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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Challenges and Opportunities in State‐of‐the‐Art Proteomics Analysis for Biomarker Development From Plasma Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-bound particles secreted by cells, playing crucial roles in intercellular communication. The composition of EVs can undergo changes in response to stress and disease conditions, making them excellent biomarker candidates. However, extracting protein information from EVs can be challenging due to their low abundance in complex biofluids and copurification with contaminant proteins and particles. Techniques to enrich EVs have their strengths and limitations, without one being able to purify EVs to complete homogeneity. This can lead to compromised recovery rates and increased complexity, making data interpretation difficult. In this viewpoint article, we explore the concept that better characterization of EV composition, followed by quantification of EV proteins in complex samples, might be a more viable route for biomarker development. Mass spectrometers can provide reproducible deep coverage of the EV proteome, despite sample impurities. This paradigm shift presents opportunities to integrate advanced bioinformatics tools to refine the EV proteome landscape, identify novel biomarkers, and streamline validation processes in biomarker development. By focusing on leveraging technology rather than achieving absolute purity, this approach can transform current practices and open opportunities for robust biomarker discovery. Herein, we highlight not only such opportunities but also challenges to implement this concept.

Dakup, Panshak P. [Pacific Northwest National Labo↗
DOE OSTI2023

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis

Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producingEscherichia coli(STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Enhanced Spatial Proteomics and Metabolomics from a Single Tissue Section Using MALDI-MSI and LCM-microPOTS Platforms

Spatially resolved mass spectrometry (MS)-based multi-omics workflows are becoming more utilized for revealing the complex biology that occurs within tissues. However, these approaches commonly require multiple independent tissue sections to analyze the metabolite and protein compositions of these samples. This poses a significant challenge in preserving cell- or region-specific molecular fidelity, as variations between tissue sections can compromise the accurate correlation of molecular data. Here, in this study, we developed workflows for comprehensive multi-omics profiling from a single tissue section (STS) using different MS modalities. We enhanced the functionality of an electrically insulated substrate by employing metal-assisted approaches that enabled both MS-based untargeted spatial metabolomics and proteomics from STS. This allowed metabolite imaging using matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI), without compromising it for subsequent proteome profiling with laser capture microdissection (LCM)-based technology. Specifically, implementing copper tape as a backing for polyethylene naphthalate (PEN) slides enabled the detection of >140 metabolites across a poplar root tissue section using MALDI-trapped ion mobility spectrometry time of flight (timsTOF)-MS. Afterwards, we detected 6,571 unique proteins from two distinct root regions by leveraging LCM technology coupled to our microdroplet based sample preparation approach. We also developed an alternative workflow utilizing gold-coated PEN substrates for imaging with MALDI-Fourier-transform ion cyclotron resonance (FTICR)-MS, which permitted the profiling of >170 metabolites and the identification of 6,542 unique proteins across a single poplar root tissue section. These results were comparable to using each assay independently without modifications. These approaches offer new opportunities for high-resolution molecular profiling of multiple omics-levels across biological tissues.

Veličković, Marija [Pacific Northwest National Lab↗
DOE OSTI2021

PSpecteR: A User-Friendly and Interactive Application for Visualizing Top-Down and Bottom-Up Proteomics Data in R

Visual examination of mass spectrometry data is necessary to assess data quality and to facilitate data exploration. Graphics provide the means to evaluate spectral properties, test alternative peptide/protein sequence matches, prepare annotated spectra for publication, and fine-tune parameters during wet lab procedures. Visual inspection of MS data is hindered by proprietary proteomics visualization software designed for particular workflows and academic software that lack visualization tools. We built PSpecteR, an open-source and interactive R Shiny web application to address these issues, with support for several steps of proteomics data processing, including: reading various mass spectrometry files, running open-source database search tools, labelling spectra with fragmentation patterns, testing post-translational modifications, plotting where identified fragments map to reference sequences, and visualizing algorithmic output and metadata. All figures, tables, and spectra are exportable within one easy-to-use graphical user interface. Our current software provides a flexible and modern R framework to support fast implementation of additional features. The open source code is readily available (https://github.com/EMSL-Computing/PSpecteR), and a PSpecteR Docker container (https://hub.docker.com/r/emslcomputing) is available for easy local installation.

96 KNOWLEDGE MANAGEMENT AND PRESERVATION↗
DOE OSTI2024

How Does Escherichia coli Allocate Proteome?

Microorganisms are shown to actively partition their intracellular resources, such as pro- teins, for growth optimization. Recent experiments have begun to reveal molecular com- ponents unpinning the partition; however, it remains unclear quantitatively how individual parts orchestrate to yield precise resource allocation that is both robust and dynamic. Here we developed a coarse-grained mathematical framework that centers on guanosine pentaphosphate (ppGpp)-mediated regulation, and used it to systematically uncover the design principles of proteome allocation in Escherichia coli. Our results showed that cellular ability of resource partition lies in an ultrasensitive, negative feedback control- ling topology with the ultrasensitivity arising from zero-order amino acid kinetics and the negative feedback from ppGpp-controlled ribosome synthesis. In addition, together with the time-scale separation between slow ribosome kinetics and fast turnovers of ppGpp and amino acids, the network topology confers the organism an optimization mechanism which mimics sliding mode control, a nonlinear optimization strategy that is widely used in man-made systems. We further showed that such a controlling mechanism is robust against parameter variations and molecular fluctuations, and is also efficient for biomass production over time. Furthermore, this work elucidates the fundamental controlling mechanism of E. coli proteome allocation, thereby providing insights into quantitative microbial physiology as well as the design of synthetic gene networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295 T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved p CO 2 , and requires exogenous vitamin B 12 . In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (~5.6 µM) O 2 concentrations. The abundance of a potentially more efficient CO 2 -fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb 3 -type terminal oxidase increased under O 2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O 2 -sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O 2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Model of metabolism and gene expression predicts proteome allocation in Pseudomonas putida

Abstract The genome-scale model of metabolism and gene expression (ME-model) forPseudomonas putidaKT2440,iPpu1676-ME, provides a comprehensive representation of biosynthetic costs and proteome allocation. Compared to a metabolic-only model,iPpu1676-ME significantly expands on gene expression, macromolecular assembly, and cofactor utilization, enabling accurate growth predictions without additional constraints. Multi-omics analysis using RNA sequencing and ribosomal profiling data revealed translational prioritization inP. putida, with core pathways, such as nicotinamide biosynthesis and queuosine metabolism, exhibiting higher translational efficiency, while secondary pathways displayed lower priority. Notably, the ME-model significantly outperformed the M-model in alignment with multi-omics data, thereby validating its predictive capacity. Thus,iPpu1676-ME offers valuable insights intoP. putida’s proteome allocation and presents a powerful tool for understanding resource allocation in this industrially relevant microorganism.

Mathematical & Computational Biology↗
DOE OSTI2023

A comprehensive spectral assay library to quantify the Halobacterium salinarum NRC-1 proteome by DIA/SWATH-MS

Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requires a priori generated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish, E. coli and few other organisms. However, a spectral assay library for the extreme halophilic archaeon Halobacterium salinarum NRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotated H. salinarum NRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. The H. salinarum NRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon. Data and spectral assay libraries are available via ProteomeXchange (PXD042770, PXD042774) and SWATHAtlas (SAL00312-SAL00319).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Top-down proteomics

Proteoforms arising from posttranslational modifications, genetic polymorphisms, and RNA splice variants, play a pivotal role as the key drivers in biology. Thus, a comprehensive understanding of proteoforms is essential for unraveling the intricacies of biological systems and bridging the gap between genotype and phenotype. By analyzing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and presents a next-generation approach for deciphering protein function, uncovering disease mechanisms, and advancing precision medicine. This Primer embarks on a journey into the world of TDP by encapsulating its historical context, underlying principles, recent advances, and an outlook on the future of TDP. The experimental section navigates instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques, and data collection. Results decipher raw data, visualize intact protein spectra, unravel data analysis, and explain proteoform identification, characterization, and quantitation, as well as statistical analysis. Various applications of TDP spanning the human proteoform project, biomedical, biopharmaceutical, and clinical applications are described. These are complemented by discussions on measurement reproducibility, limitations, and a forward-looking perspective outlining uncharted waters where the field can advance, and potential exciting future applications of TDP.

Roberts, David S.↗
DOE OSTI2020

Potential pathogenicity determinants identified from structural proteomics of SARS-CoV and SARS-CoV-2

Despite SARS-CoV and SARS-CoV-2 being equipped with highly similar protein arsenals, the corresponding zoonoses have spread among humans at extremely different rates. The specific characteristics of these viruses that led to such distinct outcomes remain unclear. Here, we apply proteome-wide comparative structural analysis aiming to identify the unique molecular elements in the SARS-CoV-2 proteome that may explain the differing consequences. By combining protein modeling and molecular dynamics simulations, we suggest non-conservative substitutions in functional regions of the spike glycoprotein (S), nsp1, and nsp3 that are contributing to differences in virulence. Particularly, we explain why the substitutions at the receptor-binding domain of S affect the structure-dynamics behavior in complexes with putative host receptors. Conservation of functional protein regions within the two taxa is also noteworthy. We suggest that the highly conserved main protease, nsp5, of SARS-CoV and SARS-CoV-2 is part of their mechanism of circumventing the host interferon antiviral response. Overall, most substitutions occur on the protein surfaces and may be modulating their antigenic properties and interactions with other macromolecules. Our results imply that the striking difference in the pervasiveness of SARS-CoV-2 and SARS-CoV among humans seems to significantly derive from molecular features that modulate the efficiency of viral particles in entering the host cells and blocking the host immune response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved

Abstract Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Top-Down Proteomics of Mouse Islets With Beta Cell CPE Deletion Reveals Molecular Details in Prohormone Processing

Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a β-cell-specific Cpe knockout mouse model. Increases in dibasic residue–containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural models and functional annotations for the Sphagnum divinum proteome

This dataset contains the structural models for the primary transcripts of the Sphagnum divinum proteome. Additionally, for a subset of these proteins, sequence and structural alignment results are provided. This dataset represents the most thorough structural study of a Sphagnum species, also known as peat mosses, by providing three-dimensional atomic resolution structures of the majority of the encoded proteins as well as structural alignment results used in the application of annotating the proteome. References (DOI) AlphaFold v2 Monomer: https://doi.org/10.1038/s41586-021-03819-2. References (DOI) US-align2: https://doi.org/10.1038/s41592-022-01585-1

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural Models and Sequence Alignment Results of the Rhodospirillum rubrum Proteome

This dataset contains the structural models for the primary transcripts of the Rhodospirillum rubrum proteome as well as sequence alignment results for a subset of the encoded proteins. For each protein, the five models inferred from AlphaFold 2 are provided. The largest pTM-scoring model for each protein was energy minimized; this minimized structure as well as its AlphaFold pickle output file are also provided. This set of structures represent an alternate source of models for the R. rubrum proteome to those available in the AlphaFold Protein Structure Database. For proteins that have been annotated as hypothetical, sequence alignment results from the HHblits and SAdLSA alignment methods are provided. These methods are often more capable to resolve sequence homology than other methods. Therefore, the results from both HHblits and SAdLSA are provided to identify possible homologs for these challenging proteins. Numerous sequence databases are utilized for these alignments. References AlphaFold v2 Multimer: https://doi.org/10.1101/2021.10.04.463034. References HHBlits: https://doi.org/10.1186/s12859-019-3019-7. References SAdLSA: https://doi.org/10.3389/fbinf.2021.689960.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural Models of the Rhodopseudomonas palustris Proteome

This dataset contains the structural models for the primary transcripts of the Rhodopseudomonas palustris proteome. For each protein, the five models inferred from AlphaFold 2 are provided. The largest pTM-scoring model for each protein was energy minimized; this minimized structure as well as its AlphaFold pickle output file are also provided. This set of structures represent an alternate source of models for the R. palustris proteome to those available in the AlphaFold Protein Structure Database.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region

Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments. We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways. We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Device and Method for Parallel Measurement of Phosphoproteome and Proteome from Single Cells

We present the development of an immobilized metal affinity chromatography (IMAC) chip designed to enable nanoscale phosphopeptide enrichment within microfabricated nanowells. This novel platform leverages surface chemistry to immobilize high-density Nickel-Nitrilotriacetic Acid (Ni-NTA) molecules on nanowells, followed by applying Fe 3+ . The nanowell surface serves as a capture media to enrich phosphopeptides based on IMAC. The system's efficiency was validated using ß-casein as a model protein, demonstrating the chip’s capability to significantly enrich phosphopeptides. Future applications of this technology are anticipated to enable the detection of over 100 phosphopeptides from individual cells and more than 500 phosphopeptides from pools of 100 cells, offering exciting potential for single-cell phosphoproteomics. We will next apply an integrated proteomics workflow to perform multi-omics measurements, including single-cell isolation, protein digestion, and phosphopeptide enrichment, followed by LC-MS analysis of both the global proteome and phosphoproteome. Future research will explore the use of this technology to study phosphorylation dynamics in cancer cells, enhancing our understanding of cellular signaling and disease mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Microbial Co-Culture Control Proteomics (MC-DP1)

Co-cultured S. elongatus PCC 7942 CscB/SPS and R. toruloides IFO0880 presented as an effective photosynthesis-driven biofuel production platform. The goal of this experiment was to understand the molecular mechanism of this co-culture system at redox post-translational modification level. The redox proteome of the co-cultured strains was compared to a mono-cultured S. elongatus in light or dark conditions. Samples were processed using a resin-assisted capture (RAC) workflow with TMT labeling to enrich and quantify modified cysteines at proteome level. The datasets were generated by a Q Exactive Plus Orbitrap Mass Spectrometer coupled with a Waters nanoAcquity UPLC, then searched by MSGF+ for downstream redox PTM analysis.

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