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Results for “low-abundance proteins”

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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8 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Enhancing Sensitivity in Targeted Single-Cell Proteomics by Coupling a Dual Ion Funnel Interface with Triple Quadrupole Mass Spectrometer

Single-cell proteomics (SCP) has emerged as a powerful approach for understanding cellular heterogeneity and biological processes at unprecedented resolution. However, the extremely limited protein content of individual cells (femtogram to picogram levels) pushes current mass spectrometry instrumentation to its sensitivity limits, creating a critical analytical bottleneck. While selected reaction monitoring (SRM) using triple quadrupole (QqQ) instruments 1 offers advantages in sensitivity and reproducibility for targeted proteomics quantification, SRM still struggles with sensitivity for quantification of moderate- or low-abundance proteins from single-cell sample amounts. Here, we report the development and systematic evaluation of a dual ion funnel interface designed to address the sensitivity limitation by significantly enhancing ion transmission efficiency in commercial QqQ mass spectrometers. The dual ion funnel interface, composed of a curved S-funnel followed by a conventional ion funnel, improves ion transmission efficiency while reducing chemical noise through selective ion focusing. The performance of the dual ion funnel interface was systematically compared to standard interface on a TSQ Vantage platform across samples with different levels of complexity. The dual funnel interface demonstrated to provide up to 25-fold improvement in sensitivity across a wide range of protein concentrations in different biological matrices (low complex mouse macrophage and high complex human cells). Critically, enhanced sensitivity was accompanied by increased analytical reproducibility with lower coefficient of variations. Most importantly, the dual funnel interface enabled reliable quantification of low-abundance proteins that were barely detectable or not detected by the standard interface, extending analysis to single-cell equivalent amounts while maintaining excellent reproducibility. These results demonstrate that the dual funnel interface addresses the critical bottleneck in quantitative targeted proteomics, providing a technological foundation for ultrasensitive targeted SCP that requires both high sensitivity and robust quantitative performance.

Min, Sehong↗
DOE OSTI2024

DNA Origami Incorporated into Solid-State Nanopores Enables Enhanced Sensitivity for Precise Analysis of Protein Translocations

The rapidly advancing field of nanotechnology is driving the development of precise sensing methods at the nanoscale, with solid-state nanopores emerging as promising tools for biomolecular sensing. Here, this study investigates the increased sensitivity of solid-state nanopores achieved by integrating DNA origami structures, leading to the improved analysis of protein translocations. Using holo human serum transferrin (holo-hSTf) as a model protein, we compared hybrid nanopores incorporating DNA origami with open solid-state nanopores. Results show a significant enhancement in holo-hSTf detection sensitivity with DNA origami integration, suggesting a unique role of DNA interactions beyond confinement. This approach holds potential for ultrasensitive protein detection in biosensing applications, offering advancements in biomedical research and diagnostic tool development for diseases with low-abundance protein biomarkers. Further exploration of origami designs and nanopore configurations promises even greater sensitivity and versatility in the detection of a wider range of proteins, paving the way for advanced biosensing technologies.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2023

Targeted Quantification of Protein Phosphorylation and Its Contributions towards Mathematical Modeling of Signaling Pathways

Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease.

mathematical modeling↗
DOE OSTI2024

Multiplexed Quantitative Proteomics in Prostate Cancer Biomarker Development

Prostate cancer (PCa) is the most common non-skin cancer among men in the United States. However, the widely used protein biomarker in PCa, prostate-specific antigen (PSA), while useful for initial detection, its use alone cannot detect aggressive PCa and can lead to overtreatment. This chapter provides an overview of PCa protein biomarker development. It reviews the state-of-the-art liquid chromatography-mass spectrometry-based proteomics technologies for PCa biomarker development, such as enhancing the detection sensitivity of low-abundance proteins through antibody-based or antibody-independent protein/peptide enrichment, enriching post-translational modifications such as glycosylation as well as information-rich extracellular vesicles, and increasing accuracy and throughput using advanced data acquisition methodologies. This chapter also summarizes recent PCa biomarker validation studies that applied those techniques in diverse specimen types, including cell lines, tissues, proximal fluids, urine, and blood, developing novel protein biomarkers for various clinical applications, including early detection and diagnosis, prognosis, and therapeutic intervention of PCa.

Prostate cancer, SRM, PRM, DIA, protein biomarker↗
DOE OSTI2022

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering

Single-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low abundance proteins. Herein, we describe an ion mobility-enhanced mass spectrometry acquisition and peptide identification method, TIFF (Transferring Identification based on FAIMS Filtering), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

LIPID DROPLET PROTEIN OF SEEDS is involved in the control of lipid droplet size in Arabidopsis seeds and seedlings

Abstract In oilseeds, energy-rich carbon is stored as triacylglycerols in organelles called lipid droplets (LDs). While several of the major biogenetic proteins involved in LD formation have been identified, the full repertoire of LD proteins and their functional roles remains incomplete. Here, we show that the low-abundance, seed-specific LD protein LIPID DROPLET PROTEIN OF SEEDS (LDPS) contains an amphipathic α-helix and proline hairpin motif that serves as an LD-targeting signal and a separate region that binds to the LD protein OLEOSIN 1 (OLEO1). Loss of LDPS function results in smaller LDs and less seed oil in comparison with wild type, while overexpression of LDPS results in an increase in LD size and seed oil content. Loss of LDPS function also results in an inability of LDs to undergo fusion during postgerminative seedling growth. Analysis of oleo1 and ldps single- and double-mutant seeds and freeze–thaw treatment of seeds revealed that OLEO1 suppresses the ability of LDPS to promote larger LDs. Collectively, our results identify LDPS as an important player in LD biology that functions together with OLEO1 to determine LD size in Arabidopsis (Arabidopsis thaliana) seeds and seedlings through a process that involves LD–LD fusion.

Biochemistry & Molecular Biology↗
DOE OSTI2023

A comparative genome analysis of the Bacillota ( Firmicutes ) class Dehalobacteriia

Dehalobacterium formicoaceticum is recognized for its ability to anaerobically ferment dichloromethane (DCM), and a catabolic model has recently been proposed. D. formicoaceticum is currently the only axenic representative of its class, the Dehalobacteriia, according to the Genome Taxonomy Database. However, substantial additional diversity has been revealed in this lineage through culture-independent exploration of anoxic habitats. Here we performed a comparative analysis of 10 members of the Dehalobacteriia, representing three orders, and infer that anaerobic DCM degradation appears to be a recently acquired trait only present in some members of the order Dehalobacteriales. Inferred traits common to the class include the use of amino acids as carbon and energy sources for growth, energy generation via a remarkable range of putative electron-bifurcating protein complexes and the presence of S-layers. The ability of D. formicoaceticum to grow on serine without DCM was experimentally confirmed and a high abundance of the electron-bifurcating protein complexes and S-layer proteins was noted when this organism was grown on DCM. We suggest that members of the Dehalobacteriia are low-abundance fermentative scavengers in anoxic habitats.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Process for ultra-sensitive quantification of target analytes in complex biological systems

Antibody-free processes are disclosed that provide accurate quantification of a wide variety of low-abundance target analytes in complex samples. The processes can employ high-pressure, high-resolution chromatographic separations for analyte enrichment. Intelligent selection of target fractions may be performed via on-line Selected Reaction Monitoring (SRM) or off-line rapid screening of internal standards. Quantification may be performed on individual or multiplexed fractions. Applications include analyses of, e.g., very low abundance proteins or candidate biomarkers in plasma, cell, or tissue samples without the need for affinity-specific reagents.

Shi, Tujin↗