochemical array (LCECA) assay, and the other used liquid chromatography-mass spectrometry (LCMS). RESULTS: The LCMS assay was more accurate than the LCECA assay for measurements of "spiked" 8OHdG levels in plasma. Neither assay demonstrated cross-sectional differences in plasma 8OHdG among controls, premanifest HD, and early symptomatic HD. Similarly, neither assay showed longitudinal changes in any disease group over 24 months. CONCLUSIONS: Plasma concentration of 8OHdG is not a biomarker of disease state or progression in HD. We recommend that future putative biomarker studies use blinded sample analysis, standard curves, independent analytical methods, and strict quality control of sample collection and storage.

Proteomic changes in cerebrospinal fluid of presymptomatic and affected persons carrying familial Alzheimer disease mutations.

OBJECTIVE: To identify cerebrospinal fluid (CSF) protein changes in persons who will develop familial Alzheimer disease (FAD) due to PSEN1 and APP mutations, using unbiased proteomics. DESIGN: We compared proteomic profiles of CSF from individuals with FAD who were mutation carriers (MCs) and related noncarriers (NCs). Abundant proteins were depleted and samples were analyzed using liquid chromatography-electrospray ionization-mass spectrometry on a high-resolution time-of-flight instrument. Tryptic peptides were identified by tandem mass spectrometry. Proteins differing in concentration between the MCs and NCs were identified. SETTING: A tertiary dementia referral center and a proteomic biomarker discovery laboratory. PARTICIPANTS: Fourteen FAD MCs (mean age, 34.2 years; 10 are asymptomatic, 12 have presenilin-1 [PSEN1 ] gene mutations, and 2 have amyloid precursor protein [APP ] gene mutations) and 5 related NCs (mean age, 37.6 years). RESULTS: Fifty-six proteins were identified, represented by multiple tryptic peptides showing significant differences between MCs and NCs (46 upregulated and 10 downregulated); 40 of these proteins differed when the analysis was restricted to asymptomatic individuals. Fourteen proteins have been reported in prior proteomic studies in late-onset AD, including amyloid precursor protein, transferrin, α(1)ß-glycoprotein, complement components, afamin precursor, spondin 1, plasminogen, hemopexin, and neuronal pentraxin receptor. Many other proteins were unique to our study, including calsyntenin 3, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) 4 glutamate receptor, CD99 antigen, di- N-acetyl-chitobiase, and secreted phosphoprotein 1. CONCLUSIONS: We found much overlap in CSF protein changes between individuals with presymptomatic and symptomatic FAD and those with late-onset AD. Our results are consistent with inflammation and synaptic loss early in FAD and suggest new presymptomatic biomarkers of potential usefulness in drug development.

Development and SAR of functionally selective allosteric modulators of GABAA receptors.

Positive modulators at the benzodiazepine site of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Through oocyte voltage clamp studies, we have discovered two series of compounds that are positive modulators at α2-/α3-containing GABA(A) receptors and that show no functional activity at α1-containing GABA(A) receptors. We report studies to improve this functional selectivity and ultimately deliver clinical candidates. The functional SAR of cinnolines and quinolines that are positive allosteric modulators of the α2- and α3-containing GABA(A) receptors, while simultaneously neutral antagonists at α1-containing GABA(A) receptors, is described. Such functionally selective modulators of GABA(A) receptors are expected to be useful in the treatment of anxiety and other psychiatric illnesses.

Molecular weight distributions of asphaltenes and deasphaltened oils studied by laser desorption ionization and ion mobility mass spectrometry.

Laser desorption ionization (LDI) and ion mobility mass spectrometry (IM-MS) are applied to study molecular weight distribution and cross sections of petroleum asphaltene (ASPH) and deasphaltened oils (DAO). Ion mobility data confirmed the presence of gas-phase aggregation in LDI experiments. Most of the molecules with MW > 3000 g/mol in LDI result from gas-phase aggregation. Two-dimensional (2D) IM-MS trend lines are compared with model polymer systems to confirm the order of cross sections (polywax > polystyrene >> DAO > ASPH >> fullerenes), and these data illustrate that ASPH has a more condensed average structure than DAO.

Propofol for emergency department procedural sedation and analgesia: a tale of three centers.

OBJECTIVES: To characterize propofol procedural sedation and analgesia (PSA) encounters for a large patient population at multiple emergency department (ED) sites. The authors sought to assess the frequency of respiratory and cardiovascular events during propofol PSA within these settings. METHODS: This study was a prospective, descriptive series of a consecutive sample of ED patients receiving propofol for PSA at three study sites. Patients were monitored for PSA-related events, including predefined clinically relevant cardiovascular and respiratory events. Data collection was performed during PSA with a standardized data collection sheet unique to each site. RESULTS: Propofol was administered during PSA to 792 patients during the respective reporting period at each center. Indications for sedation included dislocation reduction (38%), cardioversion (10%), fracture reduction (35%), abscess incision and drainage (8%), computed tomography imaging (2%), and tube thoracostomy (1%). The cumulative rate of oxygen desaturation events for all study sites was 7.7% with a brief period of assisted ventilation with bag-valve mask in 3.9%. The cumulative rate of PSA-related hypotensive events was 3.5%. Increasing patient age and specific clinical procedure were clinical variables most associated with any propofol-related respiratory event. All PSA-related events resolved with supportive interventions during the PSA encounter. No patients required endotracheal intubation, prolonged observation, or admission for PSA-related complications. CONCLUSIONS: Propofol typically confers a deep sedation experience for ED PSA. The most common PSA events associated with propofol are respiratory related and appear consistent across these three practice settings. All propofol-related PSA events resolved with brief supportive interventions in the ED with no adverse sequelae.