Automation and low-cost proteomics for characterization of the protein corona: experimental methods for big data

Poulsen, Karsten M.; Pho, Thomas; Champion, Julie A.; Payne, Christine K.

doi:10.1007/s00216-020-02726-1

Cited by 17 publications

(12 citation statements)

References 64 publications

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“…The low toxicity of these compounds thus favor them over the traditional methyl salicylate and Permount method for insect tissue, with less risk of exposure to either the noxious smelling and toxic methyl salicylate or the toluene in Permount, which must be handled at all times in a fume cupboard. The fact that these clearing media have low toxicity and are safe to use on the open bench makes it easier to think in terms of robotic automation of experimental protocols which is becoming possible even for labs with lower sample output frequency (Savall et al, 2015;Gome et al, 2019;Poulsen et al, 2020).…”

Section: Toxicitymentioning

confidence: 99%

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

et al. 2020

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Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (“MS/P”) with several more recently applied clearing media that offer tunable refractive index (n): 2,2′-thiodiethanol (TDE), “SeeDB2” (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

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Section: Toxicitymentioning

confidence: 99%

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

et al. 2020

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“…Although since this is a commonly used additive in many of the immunohistochemical procedures frequently carried out in insect brain research prior to clearing anyway, it requires no special precautions beyond those generally already employed in most labs. The fact that these clearing media have low toxicity and are safe to use on the open bench makes it easier to think in terms of robotic automation of experimental protocols which is becoming possible even for labs with lower sample output frequency (Savall et al, 2015; Gome et al, 2019; Poulsen et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Comparison of transparency and shrinkage of insect brains using novel and traditional clearing methods

Bekkouche

Fritz

Rigosi

et al. 2020

Preprint

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Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (‘MS/P’) with several more recently applied clearing media that offer tunable refractive index (n): 2,2-thiodiethanol (TDE), ‘SeeDB2’ (in variants SeeDB2S & SeeDB2G matched to oil and glycerol immersion, n=1.52 & 1.47 respectively) and Rapiclear (also with n=1.52 & 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, these newly developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

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“…28,34,38,39 Yet, knowledge of the proteins adsorbed in this corona phase would enable better prediction of the biological identity, and thus fate, of the applied nanotechnologies. 40,41 Experimental testing to fully characterize the protein corona on all synthesized nanoparticle constructs within all intended biological environments is laborious and costly: while recent work has made headway toward high-throughput experimental methods, 42,43 the most common strategies rely on labor-intensive mass spectrometry-based proteomics. 38,44,45 The ability to predict the protein corona that will form on nanoparticles in vivo remains a challenge that, if overcome, would improve applied nanotechnology performance.…”

Section: Introductionmentioning

confidence: 99%

Supervised Learning Model Predicts Protein Adsorption to Carbon Nanotubes

Pinals

Ouassil

Bonis-O’Donnell

et al. 2021

Preprint

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Engineered nanoparticles are advantageous for numerous biotechnology applications, including biomolecular sensing and delivery. However, testing the compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable biofouling often prevents effective implementation. Such biofouling is the result of spontaneous protein adsorption to the nanoparticle surface, forming the "protein corona" and altering the physicochemical properties, and thus intended function, of the nanotechnology. To better apply engineered nanoparticles in biological systems, herein, we develop a random forest classifier (RFC) trained with proteomic mass spectrometry data that identifies which proteins adsorb to nanoparticles. We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)-based optical nanosensor. We optimize the classifier and characterize the classifier performance against other models. To evaluate the predictive power of our model, we then apply the classifier to rapidly identify and experimentally validate proteins with high binding affinity to SWCNTs. Using protein properties based solely on amino acid sequence, we further determine protein features associated with increased likelihood of SWCNT binding: proteins with high content of solvent-exposed glycine residues and non-secondary structure-associated amino acids. Furthermore, proteins with high leucine residue content and beta-sheet-associated amino acids are less likely to form the SWCNT protein corona. The classifier presented herein provides an important tool to undertake the otherwise intractable problem of predicting protein-nanoparticle interactions, which is needed for more rapid and effective translation of nanobiotechnologies from in vitro synthesis to in vivo use.

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Automation and low-cost proteomics for characterization of the protein corona: experimental methods for big data

Cited by 17 publications

References 64 publications

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

Comparison of transparency and shrinkage of insect brains using novel and traditional clearing methods

Supervised Learning Model Predicts Protein Adsorption to Carbon Nanotubes

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