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Unbiased identification of the liposome protein corona using photoaffinity based chemoproteomics

ABSTRACT: Determination of protein adsorption to the surface of a nanoparticle determined by photoaffinity based chemoproteomics.

INSTRUMENT(S): MALDI Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

SUBMITTER: Roy Pattipeiluhu

LAB HEAD: Alexander Kros

PROVIDER: PXD016229 | Pride | 2020-04-02

REPOSITORIES: Pride

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	20190311_RP_Amin_02.zip.raw	Raw
	20190311_RP_Amin_03.zip.raw	Raw
	20190311_RP_Amin_04.zip.raw	Raw
	20190311_RP_Amin_05.zip.raw	Raw
	20190311_RP_Amin_06.zip.raw	Raw

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Publications

Unbiased Identification of the Liposome Protein Corona using Photoaffinity-based Chemoproteomics.

Pattipeiluhu Roy R Crielaard Stefan S Klein-Schiphorst Iris I Florea Bogdan I BI Kros Alexander A Campbell Frederick F

ACS central science 20200401 4

Protein adsorption to the surface of a nanoparticle can fundamentally alter the character, behavior, and fate of a nanoparticle in vivo. Current methods to capture the protein corona rely on physical separation techniques and are unable to resolve key, individual protein-nanoparticle interactions. As a result, the precise link between the "synthetic" and the "biological" identity of a nanoparticle remains unclear. Herein, we report an unbiased photoaffinity-based approach to capture, characteriz ...[more]

PMID: 32342003

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Unbiased Identification of the Liposome Protein Corona using Photoaffinity-based Chemoproteomics.

Project description:Protein adsorption to the surface of a nanoparticle can fundamentally alter the character, behavior, and fate of a nanoparticle in vivo. Current methods to capture the protein corona rely on physical separation techniques and are unable to resolve key, individual protein-nanoparticle interactions. As a result, the precise link between the "synthetic" and the "biological" identity of a nanoparticle remains unclear. Herein, we report an unbiased photoaffinity-based approach to capture, characterize, and quantify the protein corona of liposomes in their native state. Compared to conventional methods, our photoaffinity approach reveals markedly different interacting proteins as well as reduced total protein binding to liposome surfaces. Identified proteins do not follow protein abundancy patterns of human serum, as has been generally reported, but are instead dominated by soluble apolipoproteins-endogenous serum proteins that have evolved to recognize the lipidic surface of circulating lipoproteins. We believe our findings are the most accurate characterization of a liposome's biological identity but, more fundamentally, reveal liposome-protein binding is, in many cases, significantly less complex than previously thought.

| S-EPMC7181318 | biostudies-literature

photoaffinity for Puerarin and mouse brainstem

Project description:To elucidate the molecular mechanism mediating the inactivated effect of DMV neurons on fat absorption, we performed an activity-based protein profiling strategy, using Puerarin as a “bait”. The Puerarin-tag probe was synthesized with a photoreactive tag to enrich and visualize target proteins via a photoaffinity chemistry reaction. We verified that probe-tagged Puerarin retains the same effects of increasing fecal lipid excretion as non-tagged Puerarin. Probe-tagged Puerarin was added to the freshly isolated brainstem sample, and 10 doses of non-tagged Puerarin was used as a competitor of probe-tagged Puerarin. Following the photoaffinity reaction, targeted proteins were subsequently assessed by liquid chromatography tandem mass spectrometry (LC-MS).

2024-09-06 | PXD052140 | Pride

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

Project description:RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enables the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.

2020-08-17 | GSE156312 | GEO

Tuning Liposome Composition to Modulate the Corona Forming in Human Serum and Uptake by Cells

Project description:Nano-sized objects such as liposomes are modified by adsorption of biomolecules in biological fluids. The resulting corona critically changes nanoparticle behavior at cellular level. A better control of corona composition could allow to modulate uptake by cells. Within this context, in this work, liposomes of different charge were prepared by mixing negatively charged and zwitterionic lipids to different ratios. The series obtained was used as a model system with tailored surface properties to modulate corona composition and determine the effects on liposome interactions with cells. Uptake efficiency and uptake kinetics of the different liposomes were determined by flow cytometry and fluorescence imaging. Particular care was taken in optimizing the methods to isolate the corona forming in human serum to prevent liposome agglomeration and to exclude residual free proteins which could confuse the results. Thanks to the optimized methods, mass spectrometry of replicate corona isolations showed excellent reproducibility and this allowed semi-quantitative analysis to determine for each formulation the most abundant proteins in the corona. The results showed that by changing the fraction of zwitterionic and charged lipids in the bilayer, the amount and identity of the most abundant proteins adsorbed from serum differed. Interestingly, the formulations also showed very different uptake kinetics. Similar approaches can be used to tune lipid composition in a systematic way in order to obtain formulations with the desired corona and cell uptake behavior.

2020-02-28 | PXD017776 | Pride

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

Project description:PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

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Proteome selectivity profiling of photoaffinity probes derived from imidazopyrazine-kinase inhibitors

Project description:Kinases are attractive drug targets, but the design of highly selective kinase inhibitors remains challenging. Selectivity may be evaluated against a panel of kinases, or – preferred – in a complex proteome. Probes that allow photoaffinity-labeling of their targets can facilitate this process. Here, we report photoaffinity probes based on the imidazopyrazine scaffold, which is found in several kinase inhibitors and drugs or drug candidates. By chemical proteomics experiments, we find a range of off-targets, which vary between the different probes.

2024-10-28 | PXD053278 | Pride

Development of nucleus-targeted synthetic histone photoaffinity probes

Project description:Dissection of the physiological interactomes of histone post-translational modifications (hPTMs) is crucial for understanding fundamental epigenetic regulatory pathways. Current peptide- or protein-based histone photoaffinity probes have greatly expanded the ability to probe the epigenetic interactome, but in situ profiling in the context of native cells (live cells without genetic manipulations) remains challenging. Here, we developed a nucleus-targeted histone photoaffinity probe with a modular design that integrates a cell-permeable and nuclear localization peptide modules into an hPTM peptide equipped with a photoreactive moiety for profiling the hPTM-mediated interactome in native cells. This probe not only enables the probing of epigenetic interactomes in hard-to-transfect macrophage RAW264.7 cells, but also offers opportunities to characterize the interactome of newly emerging hPTM, such as lactylation, thus expanding the toolbox for profiling of hPTM-mediated PPIs in live cells.

2024-11-21 | PXD045620 | Pride

Design and evaluation of a cyclobutane diazirine alkyne tag for photoaffinity labeling in cells

Project description:Alkyl diazirines are frequently used in photoaffinity labeling to trap and identify small molecule–protein interactions. How-ever, the alkyl diazirine can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a long-lived alkyl diazo intermediate. To alter this reactivity preference, we developed PALBOX, a novel cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity. We show that PALBOX is readily derivatized and attached to small molecules to profile their binding interactions in cells. Systematic experiments with biological sub-strates show that the cyclobutane diazirine scaffold does not exhibit reactivity characteristic of unconstrained alkyl diazo in-termediates. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label known protein targets in cells with higher specificity. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule–protein binding sites. Thus, PALBOX is a versatile diazirine-based pho-toaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule–protein interactions.

2024-01-04 | PXD035954 | Pride

Activity-based protein profiling in human M2 macrophages using DHA-based photoaffinity probes

Project description:17-Hydroxydocosahexaenoic acid (17-HDHA), an oxidative metabolite of the fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3), is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of 17-HDHA are poorly understood. Here, we report the design, synthesis and application of a complementary pair of bioorthogonal photoreactive probes based on the polyunsaturated scaffold of 17-HDHA and DHA. In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific protein interaction partners of 17-HDHA in primary human macrophages, which led to the identification of prostaglandin reductase 1 (PTGR1) as a target of 17-HDHA, but not DHA. Ensuing biochemical studies revealed that PTGR1 converted 17-HDHA into 17-oxo-DHA, which inhibited the biosynthesis of the pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils.

2023-04-28 | PXD027600 | Pride

Peptidoglycan metabolite photoaffinity reporters reveal direct binding to intracellular pattern recognition receptors and Arf GTPases

Project description:Extensive structure-activity studies of iE-DAP and MDP have demonstrated their selective activation of NOD1- and NOD2-expressing cells, respectively. Nonetheless, the direct binding of iE-DAP and MDP to NOD1 and NOD2, respectively, as well as other proteins in mammalian cells has not been systematically investigated. To address the direct interaction of iE-DAP and MDP with NOD1 and NOD2 in living cells, we synthesized a series of peptidoglycan metabolite photoaffinity chemical reporters containing a diazirine for photocrosslinking in cells and an alkyne tag for bioorthogonal detection of covalently-labeled proteins. The analysis of these peptidoglycan metabolite photoaffinity reporters demonstrate that iE-DAP and MDP can directly bind to NOD1 and NOD2 in mammalian cells. The crosslinking of these peptidoglycan metabolite photoaffinity reporters only occur with active variants of NOD1 and NOD2. Beyond direct binding to NOD2, the active MDP photoaffinity reporter selectively crosslinked Arf GTPases in NOD2-expressing cells and did so most prominently with GTP-bound Arf6, revealing the Arf proteins as novel direct pattern recognition receptors. Additional labeling and co-precipitation experiments suggest that MDP induces a complex between NOD2 and GTP-Arf6. This study highlights the utility of peptidoglycan metabolite photoaffinity reporters for characterizing their direct binding to intracellular pattern recognition receptors and identifying unpredicted cellular cofactors. The applications of these microbial-specific metabolite reporters should enable the discovery of other receptors, transporters and regulatory enzymes in host cells and microbes.

2020-05-26 | PXD012350 | Pride

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