Academic literature on the topic '030403 Characterisation of Biological Macromolecules'

Advances in sample preparation, ion sources and mass spectrometer technology have enabled the detection and characterisation of intact proteins. The challenges associated include an appropriately soft ionisation event, efficient transmission and detection of the often delicate macromolecules. Ambient ion sources, in particular, offer a wealth of strategies for analysis of proteins from solution environments, and directly from biological substrates. The last two decades have seen rapid development in this area. Innovations include liquid extraction surface analysis, desorption electrospray ionisation and nanospray desorption electrospray ionisation. Similarly, developments in native mass spectrometry allow protein–protein and protein–ligand complexes to be ionised and analysed. Identification and characterisation of these large ions involves a suite of hyphenated mass spectrometry techniques, often including the coupling of ion mobility spectrometry and fragmentation techniques. The latter include collision, electron and photon-induced methods, each with their own characteristics and benefits for intact protein identification. In this review, recent developments for in situ protein analysis are explored, with a focus on ion sources and tandem mass spectrometry techniques used for identification.

: Proteins are versatile macromolecules that perform a variety of functions and participate in virtually all cellular processes. The functionality of a protein greatly depends on its structure and alterations may result in the development of diseases. Most well-known of these are protein misfolding disorders, which include Alzheimer’s and Parkinson’s diseases as well as type 2 diabetes mellitus, where soluble proteins transition into insoluble amyloid fibrils. Atomic Force Microscopy (AFM) is capable of providing a topographical map of the protein and/or its aggregates, as well as probing the nanomechanical properties of a sample. Moreover, AFM requires relatively simple sample preparation, which presents the possibility of combining this technique with other research modalities, such as confocal laser scanning microscopy, Raman spectroscopy and stimulated emission depletion microscopy. In this review, the basic principles of AFM are discussed, followed by a brief overview of how it has been applied in biological research. Finally, we focus specifically on its use as a characterisation method to study protein structure at the nanoscale in pathophysiological conditions, considering both molecules implicated in disease pathogenesis and the plasma protein fibrinogen. In conclusion, AFM is a userfriendly tool that supplies multi-parametric data, rendering it a most valuable technique.

Non-extractable polyphenols (NEPs), or bound polyphenols, are a significant fraction of polyphenols that are retained in the extraction residues after conventional aqueous organic solvent extraction. They include both high molecular weight polymeric polyphenols and low molecular weight phenolics attached to macromolecules. Current knowledge proved that these bioactive compounds possess high antioxidant, antidiabetic, and other biological activities. Plant-based food by-products, such as peels, pomace, and seeds, possess high amount of NEPs. The recovery of these valuable compounds is considered an effective way to recycle food by-products and mitigate pollution, bad manufacturing practice, and economic loss caused by the residues management. The current challenge to valorise NEPs from plant-based by-products is to increase the extraction efficiency with proper techniques, choose appropriate characterising methods, and explore potential functions to use in some products. Based on this scenario, the present review aims to summarise the extraction procedure and technologies applied to recover NEPs from plant-based by-products. Furthermore, it also describes the main techniques used for the characterisation of NEPs and outlines their potential food, pharmaceutical, nutraceutical, and cosmetic applications.

AbstractBiomaterials have had an increasingly important role in recent decades, in biomedical device design and the development of tissue engineering solutions for cell delivery, drug delivery, device integration, tissue replacement, and more. There is an increasing trend in tissue engineering to use natural substrates, such as macromolecules native to plants and animals to improve the biocompatibility and biodegradability of delivered materials. At the same time, these materials have favourable mechanical properties and often considered to be biologically inert. More importantly, these macromolecules possess innate functions and properties due to their unique chemical composition and structure, which increase their bioactivity and therapeutic potential in a wide range of applications. While much focus has been on integrating these materials into these devices via a spectrum of cross-linking mechanisms, little attention is drawn to residual bioactivity that is often hampered during isolation, purification, and production processes. Herein, we discuss methods of initial material characterisation to determine innate bioactivity, means of material processing including cross-linking, decellularisation, and purification techniques and finally, a biological assessment of retained bioactivity of a final product. This review aims to address considerations for biomaterials design from natural polymers, through the optimisation and preservation of bioactive components that maximise the inherent bioactive potency of the substrate to promote tissue regeneration.

Introduction: Glycation of biological macromolecules particularly protein leads to the generation of early and Advanced Glycation End (AGE) products. The interest in early glycation of protein is driven due to the fact of Amadori modified proteins having role in diabetic complications. Aim: To analyse the biophysical characterisation of Amadori modified human serum albumin as a prognostic biomarker for diabetic complications. Materials and Methods: This in-vitro experimental study was conducted at Department of Biochemistry, J.N. Medical College, Aligarh Muslim University, Uttar Pradesh, India, from May 2010 to December 2012. The structural characterisation of EGPs was generated by incubating Human Serum Albumin (HSA) with glucose for about a week. The generation of EGPs of HSA was quantitated as Hydroxy Methyl Furfural (HMF) by ThioBarbituric Acid (TBA) assay and authenticated by boronate affinity chromatography. Moreover, High Performance Liquid Chromatography (HPLC) and Electro Spray Ionisation/Mass Spectrometry (ESI/MS) was carried out to validate the presence of Amadori product formed. Additionally, Circular Dichroism (CD) and thermal denaturation studies were used to investigate the structural changes in Amadori albumin. Results: Glycated HSA was obtained as detected by the presence of HMF and chromatography peaks. On stratification, the structural perturbation was observed in Amadori HSA. Furthermore, the generation of furosine was also confirmed by obtaining a new peak in the HPLC profile of glycated HSA. The ESI/MS result also substantiated the presence of Amadori products. Conclusion: The therapeutic strategies that negate the Amadori modification of albumin might be a logical approach in the prevention of diabetic complications.

Understanding the function and structure of a protein is crucial for informing on rational drug design and for developing successful drug candidates. However, this understanding is often limited by the protein pipeline, i.e. the necessary steps to go from developing protein constructs to generating high-resolution structures of macromolecules. Because each step of the protein pipeline requires successful completion of the prior step, bottlenecks are often created and therefore this process can take up to several years to complete. Addressing current limitations in the protein pipeline can help to reduce the time required to successfully solve the structure of a protein.

The field of nonlinear optical (NLO) microscopy provides a potential solution to many issues surrounding the detection and characterization of protein crystals. Techniques such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) have already been shown to be effective methods for the detection of proteins with high selectivity and sensitivity. Efforts to improve high throughput capabilities of SHG microscopy for crystallization trials resulted in development of a custom microretarder array (μRA) for depth of field (DoF) extension, therefore eliminating the need for z-scanning and reducing the overall data acquisition time. Further work was done with a commercially available μRA to allow for polarization dependent TPE-UVF. By placing the μRA in the rear conjugate plane of the beam path, the patterned polarization was mapped onto the field of view and polarization information was extracted from images by Fourier analysis to aid in discrimination between crystalline and aggregate protein.

Additionally, improvements to X-ray diffraction (XRD), the current gold standard for macromolecular structure elucidation, can result in improved resolution for structure determination. X-ray induced damage to protein crystals is one of the greatest sources of loss in resolution. Previous work has been done to implement a multimodal nonlinear optical (NLO) microscope into the beamline at Argonne National Lab. This instrument aids in crystal positioning for XRD experiments by eliminating the need for X-ray rastering and reduces the overall X-ray dosage to the sample. Modifications to the system to continuously improve the capabilities of the instrument were done, focusing on redesign of the beam path to allow for epi detection of TPE-UVF and building a custom objective for improved throughput of 1064 nm light. Furthermore, a computational method using non-negative matrix factorization (NMF) was employed for isolation of unperturbed diffraction peaks and provided insight into the mechanism by which X-ray damage occurs. This work has the potential to improve the resolution of diffraction data and can be applied to other techniques where X-ray damage is of concern, such as electron microscopy.

Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3‑butanediol, a product formed in an alternative pathway to mixed acid fermentation.

While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism.

Here, the ALS and AHAS enzymes from Klebsiella pneumoniae were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site‑saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS in vivo.

Zein is a major storage protein of corn, with unique amphiphilic film forming properties. It is insoluble in water, but soluble in 70% ethanol and acetic acid, and has been declared ‘generally recognized as safe’ (GRAS) by the FDA. Due to new advances in food nanotechnology, zein is being investigated for various applications such as biodegradable packaging, oral delivery of proteins and peptides, scaffold for tissue engineering, as well as biodegradable sensor platforms. The time consuming and highly complicated methods for toxin and allergen analysis in the food industry necessitates the need for a rapid, selective, compact and easy-to-use method of detection for analytes. In the scope of this dissertation, we investigated the feasibility of functional zein nanocomposite films and formation of a zein nanocomposite sensor assembly for rapid and highly selective electrochemical measurements of food toxins and allergens. Fabrication of a zein based electrochemical amperometric sensor assembly was studied, first through the comparison of various zein film characteristics changes with the application of Laponite®, graphene oxide and carbon nanotube nanoparticles, followed by a proof-of-concept study by detecting the gluten allergen protein gliadin.

To mechanistically study the functional zein nanocomposite films, Laponite®, a silica nanoparticle, was added in the presence of 70% ethanol solvent and oleic acid plasticizer. The films were studied using various characterization techniques like transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), water contact angle measurements etc. Through Si-N bond formation between Laponite® and zein, fabricated zein nanocomposite films showed increase in surface hydrophobicity, water vapor barrier properties, tensile strength and Young’s modulus. Graphene oxide (GO), a carbon nanoparticle, was also incorporated into zein through the solvent casting process. Uniform dispersion of GO nanoparticles within zein matrix were confirmed up to 1% GO loading, and covalent and hydrogen bonding mechanisms were proposed. Similar to zein-Laponite® (Z-LAP) nanocomposites, zein-GO (Z-GO) showed increase in hydrophobic tendencies, rougher surface and a 300% improvement in Young’s modulus and 180% improvement in tensile strength at only 3% GO loading. Both nanoparticles increased tensile strength, thermal stability and water vapor barrier property of the films, indicating a potential for food packaging as an alternative application for the nanocomposite films.

Finally, the research focused on the fabrication of an electrochemical amperometric sensor, capable of detecting the protein gliadin, which is responsible for the allergic reaction with people having celiac disease. Novel biodegradable coatings made from zein nanocomposites: zein-graphene oxide, zein-Laponite® and zein-multiwalled carbon nanotubes (Z-CNT) using drop casting technique were tested for fabricating the electrochemical sensors using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) techniques. As Z-CNT produced the strongest signals compared to other nanomaterials, the active tip of the electrochemical sensor was functionalized through a sequence of layer by layer deposition of Z-CNT nanocomposite, antibody and target analyte. Here, Z-CNT acts as a natural linker molecule with large number of functional groups, that causes immobilization of capture antibody and target, to ensure high sensor performance. Both CV curves and SWV curves indicated successful sequential immobilization of gliadin antibody onto the Z-CNT coated electrode. The Z-CNT biosensor was successfully able to give CV signals for gliadin toxins for as low as 0.5 ppm and was highly specific for gliadin in the presence of other interfering molecules, and remained stable over a 30-day period. The low-cost, thin, conductive zein films offered a promising alternative for protein immobilization platforms used in sensors and can be extended to other matrices in biosensors as well as other functional film applications

The objective of this research is to develop and apply a HILIC UHPLC stationary phase that allows for separation of intact glycoproteins. In Chapter 1 I give an overview of the problems of current glycosylation profiling with regards to biotherapeutics, and my strategy to separate the intact glycoprotein with HILIC. Chapter 2 describes the methods used to produce the nonporous packing material and stationary phase. In Chapter 3 I describe previous work in developing a HILIC polyacrylamide stationary phase, and further improvements I have made. Chapter 4 describes development of an assay in collaboration with Genentech of therapeutic mAb glycosylation. In Chapter 5, I show HILIC-MS of digested ribonuclease B as a beginning step to analyze glycosylated biomarkers.