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Results for “bottom-up proteomics”

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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45 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes ( e . g ., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5–2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure and identification of the native PLP synthase complex from Methanosarcina acetivorans lysate

Many protein-protein interactions behave differently in biochemically purified forms as compared to their in vivo states. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here, we apply the bottom-up structural proteomics method, cryoID , toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through the cryoID approach, we successfully reconstructed and identified the native Methanosarcina acetivorans pyridoxal 5′-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryo-EM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8α/8β TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryo-EM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37-amino-acid insertion present in these prior cases. This study demonstrates the potential of applying the cryoID workflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial.

Methanosarcina acetivorans↗
DOE OSTI2025

MassIVE MSV000095714

Proteomic analysis reveals translation-related proteins are significantly altered in response to stabilized G4s. Identification of the importance of translation factors in overcoming G4s led to the question of how E. coli cells generally respond to chemicals that stabilize such structures. As a first step in addressing this question, a proteomic analysis was carried out to measure the quantitative effects of NMM on the levels of individual proteins in E. coli. Protein levels from early log-phase cultures of delta-tolC and delta-tolC tufA::kan strains grown in the presence or absence of NMM were measured to assess how reduced EF- Tu levels and NMM impacted expression.

Bottom-up proteomics↗
DOE OSTI2022

Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles within the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS) based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q-Exactive HF Orbitrap MS upgraded with ultra-high mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass measurements of proteoforms directly from tissue using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a vastly improved duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. Further, the use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps towards high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI

MALDI imaging allows for the near-cellular profiling of proteoforms directly from microbial, plant, and mammalian samples. Despite detecting hundreds of proteoforms, identification of unknowns with only intact mass information remains a distinct challenge, even with high mass resolving power and mass accuracy. To this end, many supplementary methods have been used to create experimental databases for accurate mass matching, including bulk or spatially resolved bottom-up and/or top-down proteomics. Herein, we describe the application of 193 nm ultraviolet photodissociation (UVPD) for fragmentation of quadrupole isolated singly charged ubiquitin (m/z 8565) by MALDI-UVPD on a UHMR HF Orbitrap. This platform permitted the high-resolution accurate mass measurement of not just terminal fragments but also large internal fragments. Finally, the outlined workflow demonstrates the feasibility of top-down analyses of isolated MALDI protein ions and the potential toward more comprehensive characterization of proteoforms in MALDI imaging applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Spatial Proteomics towards cellular Resolution

Introduction: Spatial biology is an emerging interdisciplinary field facilitating biological discoveries through the use of spatial omics technologies. Recent advancements in spatial transcriptomics, spatial genomics (e.g. genetic mutations and epigenetic marks), multiplexed immunofluorescence, and spatial metabolomics/lipidomics have enabled high-resolution spatial profiling of gene expression, genetic variation, protein expression, and metabolites/lipids profiles in tissue. These developments contribute to a deeper understanding of the spatial organization within tissue microenvironments at the molecular level. Areas covered: This report provides an overview of the untargeted, bottom-up mass spectrometry (MS)-based spatial proteomics workflow. It highlights recent progress in tissue dissection, sample processing, bioinformatics, and liquid chromatography (LC)-MS technologies that are advancing spatial proteomics toward cellular resolution. Expert opinion: The field of untargeted MS-based spatial proteomics is rapidly evolving and holds great promise. To fully realize the potential of spatial proteomics, it is critical to advance data analysis and develop automated and intelligent tissue dissection at the cellular or subcellular level, along with high-throughput LC-MS analyses of thousands of samples. In conclusion, achieving these goals will necessitate significant advancements in tissue dissection technologies, LC-MS instrumentation, and computational tools.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Enhancing Top-Down Proteomics of Brain Tissue with FAIMS

Proteomic investigations of Alzheimer’s and Parkinson’s disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein’s “intact” state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Using a high complexity sample derived from Alzheimer’s disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at -50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 ± 17, n = 3), compared to without (754 ± 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aß) proteoforms, including the aggregation-prone Aß1-42 variant which is strongly linked to Alzheimer’s disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials has potential applications in sensing, catalysis and medicine. Whereas numerous designs have been implemented for the bottom-up construction of novel protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. Here, the site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small-molecule- and protein-functionalized surfaces with no loss in crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces towards the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product’s primary structure reliably and reproducibly. The major objective of the present study was to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up approaches, such as confirming protein integrity, sequencing variable domains, ruling out artefacts, and revealing modifications and their locations. A panel of three mAbs was selected and centrally provided to twenty laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). A variety of different MS instrument platforms and ion dissociation techniques were employed. Overall, the present study confirms that MD/TD MS strategies are valuable and readily available tools in laboratories worldwide. They provide complementary information to the bottom-up approach and can add unique information, which may be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. One of the limitations revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS, but the number of TD/MD MS experts is still very low.

59 BASIC BIOLOGICAL SCIENCES↗