Protein corona: Dr. Jekyll and Mr. Hyde of nanomedicine.

Nanomedicine is an interdisciplinary field of research, comprising science, engineering, and medicine. Many are the clinical applications of nanomedicine, such as molecular imaging, medical diagnostics, targeted therapy, and image-guided surgery. Despite major advances during the past 20 years, many efforts must be done to understand the complex behavior of nanoparticles (NPs) under physiological conditions, the kinetic and thermodynamic principles, involved in the rational design of NP. Once administrated in physiological environment, NPs interact with biomolecules and they are surrounded by protein corona (PC) or biocorona. PC can trigger an immune response, affecting NPs toxicity and targeting capacity. This review aims to provide a detailed description of biocorona and of parameters that are able to control PC formation and composition. Indeed, the review provides an overview about the role of PC in the modulation of both cytotoxicity and immune response as well as in the control of targeting capacity.

Proteomic exploration of soft and hard biocorona onto PEGylated multiwalled carbon nanotubes.

In nanomedicine, carbon nanotubes (CNTs) are considered potential candidates as drug delivery systems. The absorption of proteins onto CNTs, after their administration in physiological environment, forms the protein corona or biocorona, which is able to influence their biological properties and biocompatibility. For this reason, characterization of protein corona is a crucial aspect in the research to control CNTs toxicity and capability to target cells. Multiwalled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG), chosen considering its well-known biocompatibility, and then incubated in human plasma to create the biocorona. Plasma proteins, which bound around PEGylated CNTs, were detached using five different solutions, grouped into native and denaturant buffers, and used to characterize the two components of biocorona. The proteomic fingerprinting of biocorona was performed by SDS-PAGE and 2D-PAGE separation and mass spectrometry analysis. Native eluents were able to capture proteins of soft corona, characterized by complex secondary structures, and formed by both ß-sheet and α-helices domains. Denaturant buffers have eluted many proteins with a high percentage of the α-helix structure that could be involved in specific interactions responsible for the formation of hard corona.

Proteomic fingerprinting of apple fruit, juice, and cider via combinatorial peptide ligand libraries and MS analysis.

Combinatorial peptide ligand libraries coupled to MS was applied to extensively map the proteome of apple fruit, and to detect its presence in commercial apple juice and cider to evaluate their authenticity and genuineness. Using the Uniprot_Malus database, 96 proteins were detected in apples, among which 30 proteins were specifically captured via combinatorial peptide ligand libraries. Next, three proteins, previously recognized in fruits, were found in apple juice, which were involved in cellular metabolism of fruit maturation and in allergenic reactions. On the other hand, only one Malus allergen was identified in cider beads eluate, demonstrating that the industrial processes did not prevent any negative effects in sensitive subjects. Thus, the present study not only increases the knowledge of the apple proteome but also offers a reliable analytical method to assess quality and genuineness of commercial products, which could be also used to inform consumers about the presence of allergens.

Proteomic investigation on bio-corona of functionalized multi-walled carbon nanotubes.

BACKGROUND: The formation of bio-corona, due to adsorption of biomolecules onto carbon nanotubes (CNTs) surface in a physiological environment, may lead to a modified biological "identity" of CNTs, contributing to determination of their biocompatibility and toxicity. METHODS: Multi-walled carbon nanotubes surfaces (f-MWCNTs) were modified attaching acid and basic chemical functions such as carboxyl (MWCNTs-COOH) and ammonium (MWCNTs-N) groups respectively. The investigation of interactions between f-MWCNTs and proteins present in biological fluids, like human plasma, was performed by electrophoretic separation (SDS-PAGE) and mass spectrometry analysis (nLC-MS/MS). RESULTS: A total of 52 validated proteins was identified after incubation of f-MWCNTs in human plasma. 86% of them was present in bio-coronas formed on the surface of all f-MWCNTs and 29% has specifically interacted with only one type of f-MWCNTs. CONCLUSIONS: The evaluation of proteins primary structures, present in all bio-coronas, did not highlight any correlation between the chemical functionalization on MWCNTs and the content of acid, basic and hydrophobic amino acids. Despite this, many proteins of bio-corona, formed on all f-MWCNTs, were involved in the inhibitor activity of serine- or cysteine- endopeptidases, a molecular function completely unrevealed in the human plasma as control. Finally, the interaction with immune system's proteins and apolipoproteins has suggested a possible biocompatibility and a favored bio-distribution of tested f-MWCNTs. GENERAL SIGNIFICANCE: Considering the great potential of CNTs in the nanomedicine, a specific chemical functionalization onto MWCNTs surface could control the protein corona formation and the biocompatibility of nanomaterials.

A sarabande of tropical fruit proteomics: Avocado, banana, and mango.

The present review highlights the progress made in plant proteomics via the introduction of combinatorial peptide ligand libraries (CPLL) for detecting low-abundance species. Thanks to a novel approach to the CPLL methodology, namely, that of performing the capture both under native and denaturing conditions, identifying plant species in the order of thousands, rather than hundreds, is now possible. We report here data on a trio of tropical fruits, namely, banana, avocado, and mango. The first two are classified as "recalcitrant" tissues since minute amounts of proteins (in the order of 1%) are embedded on a very large matrix of plant-specific material (e.g., polysaccharides and other plant polymers). Yet, even under these adverse conditions we could report, in a single sweep, from 1000 to 3000 unique gene products. In the case of mango the investigation has been extended to the peel too, since this skin is popularly used to flavor dishes in Far East cuisine. Even in this tough peel 330 proteins could be identified, whereas in soft peels, such as in lemons, one thousand unique species could be detected.

According to the CPLL proteome sheriffs, not all aperitifs are created equal!

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called "Amaro Branzi", stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products.

Extensive heterogeneity of human urokinase, as detected by two-dimensional mapping.

Urokinase (uPA, urinary plasminogen activator) is a serine protease belonging to the peptidase S1 family. Specifically, uPA cleaves the zymogen plasminogen into the active form (plasmin), which then degrades the fibrin clots. It is widely used as a fibrinolytic agent in thrombolytic therapy and it is also used clinically as a thrombolytic agent. It can be administered to improve the drainage of complicated pleural effusions and empyemas and it is the most effective drug in myocardial infarction. The enzyme was originally identified in human urine for its ability to catalyze the transformation of plasminogen into its active form (plasmin), which degrades fibrin and extracellular matrix components. The present report deals with the analysis and characterization of this preparation.

The peel and pulp of mango fruit: a proteomic samba.

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of mango peel and pulp as well their peptidome content (the latter as captured with a C18 resin). The aim of this study was not only to perform the deepest investigation so far of the mango proteome, but also to assess the potential presence of allergens and of peptides endowed with biological activities. The proteins of peel and pulp have been captured under both native and denaturing extraction techniques. A total of 334 unique protein species have been identified in the peel vs. 2855 in the pulp, via capture with CPLLs at different pH values (2.2 and 7.2).

Artichoke and Cynar liqueur: two (not quite) entangled proteomes.

Combinatorial peptide ligand libraries (CPLLs) have been adopted to investigate the proteome of artichoke extracts, of a home-made alcoholic infusion and of the Italian Cynar liqueur. The aim of study was not only to perform the deepest investigation so far of the artichoke proteome but also to assess the genuineness of the commercial aperitif via a three-pronged attack. First, different extraction techniques have been used for the characterization of the artichoke's proteome, secondly a home-made infusion has been analyzed and finally the proteome of the commercial drink was checked. The artichoke proteome has been evaluated via prior capture with CPLLs at four different pH (2.2, 4.0, 7.2 and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 876 unique gene products in the artichoke extracts, 18 in the home-made infusion and no proteins at all in the Italian Cynar liqueur, casting severe doubts on the procedure stated by the manufacturer (that should be made by an infusion of artichoke leaves plus thirteen different herbs). This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.

Lemon peel and Limoncello liqueur: a proteomic duet.

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of lemon peels and pulp, of a home-made alcoholic infusion of peels and of a very popular Italian liqueur called "Limoncello", stated to be an infusion of the flavedo (the outer, yellow skin of lemons). The aim of this study was not only to perform the deepest investigation so far of the lemon peel proteome but also to assess the genuineness of the commercial liqueur via a three-pronged attack. First, different extraction techniques have been used for the characterization of the peel (and additionally of the pulp) proteome, secondly a home-made infusion has been analysed and finally the proteome of the commercial drink was checked. The peel (the flavedo, not the underlying layer called albedo) proteome has been evaluated via prior capture with CPLLs at different pH values (2.2 and 7.2). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 1011 unique gene products in the peel extracts and 674 in the pulp, 264 proteins in the home-made infusion and just 8 proteins (and protein fragments), together with 12 peptides, in one Italian Limoncello produced in the Sorrento Region, thus proving the genuineness of this product. On the contrary, cheaper Limoncellos were devoid of any protein/peptide, casting doubts on their production from vegetable extracts. This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.

The D-amino acid oxidase-carbon nanotubes: evaluation of cytotoxicity and biocompatibility of a potential anticancer nanosystem.

The 'enzyme prodrug therapy' represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric flavoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing five aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efficacy under low O2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O2 concentration. In order to evaluate the nanocarriers' biocompatibility, PEG-MWCNTs-DAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has influenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic effect controlled by the substrate addition, against different tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug's biodistribution. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03568-1.

Unveiling the Bio-corona Fingerprinting of Potential Anticancer Carbon Nanotubes Coupled with D-Amino Acid Oxidase.

The oxidation therapy, based on the controlled production of Reactive Oxygen Species directly into the tumor site, was introduced as alternative antitumor approach. For this purpose, d-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, an enzyme able to efficiently catalyze the production of hydrogen peroxide from d-amino acids, was adsorbed onto multi-walled carbon nanotubes (MWCNTs), previously functionalized with polylactic-co-glycolic acid (PLGA) or polyethylene glycol (PEG) at different degrees to reduce their toxicity, to be targeted directly into the tumor. In vitro activity and cytotoxicity assays demonstrated that DAAO-functionalized nanotubes (f-MWCNTs) produced H2O2 and induced toxic effects to selected tumor cell lines. After incubation in human plasma, the protein corona was investigated by SDS-PAGE and mass spectrometry analysis. The enzyme nanocarriers generally seemed to favor their biocompatibility, promoting the interaction with dysopsonins. Despite this, PLGA or high degree of PEGylation promoted the adsorption of immunoglobulins with a possible activation of immune response and this effect was probably due to PLGA hydrophobicity and dimensions and to the production of specific antibodies against PEG. In conclusion, the PEGylated MWCNTs at low degree seemed the most biocompatible nanocarrier for adsorbed DAAO, preserving its anticancer activity and forming a bio-corona able to reduce both defensive responses and blood clearance.

Mehercules, adhuc Bacchus! The debate on wine proteomics continues.

The proteome of untreated white wines (a Recioto made with Garganega grapes from the Veneto region) was explored in depth via capture with combinatorial peptide ligand libraries (CPLL) at four different pH values: pH 2.2, 3.8, 7.2, and 9.3. The combined data on the discoveries in the four CPLL eluates, as well as in the collected bottle sediment, allowed the identification of 106 unique gene products belonging to Vitis vinifera, as well as of an additional 11 proteins released by the S. cerevisiae used in the fermentation process. Among the residual grape proteins detected in the Recioto wine, ca. 30% were categorized as medium to high-abundance species, vs 70% low-abundance ones. The detection of so many low-abundance species suggests that proteomic (coupled to peptidomic) data might be used for typing high-quality products (grand crus) to assess their genuineness and protect them from fraudulent imitations.

Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries.

The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.

Combinatorial peptide ligand libraries: the conquest of the 'hidden proteome' advances at great strides.

The combinatorial peptide ligand library (CPLL) is compared here with the immuno-depletion method for evaluating their respective abilities in digging deeper and deeper into the low-abundance proteome. A recent report suggested in fact that immuno-subtraction for biomarkers discovery in sera does not perform so well, since it results in a meagre 25% increase in identified proteins compared with unfractionated plasma, leaving little capacity to sample lower abundance proteins. On the contrary, CPLLs permit from 300 to 600% increments in detection abilities, as amply demonstrated in several reports. Moreover, when dealing with large sample volumes, an amplification factor of up to four orders of magnitude for trace proteins could be demonstrated, with 80% capture efficiencies even in large (up to 1 L) sample volumes. At present, the lower detection ability of CPLLs has been evaluated at 1 ng/mL (traces of casein additives in white wines).

Proteomic fingerprinting of protein corona formed on PEGylated multi-walled carbon nanotubes.

In Nanomedicine, carbon-based nanomaterials, like Carbon Nanotubes (CNT), are considered potential candidates as drug delivery systems. In vivo adsorption of physiological proteins onto carbon nanotubes, through noncovalent interactions, forms a protein corona or bio corona, able to influence biological properties and biocompatibility of CNT. This study aimed to explore the composition of protein corona formed onto PEGylated Multi-Walled Carbon Nanotubes (MWCNT-PEG5k), after their incubation in human plasma. Plasma proteins were sequentially eluted in different conditions by using both native and denaturant buffers, useful to characterize soft and hard corona. Proteomic methods and mass spectrometry analysis have identified proteins in soft corona, involved in the regulation of immune response and in the CNT transport, and biomolecules in hard corona with a role in the maintenance of host homeostasis. These promising results have demonstrated the potential of PEGylated Multi-Walled Carbon Nanotubes as future candidates for drug delivery.

Les Maîtres de l'Orge: the proteome content of your beer mug.

The beer proteome has been evaluated via prior capture with combinatorial peptide ligand libraries (ProteoMiner as well as a homemade library of reduced polydispersity) at three different pH (4.0, 7.0, and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could categorize such species in 20 different barley protein families and 2 maize proteins, the only ones that had survived the brewing process (the most abundant ones being Z-serpins and lipid transfer proteins). In addition to those, we could identify 40 unique gene products from Saccharomyces cerevisiae, one from S. bayanus and one from S. pastorianus as routinely used in the malting process for lager beer. These latter species must represent trace components, as in previous proteome investigations barely two such yeast proteins could be detected. Our protocol permits handling of very large beer volumes (liters, if needed) in a very simple and user-friendly manner and in a much reduced sample handling time. The knowledge of the residual proteome in beers might help brewers in selecting proper proteinaceous components that might enrich beer flavor and texture.

Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes.

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

The proteome buccaneers: how to unearth your treasure chest via combinatorial peptide ligand libraries.

The latest advances in combinatorial peptide ligand libraries, with their unique performance in discovering low-abundance species in proteomes, are reviewed here. Explanations of mechanism, potential applications, capture of proteomes at different pH values to enhance the total catch and quantitative elutions, such as boiling in the presence of 5% sodium dodecyl sulfate and 3% dithiothreitol are included. The reproducibility of protein capture among different experiments with the same batch of beads or with different batches is also reported to be very high, with coefficient of variations in the order of 10-20%. Miniaturized operations, consisting of capture with as little as 20 or even 5 microl of peptide beads are reported, thus demonstrating that the described technology could be exploited for routine biomarker discovery in a biomedical environment. Finally, it is shown that the signal of captured proteins is linear over approximately three orders of magnitude, ranging from nM to microM, thus ensuring that differential quantitative proteomics for biomarker discovery can be fully implemented, providing species do not saturate their ligands.

Influence of tetraconazole on the proteome profile of Saccharomyces cerevisiae Lalvin T73™ strain.

This work aimed to evaluate the modifications on the proteome profile of Saccharomyces cerevisiae T73™ strain as a consequence of its adaptive response to the presence of tetraconazole molecules in the fermentation medium. Pasteurised grape juices were separately supplemented with tetraconazole or a commercial formulation containing 12.5% w/v of tetraconazole at two concentration levels. In addition, experiments without fungicides were developed for comparative purposes. Proteome profiles of yeasts cultured in the presence or absence of fungicide molecules were different. Independently of the fungicide treatment applied, the highest variations concerning the control sample were observed for those proteins involved in metabolic processes, especially in the metabolism of nitrogen compounds. Tetraconazole molecules altered the abundance of several enzymes involved in the biosynthesis of amino acids, purines, and ergosterol. Moreover, differences in the abundance of several enzymes of the TCA cycle were found. Changes observed were different between the active substance and the commercial formulation. SIGNIFICANCE: The presence of fungicide residues in grape juice has direct implications on the development of the aromatic profile of the wine. These alterations could be related to changes in the secondary metabolism of yeasts. However, the molecular mechanisms involved in the response of yeasts to fungicide residues remains quite unexplored. Through this exhaustive proteomic study, alterations in the amino acids biosynthesis pathways due to the presence of the tetraconazole molecules were observed. Amino acids are precursors of some important higher alcohols and ethyl acetates (such as methionol, 2-phenylethanol, isoamyl alcohol or 2-phenylacetate). Besides, the effect of tetraconazole on the ergosterol biosynthesis pathway could be related to a higher production of medium-chain fatty acids and their corresponding ethyl acetates.