Chemical Modification Of Proteins Research Articles

Staudinger reaction using 2,6-dichlorophenyl azide derivatives for robust aza-ylide formation applicable to bioconjugation in living cells.

Efficient formation of water- and air-stable aza-ylides has been achieved using the Staudinger reaction between electron-deficient aromatic azides such as 2,6-dichlorophenyl azide and triarylphosphines. The reaction proceeds rapidly and has been successfully applied to chemical modification of proteins in living cells.

Modern Approaches to Chemical Modification of Proteins in Biological Tissues: Consequences and Application

Products made of biomaterials, such as heart valve prostheses, vascular grafts, and patches for vascular and intracardiac plastics, are currently used in cardiovascular surgery. The biological tissue used for prosthetics is the alternation of transverse and longitudinal layers of collagen fibers consisting of type I collagen (75%), elastin (<5%), cell elements, as well as glycoproteins, glycosaminoglycans, and other components of the cell matrix. Chemical modifications of components of a biological tissue allow for retention of its natural architectonics and stability of collagen structure over time, while simultaneously increasing the collagen resistance to enzymatic and mechanical destruction and preventing cellular and immune effects on the part of the recipient organism. Proteins in biological tissues are chemically modified (preserved) by the formation of intramolecular and intermolecular cross-links between the amino groups of amino acid residues in collagen molecules. However, cross-linking increases the calcification of biomaterial, making the tissue more rigid and leading to the rupture of the valve flaps, stenosis (reduced clearance), or insufficiency (a decrease in the closure function) of the heart valves. Calcification can also result from specific physiological features of recipient (the patient who received the artificial organ), the nature of the preserving agent, components of the dead cells, defects of collagen structure, cavities in tissues, and the presence of lipids, elastin fibers, glycosaminoglycans, and so on. The factors that induce calcification of the materials used for prosthetic repair and the corresponding methods for its prevention are reviewed. All methods are conventionally divided into three groups: chemical pretreatment of tissues, modification of the preservation method, and posttreatment of preserved tissues with chemical agents. The mechanisms of the processes underlying the effect of chemical agents on the structures of biological tissues are described. The results of their use in clinical practice and prospects for methods still under development and in preclinical trials are discussed, as well as the reasons why some methods have failed. The advantages and disadvantages of various types of treatments are considered. Variants of new methods for chemical modification of biological materials potentially effective in reducing the risk of calcification are proposed.

Native chemical ligation at methionine bioisostere norleucine allows for N-terminal chemical protein ligation.

The development of γ-thionorleucine (ThioNle) as a handle for native chemical ligation-desulfurization is reported here. ThioNle is a new addition to the expanding thiolated amino acid toolbox and serves as a methionine substitute in NCL with the advantage that it lacks the undesirable oxidation-prone thioether moiety. Its usefulness for N-terminal ubiquitination is demonstrated by efficient preparation of fully synthetic linear diubiquitin with preserved protein folding compared to the expressed material. Interestingly, gel-based deubiquitinating assays revealed that the methionine to norleucine substitution did affect diubiquitin cleavage, which may indicate a more profound role for methionine in the interaction between ubiquitin and the deubiquitinating enzymes than has been known so far.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery.

Proteins can undergo oxidative cleavage by in vitro metal-catalyzed oxidation (MCO) in either the α-amidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal α-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-α-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein’s structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.

Role of Oxidized Low-Density Lipoprotein in Hepatitis C Related Chronic Liver Disease

Hepatitis C virus(HCV) lifecycle is closely connected to host cell lipid metabolism, inducing oxidative stress and oxidation reaction induce chemical modification of proteins and lipids in plasma LDL transforming it to abnormal oxidized-LDL (Ox-LDL). Ox-LDL is more stable marker molecule with longer half-life than free radicals. This study assessed its contribution to the pathogenesis of liver diseases, and liver cirrhosis. This work studied serum level of ox-L L to evaluate its role in chronic hepatitis C patients and its association with liver cirrhosis. Also, the level of serum total antioxidant status was determined to evaluate their role in the treatment. The study included 20 chronic hepatitis C patients, 20 chronic hepatitis C patients with liver cirrhosis and 20 controls (age, sex & anthropometric measures were matched). All patients and controls were males. Serum was used to estimate Ox-LDL total direct bilirubin, albumin, aminotransferases, total antioxidant capacity, Urea and creatinine.

Statistical characterization of therapeutic protein modifications

Peptide mapping with liquid chromatography–tandem mass spectrometry (LC-MS/MS) is an important analytical method for characterization of post-translational and chemical modifications in therapeutic proteins. Despite its importance, there is currently no consensus on the statistical analysis of the resulting data. In this manuscript, we distinguish three statistical goals for therapeutic protein characterization: (1) estimation of site occupancy of modifications in one condition, (2) detection of differential site occupancy between conditions, and (3) estimation of combined site occupancy across multiple modification sites. We propose an approach, which addresses these goals in terms of summarizing the quantitative information from the mass spectra, statistical modeling, and model-based analysis of LC-MS/MS data. We illustrate the approach using an LC-MS/MS experiment from an antibody-drug conjugate and its monoclonal antibody intermediate. The performance was compared to a ‘naïve’ data analysis approach, by using computer simulation, evaluation of differential site occupancy in positive and negative controls, and comparisons of estimated site occupancy with orthogonal experimental measurements of N-linked glycoforms and total oxidation. The results demonstrated the importance of replicated studies of protein characterization, and of appropriate statistical modeling, for reproducible, accurate and efficient site occupancy estimation and differential analysis.

Palladium in the Chemical Synthesis and Modification of Proteins.

The field of site-specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium-mediated C-C bond-forming and C-O bond-cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.

Palladium in der chemischen Synthese und Modifizierung von Proteinen

AbstractIn den letzten Jahren hat das Gebiet der ortsspezifischen Modifizierung von Proteinen aufgrund seiner Bedeutung für die Entwicklung neuer Therapeutika, das Verständnis biochemischer und zellulärer Verhaltensweisen von Proteinen und verwandte Forschungsgebiete erhebliche Beachtung gefunden. Durch die Gegenwart einer großen Anzahl reaktiver funktioneller Gruppen im betreffenden Protein und in der zellulären Umgebung stellt die Modifizierung einer spezifischen Stelle eine besonders schwierige Aufgabe dar. Mit der Entwicklung ausgeklügelter chemischer Methoden ist es nun möglich, eine bestimmte Stelle eines Proteins hinsichtlich einer gewünschten Modifizierung zielgerichtet anzugehen, obgleich noch nicht alle Probleme gelöst sind. In diesem Zusammenhang haben Übergangsmetall‐gesteuerte Reaktionen, insbesondere Palladium‐vermittelte C‐C‐Bindungsbildungen und C‐O‐Bindungsspaltungen, aufgrund der einzigartigen katalytischen Eigenschaften von Palladium breites Interesse gewonnen. Palladium‐Chemie wird derzeit intensiv für Protein‐Modifizierungen in vitro, auf der Zelloberfläche und im Zellinneren untersucht. Erst kürzlich wurden Palladium‐Komplexe für die schnelle Entfernung mehrerer weithin eingesetzter Cystein‐Schutzgruppen sowie für die Entfernung von Löslichkeits‐Tags zur Ermöglichung einer chemischen Protein‐Synthese eingesetzt. Dieser Kurzaufsatz beleuchtet die Fortschritte und erörtert, wie vorhandene Kenntnisse der Palladium‐Chemie kleiner Moleküle eindrucksvoll auf das Gebiet der chemischen Protein‐Synthese und ‐Modifizierung übertragen werden können.

Use of MALDI-MS Combined with Differential Hydrogen-Deuterium Exchange for Semiautomated Protein Global Conformational Screening.

Matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF) mass-spectrometry (MS) detector is acknowledged to be very useful for analysis of biological molecules. At the same time, hydrogen-deuterium exchange (HDX) is a well-known technique for studying protein higher-order structure. However, coupling MALDI with HDX has been challenging because of undesired back-exchange reactions during analysis. In this report, we survey an approach that utilizes MALDI coupled with an automated sample preparation to compare global conformational changes of proteins under different solution conditions using differential HDX. A nonaqueous matrix was proposed for MALDI sample preparation to minimize undesirable back-exchange. An automated experimental setup based on the use of a liquid-handling robot and automated data acquisition allowed for tracking protein conformational changes as a difference in the number of protons exchanged to deuterons at specified solution conditions. Experimental time points to study the deuteration-labeling kinetics were obtained in a fully automated manner. The use of a nonaqueous matrix solution allowed experimental error to be minimized to within 1% RSD. We applied this newly developed MALDI-HDX workflow to study the effect of several common excipients on insulin folding stability. The observed results were corroborated by literature data and were obtained in a high-throughput and automated manner. The proposed MALDI-HDX approach can also be applied in a high-throughput manner for batch-to-batch higher-order structure comparison, as well as for the optimization of protein chemical modification reactions.

Controlled modification of biomolecules by ultrashort laser pulses in polar liquids

Targeted chemical modification of peptides and proteins by laser pulses in a biologically relevant environment, i.e. aqueous solvent at room temperature, allows for accurate control of biological processes. However, the traditional laser methods of control of chemical reactions are applicable only to a small class of photosensitive biomolecules because of strong and ultrafast perturbations from biomolecule-solvent interactions. Here, we report excitation of harmonics of vibration modes of solvent molecules by femtosecond laser pulses to produce controlled chemical modifications of non-photosensitive peptides and proteins in polar liquids under room conditions. The principal modifications included lysine formylation and methionine sulfoxidation both of which occur with nearly 100% yield under atmospheric conditions. That modification occurred only if the laser irradiance exceeded certain threshold level. The threshold, type, and extent of the modifications were completely controlled by solvent composition, laser wavelength, and peak irradiance of ultrashort laser pulses. This approach is expected to assist in establishing rigorous control over a broad class of biological processes in cells and tissues at the molecular level.

High-Yield Site-Specific Conjugation of Fibroblast Growth Factor 1 with Monomethylauristatin E via Cysteine Flanked by Basic Residues.

Site-specific conjugation is a leading trend in the development of protein conjugates, including antibody-drug conjugates (ADCs), suitable for targeted cancer therapy. Here, we present a very efficient strategy for specific attachment of a cytotoxic drug to fibroblast growth factor 1 (FGF1), a natural ligand of FGF receptors (FGFRs), which are over-expressed in several types of lung, breast, and gastric cancers and are therefore an attractive molecular target. Recently, we showed that FGF1 fused to monomethylauristatin E (vcMMAE) was highly cytotoxic to cells presenting FGFRs on their surface and could be used as a targeting agent alternative to an antibody. Unfortunately, conjugation via maleimide chemistry to endogenous FGF1 cysteines or a cysteine introduced at the N-terminus proceeded with low yield and led to nonhomogeneous products. To improve the conjugation, we introduced a novel Lys-Cys-Lys motif at either FGF1 terminus, which increased cysteine reactivity and allowed us to obtain an FGF1 conjugate with a defined site of conjugation and a yield exceeding 95%. Using FGFR-expressing cancer lines, we confirmed specific cytotoxity of the obtained C-terminal FGF1-vcMMAE conjugate and its selective endocytososis as compared with FGFR1-negative cells. This simple and powerful approach relying on the introduction of a short sequence containing cysteine and positively charged amino acids could be used universally to improve the efficiency of the site-specific chemical modification of other proteins.

Molecular modification associated with the heat treatment of bovine milk

Raw bovine milk was heated using common industrial heat treatment conditions to determine how treatment conditions modify proteins and lipids. Processing temperature and time were found to affect both the degree and type of molecular modification. An early and consistent protein marker of heating was identified, namely the presence of the Maillard modification (carboxymethyl or carboxyethyl) at the Lys22 residue in the whey protein glycosylation-dependent cell adhesion molecule 1 (GLCM1). Observable chemical protein modifications generally initially increased with increasing temperature but decreased then under more extreme conditions. For lipids, the concentrations of free fatty acids, methylketones, and oxidised fatty acids were directly correlated with pasteurisation temperature. However, mirroring the trend for protein modification, phosphatidylcholine hydroperoxide levels first increased and then decreased at higher temperature. These protein and lipid modifications are potential markers of milk modification during processing and product development.

Epigenetic Understanding of Gene-Environment Interaction in Autism Spectrum Disorder

AbstractThe number of children with autism spectrum disorder (ASD) has increased in many countries over the past 10 years. Genetic studies have revealed that ASD is caused by mutations in the genes coding proteins related to neuronal function. However, such genetic abnormalities cannot underlie the increase in this disorder since mutations do not accumulate among children in the short-term. Epigenetics is a mechanism that is involved in gene regulation not by changing DNA sequence (mutations) but by changing the chemical modifications of DNA and histone proteins. Current studies suggest that mental stress and other forms of environmental stress in early life alter the epigenetic status of genes and change the neuronal gene function, resulting in persistent behavioral abnormalities. Therefore, it can be speculated that the current increase in the prevalence of ASD is partially caused by epigenetic changes in the brains of children induced by some recent socio-environmental conditions. However, epigenetic changes are reversible. Indeed, it has been demonstrated that some drugs for mental disorders reverse the altered epigenetic state and recover the expression of neuronal genes. It has also been demonstrated that offering an appropriate nurturing environment in early life reverses the altered epigenetic state and recovers the neurological gene function in mouse models of ASD. Therefore, from the epigenetic point of view, early medical and educational interventions may be important for children with ASD.

Extrusion cooking technology: Principal mechanism and effect on direct expanded snacks – An overview

The snack industry is one of the fastest growing food sectors and is an important contributor within the global convenience food market. Nowadays snacks and convenience foods are also consumed regularly in India. Properly designed convenience foods can make an important contribution to nutrition in societies where social changes are altering traditional patterns of food preparation. Extrusion cooking as a popular means of preparing snack foods based on cereals and plant protein foodstuff has elicited considerable interest and attention over the past 30 years. Several studies on the extrusion of cereals and pulses, using various proportions, have been conducted because blends of cereals and pulses produce protein enriched products. Special importance is placed on the physicochemical and chemical modifications of protein, starch and dietary fibre. Extruded products can be categorized for a particular application based on their functional properties such as water absorption and water solubility index, expansion ratio, bulk density and viscosity of the dough. Therefore, the literature was reviewed for effect of extrusion processing on product parameters, and nutritional and anti-nutritional properties of extruded products.

Extrusion cooking technology: Principal mechanism and effect on direct expanded snacks – An overview

The snack industry is one of the fastest growing food sectors and is an important contributor within the global convenience food market. Nowadays snacks and convenience foods are also consumed regularly in India. Properly designed convenience foods can make an important contribution to nutrition in societies where social changes are altering traditional patterns of food preparation. Extrusion cooking as a popular means of preparing snack foods based on cereals and plant protein foodstuff has elicited considerable interest and attention over the past 30 years. Several studies on the extrusion of cereals and pulses, using various proportions, have been conducted because blends of cereals and pulses produce protein enriched products. Special importance is placed on the physicochemical and chemical modifications of protein, starch and dietary fibre. Extruded products can be categorized for a particular application based on their functional properties such as water absorption and water solubility index, expansion ratio, bulk density and viscosity of the dough.Therefore, the literature was reviewed for effect of extrusion processing on product parameters, and nutritional and anti-nutritional properties of extruded products.

Single-molecule electrometry.

Mass and electrical charge are fundamental properties of biological macromolecules. Although molecular mass has long been determined with atomic precision, a direct and precise determination of molecular charge remains an outstanding challenge. Here we report high-precision (<1e) measurements of the electrical charge of molecules such as nucleic acids, and globular and disordered proteins in solution. The measurement is based on parallel external field-free trapping of single macromolecules, permits the estimation of a dielectric coefficient of the molecular interior and can be performed in real time. Further, we demonstrate the direct detection of single amino acid substitution and chemical modifications in proteins. As the electrical charge of a macromolecule strongly depends on its three-dimensional conformation, this kind of high-precision electrometry offers an approach to probe the structure, fluctuations and interactions of a single molecule in solution.

Synthesis and Applications of Protein/Peptide-Polymer Conjugates

Recent advances in synthetic methodologies have brought us closer than ever to the precision conferred by nature. For example, the control possible in reversible deactivation radical polymerization enables us to design and synthesize macromolecules with unprecedented control over not only the polymer chain ends, but also the side chain functionality. Furthermore, this functionality can be exploited to afford chemical modification of peptides and proteins, with ever-improving site-specificity, yielding a range of well-defined protein/peptide-hybrid materials. Such materials benefit from the amalgamation of the properties of proteins/peptides with those of the synthetic (macro)molecules in question. Here, the latest developments in the synthesis of functional polymers and their use for preparation of well-defined protein/peptide-polymer conjugates will be discussed, with particular attention focused on modulating the stability, efficacy and/or administration of therapeutic peptides.