Proteomics For Studying Protein Coronas Of Nanoparticles

Questo libro ha vinto il Premio Tesi di Dottorato istituito dalla Sapienza Università di Roma, nell’edizione 2013. In nanomedicina, la terapia genica rappresenta un fronte di ricerca promettente. Le limitazioni e i rischi associati ai vettori virali hanno stimolato lo sviluppo di vettori alternativi, in particolare i liposomi cationici. Al momento della somministrazione, l’organismo reagisce formando una “corona proteica”, uno strato di proteine la cui composizione dipende dalle dimensioni dei liposomi e dalle loro caratteristiche chimico-fisiche. Si tratta di un evento critico per la biodistribuzione in vivo, nonché per l’attivazione della risposta del sistema del reticolo-endoteliale, che regola la rimozione dall’organismo. Lo scopo del lavoro è stato quello di identificare e comparare, mediante studi proteomici basati sull’utilizzo di sistemi cromatografici e mass-spettrometrici ad elevata risoluzione, le proteine plasmatiche adsorbite su varie tipologie di liposomi, al fine di caratterizzarne le differenze, comprendere l’impatto biologico dei vettori e migliorare la loro progettazione per aumentare l’efficienza di trasfezione di sistemi già esistenti e poterne ingegnerizzare di nuovi.

Nanoparticles have numerous biomedical applications including drug delivery, bone implants and imaging. A protein corona is formed when proteins existing in a biological system cover the nanoparticle surface. The formation of a nanoparticle–protein corona, changes the behaviour of the nanoparticle, resulting in new biological characteristics and influencing the circulation lifetime, accumulation, toxicity, cellular uptake and agglomeration. This book provides a detailed understanding of nanoparticle–protein corona formation, its biological significance and the factors that govern the formation of coronas. It also explains the impact of nanoparticle–protein interactions on biological assays, ecotoxicity studies and proteomics research. It will be of interest to researchers studying the application of nanoparticles as well as toxicologists and pharmaceutical chemists.

In recent years, the fabrication of nanomaterials and exploration of their properties have attracted the attention of various scientific disciplines such as biology, physics, chemistry, and engineering. Although nanoparticulate systems are of significant interest in various scientific and technological areas, there is little known about the safety of these nanoscale objects. It has now been established that the surfaces of nanoparticles are immediately covered by biomolecules (e.g. proteins, ions, and enzymes) upon their entrance into a biological medium. This interaction with the biological medium modulates the surface of the nanoparticles, conferring a “biological identity” to their surfaces (referred to as a “corona”), which determines the subsequent cellular/tissue responses. The new interface between the nanoparticles and the biological medium/proteins, called “bio-nano interface,” has been very rarely studied in detail to date, though the interest in this topic is rapidly growing. In this book, the importance of the physiochemical characteristics of nanoparticles for the properties of the protein corona is discussed in detail, followed by comprehensive descriptions of the methods for assessing the protein-nanoparticle interactions. The advantages and limitations of available corona evaluation methods (e.g. spectroscopy methods, mass spectrometry, nuclear magnetic resonance, electron microscopy, X-ray crystallography, and differential centrifugal sedimentation) are examined in detail, followed by a discussion of the possibilities for enhancing the current methods and a call for new techniques. Moreover, the advantages and disadvantages of protein-nanoparticle interaction phenomena are explored and discussed, with a focus on the biological impacts.

Proteomics is a study which takes the existence and activities of all proteins in cells as the research object for a more in-depth understanding of the laws of life activity. Since the concept of proteome was proposed in 1995, proteomics has achieved rapid development during the past decades. As a significant part of post-genomics, proteomics has strengthened its position and also become the forefront of scientific research in this century. Magnetic nanomaterials in proteomics analysis is an interdisciplinary field combining magnetic nanomaterials science and proteomics. From the current research, magnetic nanomaterials have shown great potential in complex proteomics research.This book focuses on the application of magnetic nanomaterials in the forefront of proteomics research, and describes in details the synthetic methods, properties, principles and performance of magnetic nanomaterials in various branches of proteomics, which includes digestion studies, enrichment of low abundance peptides, as well as the analysis of phosphoprotein and glycoprotein. Past 10 years' research on magnetic materials in proteomics is integrated, and then the application of magnetic nanomaterials in proteomics analysis are stated systematically.This book can serve as reference book for teaching in the major of proteomics analysis, and also can be reference book for researchers who are studying materials for proteomics.

In this special volume on polymer particles, recent trends and developments in the synthesis of nano- to micron-sized polymer particles by radical polymerization (Emulsion, Miniemulsion, Microemulsion, and Dispersion Polymerizations) of vinyl monomers in environmentally friendly heterogeneous aqueous and supercritical carbon dioxide fluid media are reviewed by prominent worldwide researchers. In addition to the important challenges and possibilities with regards to design and preparation of functionalized polymer particles of controlled size, the topics described are of great current interest due to the increased awareness of environmental issues.

As two relatively new fields of study, proteomics and nanotechnology have developed in parallel with each other to allow an increased precision in the identification of post-translational protein modifications as well as to provide a more automated isolation and detection of rare proteins in both serum and tissues. The Nanoproteomics: Methods and Protocols volume organizes and collects technical advances from leaders in the field to make laboratory protocols more readily available and understandable to those who are attempting to incorporate nanotechnologic techniques into their proteomic research. Conveniently divided into five sections, this detailed volume covers preliminary sample preparation, nanoscale fluidic devices and methods, nanostructured surfaces and nanomaterials, and nanoproteomic techniques to detect and understand protein and proteomic alterations specific to human pathology. Written in the highly successful series entitled Methods in Molecular BiologyTM, these chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols that are readily reproducible, and tips on troubleshooting and avoiding known pitfalls. Convenient and authoritative, Nanoproteomics: Methods and Protocols offers key procedures that are culled from the laboratories of leaders in the field of nanoproteomics with the aim of helping researchers in their standardization and proliferation of protocols that will lead to a more wide scale adoption and smoother progress in this vital field.

Proteomics is evolving as a multi-faceted tool for addressing various biochemical and biomedical queries in the field of scientific research. This involves various stages, ranging from sample preparation to data analysis and biological interpretation. Sample preparation involves isolating proteins from the sample source, purifying and digesting them to initiate shotgun proteomics. Shotgun proteomics identifies proteins by bottom-up proteomic approaches where proteins are identified from the fragmentation spectra of their own peptides. Paper I: deals with the simplification of functional characterization for nanoparticles intended for use in biomedicine. Proteomics was constructive in differentiating and semi-quantifying the surface of protein corona. This could be beneficial in predicting the interactions between nanoparticles and a biological entity like the cell or a receptor protein and provide initial valuable information related to targeting, uptake and safety. Paper II: deals with understanding effects of TiO2 nanoparticles on endothelial cells. A combinatorial approach, involving transcriptomics and proteomics was used to identify aberrations in the permeability and integrity of endothelial cells and tissues. Our study also investigated the correlation of size and how they motivated a differential cellular response. In case of intravenous entry for nanoparticles in targeted drug delivery systems, endothelial cells are the first barrier encountered by these drug carriers. This evaluation involving endothelial cell response could be very instrumental during the designing of NP based drug delivery systems. Paper III: Pharmaceuticals and its metabolites could be very hazardous, especially if its disposal is not managed properly. Since water bodies are the ultimate sink, these chemicals could end up there, culminating in toxicity and other ‘mixture effects’ in combination with other factors. To evaluate the effects of the pharmaceutical, propranolol and climatic factors like low salinity conditions, a microcosm exposure was designed and shotgun proteomics helped understand its impact on mussel gills. In this study too, a combination of transcriptomics and proteomics unveiled molecular mechanisms altered in response to stressors, both individually and in combination. Paper IV: An interplay of various factors like EBF1 and PAX5 determines B-cell lineage and commitment. This might have been materialized by direct and transient proteinprotein interactions. A unique method called BioID helped screen relevant interactions in living cells by the application of a promiscuous biotin ligase enzyme capable of tagging proteins through biotinylation based on a proximity radius. Biotinylation of endogenous proteins enabled their selective isolation by exploiting the high affinity of biotin and streptavidin on streptavidin coated agarose beads, leading to their identification by mass spectrometry. The biotinylated proteins were potential candidate interactors of EBF1 and PAX5, which were later confirmed by sequencing techniques like ChIP-Seq, ATAC seq, and visualization techniques like proximity ligation assay (PLA).