RESUMEN
Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.
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Anticuerpos , Recursos Comunitarios , Humanos , Reproducibilidad de los Resultados , Diagnóstico por ImagenRESUMEN
We introduce single cell Proteoform imaging Mass Spectrometry (scPiMS), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new scPiMS platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage.
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Espectrometría de Masas , Proteómica , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Proteómica/métodos , Humanos , Espectrometría de Masas/métodos , Proteoma/análisisRESUMEN
19F parashift probes with paramagnetically shifted reporter nuclei provide attractive platforms to develop molecular imaging probes. These probes enable ratiometric detection of molecular disease markers using a direct detection technique. Here, we describe a series of trivalent lanthanide (Ln(III)) complexes that are structural analogues of the clinically approved MR contrast agent (CA) ProHance to obtain LnL 19F parashift probes. We evaluated trans-gadolinium paramagnetic lanthanides compared to diamagnetic YL for 19F chemical shift and relaxation rate enhancement. The paramagnetic contribution to chemical shift (δPCS) for paramagnetic LnL exhibited either shifts to lower frequency (δPCS < 0 for TbL, DyL, and HoL) or shifts to higher frequency (δPCS > 0 for ErL, TmL, and YbL) compared to YL 19F spectroscopic signal. Zero-echo time pulse sequences achieved 56-fold sensitivity enhancement for DyL over YL, while developing probe-specific pulse sequences with fast delay times and acquisition times achieved 0.6-fold enhancement in limit of detection for DyL. DyL provides an attractive platform to develop 19F parashift probes for ratiometric detection of enzymatic activity.
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Elementos de la Serie de los Lantanoides , Elementos de la Serie de los Lantanoides/química , Estructura Molecular , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Imagen por Resonancia Magnética , Medios de Contraste/química , Flúor/química , HumanosRESUMEN
ProGlo is an efficient steroid receptor-targeted magnetic resonance (MR) imaging contrast agent (CA). It has been shown to bind to the progesterone receptor (PR) and produce enhanced image contrast in PR-positive cells and tissues in vitro and in vivo. However, the hydrophobicity of the steroid targeting domain of ProGlo (logP = 1.4) limits its formulation and delivery at clinically relevant doses. In this work, a hydrophobic moiety was utilized to drive efficient adsorption onto nanodiamond (ND) clusters to form a water-soluble nanoconstruct (logP = -2.4) with 80% release in 8 h under biological conditions. In cell culture, the ND-ProGlo construct delivered increased concentrations of ProGlo to target cells compared to ProGlo alone. Importantly, these results were accomplished without the use of solvents such as DMSO, providing a significant advance toward formulating ProGlo for translational applications. Biodistribution studies confirm the delivery of ProGlo to PR(+) tissues with enhanced efficacy over untargeted controls. These results demonstrate the potential for a noncovalent ND-CA construct as a general strategy for solubilizing and delivering hydrophobic targeted MR CAs.
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Neoplasias de la Mama/patología , Medios de Contraste/farmacocinética , Imagen por Resonancia Magnética/métodos , Nanoconjugados/química , Nanodiamantes/administración & dosificación , Receptores de Progesterona/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Medios de Contraste/química , Femenino , Humanos , Ratones , Nanodiamantes/química , Receptores de Progesterona/química , Solubilidad , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between â¼1 µg/mL and â¼10 pg/mL in absolute abundance, providing up to 10 5 -fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 µg/mL) were detected. The ability to monitor medium and some low abundant proteoforms through reproducible enrichment significantly extends the applicability of proteoform research by adding depth beyond albumin, immunoglobins and apolipoproteins to uncover many involved in immunity and cell signaling. As proteoforms carry unique information content relative to peptides, this report opens the door to deeper proteoform sequencing in clinical proteomics of disease or aging cohorts.
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Diagnostic medical imaging utilizes magnetic resonance (MR) to provide anatomical, functional, and molecular information in a single scan. Nanoparticles are often labeled with Gd(III) complexes to amplify the MR signal of contrast agents (CAs) with large payloads and high proton relaxation efficiencies (relaxivity, r1). This study examined the MR performance of two structurally unique cages, AaLS-13 and OP, labeled with Gd(III). The cages have characteristics relevant for the development of theranostic platforms, including (i) well-defined structure, symmetry, and size; (ii) the amenability to extensive engineering; (iii) the adjustable loading of therapeutically relevant cargo molecules; (iv) high physical stability; and (v) facile manufacturing by microbial fermentation. The resulting conjugates showed significantly enhanced proton relaxivity (r1 = 11-18 mM-1 s-1 at 1.4 T) compared to the Gd(III) complex alone (r1 = 4 mM-1 s-1). Serum phantom images revealed 107% and 57% contrast enhancements for Gd(III)-labeled AaLS-13 and OP cages, respectively. Moreover, proton nuclear magnetic relaxation dispersion (1H NMRD) profiles showed maximum relaxivity values of 50 mM-1 s-1. Best-fit analyses of the 1H NMRD profiles attributed the high relaxivity of the Gd(III)-labeled cages to the slow molecular tumbling of the conjugates and restricted local motion of the conjugated Gd(III) complex.
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Nanopartículas , Protones , Medios de Contraste/química , Gadolinio/química , Imagen por Resonancia Magnética/métodosRESUMEN
Differences in tissue pH can be diagnostic of cancer and other conditions that shift cell metabolism. Paramagnetic probes are promising tools for pH mapping in vivo using magnetic resonance spectroscopy (MRS) as they provide uniquely shifted MR signals that change with pH. Here, we demonstrate a 3-hydroxy-6-methylpyridyl coordinating group as a new pH-responsive reporter group for Ln(III) MRS probes. The pH response of the complex was observed by UV-Vis, fluorescence, and NMR spectroscopies, and modeled using DFT. These results provide insight into the observed pH-dependent NMR spectrum of the complex. The protonation state of the hydroxypyridine changes the coordinating ability of the ligand, affecting the dipolar field of the lanthanide and the chemical shift of nearby reporter nuclei. The favorable pH response and coordination properties of the 3-hydroxypyridyl group indicates its potential for further development as a dual responsive-reporter group. Incorporation into optimized scaffolds for MRS detection may enable sensitive pH-mapping in vivo.