Electrochemical processes in a wire-in-a-capillary bulk-loaded, nano-electrospray emitter

AbstractExperiments are described that illustrate solvent oxidation, emitter electrode corrosion, and analyte oxidation in positive ion mode nano-electrospray mass spectrometry using a wire-in-a-capillary, bulk-loaded nano-electrospray emitter geometry. Time-lapsed color photography of pH and metal specific indicator solutions within operating nano-electrospray emitters, as well as temporal changes in the ions observed in the nano-electrospray mass spectra, are used to probe these reactions, judge their magnitude, and study the time dependent changes in solution composition and gas-phase ion signal brought about as a result of these electrochemical reactions. The significance of these observations for analytical applications of nano-electrospray mass spectrometry are discussed. (J Am Soc Spectrom 2001, 853–862) Published by Elsevier Science Inc

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) has the potential to produce enzymes, therapeutic agents, and other proteins, while circumventing difficulties associated with in vivo heterologous expression. However, the contents of the cell-free extracts used to carry out synthesis are generally not characterized, which hampers progress toward enhancing yield or functional activity of the target protein. We explored the utility of mass spectrometry (MS)-based proteomics for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions. Full proteome experiments identified over 1000 proteins per reaction. The complete set of proteins necessary for transcription and translation were found, demonstrating the ability to define potential metabolic capabilities of the extract. Further, MS-based techniques allowed characterization of the CFPS product and provided insight into the synthesis reaction and potential functional activity of the product. These capabilities were demonstrated using two different CFPS products, the commonly used standard green fluorescent protein (GFP, 27 kDa) and the polyketide synthase DEBS1 (394 kDa). For the large, multidomain DEBS1, substantial premature termination of protein translation was observed. Additionally, MS/MS analysis, as part of a conventional full proteomics workflow, identified post-translational modifications, including the chromophore in GFP, as well as the three phosphopantetheinylation sites in DEBS1. A hypothesis-driven approach focused on these three sites identified that all were correctly modified for DEBS1 expressed in vivo but with less complete coverage for protein expressed in CFPS reactions. These post-translational modifications are essential for functional activity, and the ability to identify them with mass spectrometry is valuable for judging the success of the CFPS reaction. Collectively, the use of MS-based proteomics will prove advantageous for advancing the application of CFPS and related techniques