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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices

Sensitive, multiplexed protein quantification remains challenging despite recent advancements in LC-MS assays for targeted protein biomarker quantification. High-sensitivity protein biomarker measurements usually require immuno-affinity enrichment of target protein; a process which is highly dependent on capture reagent and limited in capability to measure multiple analytes. Herein, we report a novel antibody-free platform, which measures multiple biomarkers from complex matrices employing a strategically optimized solid-phase extraction cleanup and orthogonal multidimensional LC-MS. Eight human protein biomarkers with different specifications were spiked into canine plasma as a model investigation system. The developed strategy achieved the desired sensitivity, robustness, and throughput via the following steps: (1) post digestion mixed-mode cation exchange-reverse phase SPE enrichment cleaned up the sample initially; (2) rapid, high-pH peptide fractionation further eliminated background components efficiently while selectively enriched signature peptides (SP) to provide sufficient sensitivity for multiple targets; and (3) trapping-micro-LC-MS analysis delivered high sensitivity comparable to a nano-LC-MS method but with much better robustness and throughput for the final analysis. Compared with a conventional LC-MS assay with direct protein digestion and limited clean-up, analysis with this antibody-free platform improved the LLOQ by 1–2 orders of magnitude for the eight protein biomarkers, reaching as low as 5 ng/mL in plasma, with feasible robustness and throughput. In conclusion, this platform was applied for the quantification of biomarkers of respiratory conditions in patients with various lung diseases, demonstrating real-world applicability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Advances in mass spectrometry-enabled multiomics at single-cell resolution

We report biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogeneous origins, and within single-cells. Hence, multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogeneous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry (MS) has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regard to the breadth of biomolecules that can be analyzed. Recently, many studies have coupled MS to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this review on MS-enabled multiomic measurements at single-cell or near-single- cell resolution.

47 OTHER INSTRUMENTATION↗
DOE OSTI2021

Pregnancy is accompanied by larger high density lipoprotein particles and compositionally distinct subspecies

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identifying Receptor Kinase Substrates Using an 8000 Peptide Kinase Client Library Enriched for Conserved Phosphorylation Sites

In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The kinase-client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including integrin-linked kinase 4, for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B–like protein and G protein alpha subunit 1, which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates calcineurin B–like protein and G protein alpha subunit 1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

Circadian rhythms, the 24-hour cycles that tune organismal physiology to the daily rhythms of light and dark, optimally organize cellular processes such as metabolism and mitochondrial function. In mammals, macrophage functions are regulated by these 24-hour circadian rhythms such that the immunometabolic response is coordinated across the day, consolidating macrophage physiology into temporally distinct phases to time the cellular immune response. However, while it is known that there are time-of-day specific responses to stress in a macrophage, little has been done to determine if circadian regulation coordinates the response of a macrophage to real-world pathogens. Importantly, key proteins in the response to viral infection have been found to be under circadian control, and time of day of application is known to affect the efficacy of vaccinations, including in the case of the COVID-19 virus. Therefore, to investigate if the circadian regulation of macrophage physiology imparted a time-of-day response to viral exposure, we exposed primary mouse and human macrophages to the SARS-CoV-1 and CoV-2 spike proteins at different times over the circadian day. To establish a time-of-day effect, we performed a multi-omics analysis and in vitro tissue culture assays examining macrophage responses over circadian time. We found that, conserved across the species, the timing of spike protein exposure dictated two distinct temporal responses which were characterized by hallmarks of immunometabolic suppression and modest inflammatory activation. However, these responses were primarily influenced by central metabolic and mitochondrial changes and not by classical immune activation.

Circadian Biology↗
DOE OSTI2020

Review: The Next Steps in Crop Improvement: Adoption of Emerging Strategies to Identify Bottlenecks in Sugar Flux

We report sugar allocation in plants is the fundamental process that transports sugar from source to sink tissues and has a dramatic impact on crop yields. Controlling sugar allocation is required to increase crop yields, as well as biomass for biofuel production. Successful examples have demonstrated that genetic engineering of sugar partitioning offers a promising strategy to achieve this goal. However, improvement has thus far been limited by gaps in understanding of the underlying mechanisms controlling the allocation of sugars. The dynamics of sugar partitioning are minimally predictable under different conditions, between species, or in response to abiotic stresses. Here, we discuss four methodologies that have not been sufficiently exploited for the identification of bottlenecks in sugar flux. Furthermore, we suggest how these strategies can be used and combined to provide the insight needed to maximize crop yields or biomass, especially under conditions of environmental stress.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Coarse-grained resource allocation modeling for decoding and rewiring microbial metabolism

Microbial metabolism is a complex, emergent system driven by the coordinated interplay of intricate and dynamic molecular processes. To elucidate cellular behavior and enable biotechnological applications, quantitative models that address the inherent complexity of metabolism have been developed from a resource allocation perspective. Here, we synthesize recent advances in coarse-grained resource allocation frameworks and their applications in understanding microbial physiology and guiding gene circuit design. Here, these frameworks reveal global regulatory constraints and predict cellular adaptation to nutrient and environmental changes. In addition, they enable the quantification of metabolic costs, the dissection of circuit–host interactions, and the development of strategies for burden mitigation. Collectively, these modeling frameworks provide a powerful platform for uncovering quantitative principles of microbial growth and engineering robust synthetic biological systems.

coarse-grained modeling↗