Complex Protein Pattern Research Articles

Urinary proteome and metabolome in dogs (Canis lupus familiaris): The effect of chronic kidney disease

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands <65 kDa) proteinuria. Urine spectra acquired by NMR allowed the identification of 86 metabolites in healthy dogs, belonging to 49 different pathways mainly involved in amino acid metabolism, purine and aminoacyl-tRNA biosynthesis or tricarboxylic acid cycle. Seventeen metabolites showed significantly different concentrations when comparing healthy and CKD dogs. In particular, carnosine, trigonelline, and cis-aconitate, might be suggested as putative biomarkers of CKD in dogs. SignificanceUrine is an ideal biological sample, however few proteomics and metabolomics studies investigated this fluid in dogs and in the context of CKD (chronic kidney disease). In this research, applying a multi-omics approach, new insights were gained regarding the molecular changes triggered by this disease in canine urinary proteome and metabolome. In particular, the involvement of the tubular component was highlighted, suggesting uromodulin, trigonelline and carnosine as possible biomarkers of CKD in dogs.

Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells

Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response.

Investigation of complex protein patterns of latex proteins of various Euphorbiaceae Juss. by two dimensional gel electrophoresis

Investigation of complex protein patterns of latex proteins of various Euphorbiaceae Juss. by two dimensional gel electrophoresis

Enhancement of adaptive biological effects by nanotechnology preparation methods in homeopathic medicines

Multiple studies have demonstrated that traditional homeopathic manufacturing reagentsand processes can generate remedy source and silica nanoparticles (NPs). Homeopathically-made NPs would initiate adaptive changes in an organism as a complex adaptive system (CAS) or network. Adaptive changes would emerge from several different endogenous amplification processes that respond to exogenous danger or threat signals that manufactured nanomaterials convey, including (1) stochastic resonance (SR) in sensory neural systems and (2) time-dependent sensitization (TDS)/oscillation. SR is nonlinear coherent amplification of a weak signal by the superposition of a larger magnitude white noise containing within it the same frequencies of the weak signal. TDS is progressive response magnitude amplification and oscillatory reversal in response direction to a given low dose at physiological limits with the passage of time. Hormesis is an overarching adaptive phenomenon that reflects the observed nonlinear adaptive dose-response relationship. Remedies would act as enhanced micro- and nanoscale forms of their source material via direct local ligand-receptor interactions at very low potencies and/or by triggering systemic adaptive network dynamical effects via their NP-based electromagnetic, optical, and quantum mechanical properties at higher potencies. Manufacturing parameters including dilution modify sizes, shapes, and surface charges of nanoparticles, thereby causing differences in physico-chemical properties and biological effects. Based on surface area, size, shape, and charge, nanoparticles adsorb a complex pattern of serum proteins, forming a protein corona on contact that constitutes a unique biological identity. The protein corona may capture individualized dysfunctional biological mediator information of the organism onto the surfaces of the salient, i.e., resonant, remedy nanostructures. SR would amplify this weak signal from the salient remedy NPs with protein corona adsorbed, leading to sensitized nonlinear dynamical modulation of gene expression and associated changes in biological signaling pathways. When the system reaches its physiological limits during a homeopathic aggravation or the natural disease state, the amplified remedy signal triggers a nonlinear reversal in dynamical direction back towards health.

Imaging mass spectrometry to discriminate breast from pancreatic cancer metastasis in formalin‐fixed paraffin‐embedded tissues

Diagnosis of the origin of metastasis is mandatory for adequate therapy. In the past, classification of tumors was based on histology (morphological expression of a complex protein pattern), while supportive immunohistochemical investigation relied only on few "tumor specific" proteins. At present, histopathological diagnosis is based on clinical information, morphology, immunohistochemistry, and may include molecular methods. This process is complex, expensive, requires an experienced pathologist and may be time consuming. Currently, proteomic methods have been introduced in various clinical disciplines. MALDI imaging MS combines detection of numerous proteins with morphological features, and seems to be the ideal tool for objective and fast histopathological tumor classification. To study a special tumor type and to identify predictive patterns that could discriminate metastatic breast from pancreatic carcinoma MALDI imaging MS was applied to multitissue paraffin blocks. A statistical classification model was created using a training set of primary carcinoma biopsies. This model was validated on two testing sets of different breast and pancreatic carcinoma specimens. We could discern breast from pancreatic primary tumors with an overall accuracy of 83.38%, a sensitivity of 85.95% and a specificity of 76.96%. Furthermore, breast and pancreatic liver metastases were tested and classified correctly.

Gel2DE - A software tool for correlation analysis of 2D gel electrophoresis data

BackgroundTwo-dimensional gel electrophoresis (2DE) is a powerful technique for studying protein isoforms and their modifications. Existing commercial 2D image analysis tools rely on spot detection that limits analysis of complex protein profiles, e.g. spot appearance/disappearance or overlapping spots. Pixel-by-pixel correlation analysis, an analysis technique for identifying relations between protein patterns in gel images and external variables, can overcome such limitations in spot analysis.ResultsWe have implemented the first publically available pixel-by-pixel correlation analysis tool, the software Gel2DE. 2D immunoblot time course analysis of p53 protein stabilization in response to ionizing irradiation shows that pixel-by-pixel analysis can yield an overall activation biosignature for p53, despite changing spots shape, size and position.ConclusionsPixel-by-pixel correlation of aligned 2D images permits analysis of complex protein patterns. We anticipate that the Gel2DE correlation software will be a useful tool for future bioinformatics discoveries through 2D gel electrophoresis.

The Performing Protein: Beyond Wheat Proteomics?

ABSTRACTSubstantial progress has been made in breaking down the complex pattern of proteins in wheat, and the constant improvement of “‐omics” approaches has allowed relating the abundance of selected wheat proteins to flour and semolina performance during processing. This strategy has provided some sound molecular basis for the work of geneticists, breeders, and growers. Along with an update on work in this area, this review is meant to highlight that performance‐relevant proteins may need specific structural features to carry out performance‐relevant functions during processing. In this frame, some of the approaches used to investigate structural features of wheat proteins are discussed, with a focus on water‐insoluble gluten proteins and on those approaches that allow minimal disturbance of the protein structure itself. Selected examples of the application of some of these approaches to the study of starting materials and of structural modifications that occur at various processing stages are presented. The effects of process‐induced modifications on the various interactions among macromolecules that govern the physical and sensory features of wheat‐based foods are also briefly discussed.

Photoinduced Monolayer Patterning for the Creation of Complex Protein Patterns

This work investigates self-assembled monolayers that were formed from a glycol-terminated thiol monomer and were patterned using photoinduced monolayer desorption. Utilizing direct-write photolithography provided a facile means to generate complex protein patterns containing gradients and punctate regions. The ablated glycol monolayers were characterized using scanning probe microscopy, which allowed us to observe differences in the nanomechanical properties between the patterned and nonpatterned regions of the substrate. The patterned regions on the surface adsorbed proteins, and this process was monitored quantitatively using surface plasmon resonance imaging (SPRi). Moreover, the concentration of the protein could be controlled accurately by simply setting the gray level in the 8-bit image. Adsorbed protein was probed using a commercially available antibody binding assay, which showed significant enhancement over the background. The ability to produce complex protein patterns will contribute greatly to creating in vitro models that more accurately mimic an in vivo environment.

The soluble proteome phenotypes of ivermectin resistant and ivermectin susceptible Haemonchus contortus females compared

Anthelmintics in the absence of vaccines have underpinned a parasite control strategy for over 50 years. However, the continued development of anthelmintic resistance (AR) threatens this control. Measuring early AR is difficult as there many routes that resistance can arise from within multi-nematode populations operating complex metabolism capabilities coupled to different drug management pressures. There is an urgent need to identify and measure early resistance in the field situation. Proteomic profiling of expressed soluble proteins offers a new approach to reveal a drug resistant phenotype within a complex protein pattern. The hypothesis under test was that established differences in drug response phenotypes between nematode isolates can also be measured in their comparative proteomes. As a case study, proteomic differences were measured between an ivermectin resistant and susceptible adult female Haemonchus contortus. Adult H. contortus females were extracted from the abomasa of six lambs. The nematodes had been maintained in the lambs as monospecific isolates of either ivermectin susceptible or ivermectin resistant worms. Comparative analysis of the soluble proteome was completed along with immuno-proteomic analysis using pooled infection sera from the lambs. Following image analysis, spots of interest were excised and analysed by peptide mass fingerprinting and the proteins putatively identified using BLAST. Overall, a relative increase in the expression of proteins involved in the detoxification metabolic area was observed in the resistant isolate. In addition, Western blotting analysis also revealed differences in immuno-reactivity profiles between resistant and susceptible isolates. It can be concluded from this study that proteomic differences can be detectable between ivermectin susceptible and a resistant isolates of H. contortus, which could be further explored using other isolates to confirm if proteomic based fingerprinting offers molecular phenotyping or a new panel of resistance biomarkers.

Barriers and more: functions of tight junction proteins in the skin

Although the existence of tight junction (TJ) structures (or a secondary epidermal barrier) was postulated for a long time, the first description of TJ proteins in the epidermis (occludin, ZO-1, and ZO-2) was only fairly recent. Since then, a wealth of new insights concerning TJs and TJ proteins, including their functional role in the skin, have been gathered. Of special interest is that the epidermis as a multilayered epithelium exhibits a very complex localization pattern of TJ proteins, which results in different compositions of TJ protein complexes in different layers. In this review, we summarize our current knowledge about the role of TJ proteins in the epidermis in barrier function, cell polarity, vesicle trafficking, differentiation, and proliferation. We hypothesize that TJ proteins fulfill TJ structure-dependent and structure-independent functions and that the specific function of a TJ protein may depend on the epidermal layer where it is expressed.

Controlled Modification of Protein-Repelling Self-Assembled Monolayers by Ultraviolet Light: The Effect of the Wavelength

Exposure of protein-repelling oligo(ethylene glycol) (OEG) terminated alkanethiolate (AT) monolayers to ultraviolet (UV) light results in the damage of the OEG chains and photooxidation of the thiolate headgroups, which can be used for controlled tuning of protein-repelling properties within the so-called UV direct writing (UVDW) approach or for the preparation of mixed OEG-AT/specific-receptor films by so-called UV-promoted exchange reaction (UVPER). Using several model systems, we studied the effect of the wavelength (254–365 nm) on the course and efficiency of UVDW and UVPER applied to different OEG-AT matrices. The cross sections of the UV-induced damage were found to decrease significantly with increasing wavelength of UV light. In accordance with this behavior, the efficiencies of both UVDW and UVPER were maximal at a wavelength of 254 nm, somewhat lower at 313 nm, and lowest at 365 nm. Both UVDW and UVPER allowed a fine-tuning of protein affinity for nonspecific and specific adsorption, respectively, but UVDW did not occur below a certain, wavelength-dependent threshold dose. Performing UVPER below this dose enables us to suppress possible nonspecific adsorption of proteins even in the case of noncomplete exchange of the UV-damaged molecules of the primary OEG-AT matrix by receptor-bearing moieties. The obtained results are of direct relevance for the preparation of high-quality mixed OEG-AT/specific-receptor films and the fabrication of complex protein patterns by UVDW and UVPER lithography.

Polymer brush nanopatterns with controllable features for protein pattern applications

A novel technique is reported for preparing wafer-scale polymer brush nanopatterns by combining colloidal lithography with surface-initiated atom-transfer radical polymerization. The nanoarchitecture parameters of the polymer patterns can be fully controlled by tuning the experimental conditions and polymer nanopatterns with a lateral resolution down to 100 nm are successfully achieved. In addition, by coupling this technique with photolithography, hierarchical polymer brush nanopatterns have been fabricated. Significantly, the influences of nanodot morphologies on the feature sizes of the polymer nanopatterns are explained in detail. More importantly, complex protein patterns are prepared by conjugating proteins on the polymer brush nanopatterns. The proteins are covalently immobilized on the polymers and exhibit 3D distribution without denaturation. Cells can adhere quickly to the fibronectin patterns and elongate along the stripes of the protein patterns.

Proteomics beyond proteomics: toward clinical applications

There is an increasing interest in the application of proteomics in all areas of the life sciences, especially in the area of biomedical research wherein the analysis of biological fluids is very promising for biomarker discovery. The rapid development of proteomics as a field has depended upon substantial advances in instrumentation, methodologies, software and the availability of comprehensive sequence databases. However, progress has been slower than anticipated with respect to application in the realm of biomedicine. We review some of the areas of importance for the development of proteomics and its applications to biomedical research. One of the factors explaining the slow progress of candidate proteomics biomarkers through the translational pipeline has been the difficulty to develop validation and standardization of procedures to improve reproducibility, sensitivity and specificity. This challenge has now largely been met by large, consortium efforts, leading to the discovery of drug-specific protein biomarkers. The need for integrating complex protein patterns is now recognized in a new test classification by the US Food and Drug Administration termed 'in-vitro diagnostic multivariate index assays'. Further progress in clinical proteomics will depend upon continued collaborative efforts and infrastructure development by the proteomics community.

Complex protein patterns formation via salt-induced self-assembly and droplet evaporation

Complex and elegant protein patterns in rosette, scallop, Chinese arrow and dendrite shapes at macroscopic length scales were prepared using salt-induced molecular self-assembly and droplet evaporation methods. The direct visual observation method using fluorescence microscopy was adopted to characterize the formation of these protein patterns in situ. Further studies from an optical interferometric profiler have shown that both rosette and scalloped protein patterns are hierarchical structures of concentric rings consisting of many prism-like columnar stacks, with each of the stack having thousands of protein molecules. Systematic experimental studies were performed to investigate the influence of salt concentration, protein concentration and evaporation rate on the morphologies of protein patterns. Upon the analysis of the representative fluorescent microscope images some theoretical explanations, based on Deegan's theory on the "coffee ring" effect and the dynamic self-assembly mechanism, were proposed to illustrate the dynamics for the formation of different protein patterns. Two different evaporation modes have been found: edge-enhanced evaporation for low salt concentration solutions, i.e., the higher evaporation rate exists at the edge of the droplet; center-enhanced evaporation for high salt concentration solutions, in which faster evaporation occurs at the droplet center consisting of a lot of crystallized salts.

Proteomics translational

The proteome is a complex but precise reflection of cellular vitality and function. Proteomic techniques such as 2D gel electrophoresis (2DE) and mass spectrometry (MS) are employed to investigate pathognomonic processes and discover differentially expressed proteins when comparing normal and diseased entities. On the one hand proteomic research provides valuable findings on dysregulations in disease, while on the other it provides diagnostically relevant information which could form the basis for early detection of dysregulation and disease predisposition. 2DE is an excellent technique to detect differentially expressed proteins which are subjected to tandem mass spectrometry (MS-MS) for protein identification. Moreover, MS is applied for complex protein pattern profiling to analyze samples by means of “high throughput” screening and allocation to specific pathognomonic alterations. A series of scientific publications reporting on initial clinical applications appear promising in the search of prediction markers.

Complex Protein Patterns in Drying Droplets

AbstractWe demonstrate that deposition patterns formed during drying droplets of aqueous protein solutions are complex, characteristic, and highly reproducible. Substrate, buffer as well as protein type are important factors largely influencing the patterned structure. Specifically, multiple growth zones in what we refer to as “soccer ball pattern” are formed when a droplet of albumin solution in sodium bicarbonate buffer is dried. Each growth zone has periodically patterned, concentric ringed structures surrounding a core at the center. Different macroscopic patterns are also found for streptavidin, fibrinogen, IgG antibody as well as rhodamine B base and polystyrene beads when droplets of their aqueous solutions are dried on the substrates with different degrees of hydrophilicity/hydrophobicity. Furthermore, distinguishable deposition patterns are formed in drying droplets of aqueous protein solutions containing albumin and fibrinogen at different ratios, suggesting that even the relative abundance of multiple proteins influences the deposition patterns. Since the protein pattern is reproducible for a given protein and variable among different proteins, the protein patterns from drying droplets might be useful to potentially identify a given protein under specific conditions.

Characterization and Genetic Study of the Ovine α S2 -Casein (CSN1S2) Allele B

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine alpha ( s2 )-casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp(75) --> Tyr(75) and Ile(105) --> Val(105). The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp(75) --> Tyr(75) substitution modifies the protein electric charge, milk properties may result affected to some extent.

Low‐density lipoproteins from embryonic cerebrospinal fluid are required for neural differentiation

During development, embryonic cerebrospinal fluid(E-CSF) is involved in cell survival, proliferation, and neurogenesis of the neuroepithelial progenitor cells. We have recently identified a complex pattern of proteins in chick and rat E-CSF, which include apolipoproteins. Apolipoproteins play a critical role in the function of lipoproteins by interacting with receptors to deliver the lipid cargo to target cells. However, the function of these E-CSF apolipoproteins is unclear. Here, we characterized the chick E-CSF lipoprotein profile and analyzed the role of its lipoprotein fractions in neural differentiation. We found that the lipoprotein pattern of chick E-CSF differed significantly from that of adult plasma, with a major proportion of apoB-containing lipoproteins. Further, supplementation of lipoprotein-depleted fraction with E-CSF very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) resulted in 25% and 60%, respectively, of the neurogenesis induced by the whole E-CSF in chick neuroepithelium explants, whereas high-density lipoproteins caused the lowest induction. We further investigated the potential role of E-CSF LDL in vivo by analyzing neural differentiation in the neuroepithelium of wild-type (WT) and LDL receptor-knockout (LDLR KO) mouse embryos. E-CSF lipids were mainly associated with LDL in both WT and LDLR KO mice, and the latter exhibited a substantial increase in LDL lipids compared with WT mice. Externally, LDLR KO embryos were apparently normal, and they exhibited up to 26% reduction in the number of neural differentiating cells in comparison with WT mice, although this finding was not statistically significant. These data strongly suggest that E-CSF LDL plays a critical role during early neural differentiation.

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

Principal Cells' Apocrine Secretion of Epididymosomes and Its Function in Sperm Maturation.

During their transit along the epididymis, mammalian spermatozoa acquire new proteins involved in fertilization. Some of these epididymal proteins behave like integral membrane proteins when spermatozoa are submitted to different physico-chemical treatments, suggesting an unusual mechanism of secretion. In fact, Principal cells of the epididymal epithelium use an apocrine mode of secretion to deliver small membranous particles named «epididymosomes» in the epididymal intraluminal compartment. Apocrine secretion is common in reproductive organs such as the prostate and the mammary glands. It involves the formation at the apical pole of secretory epithelial cells of cytoplasmic blebs containing selected organelles. These blebs detach from the secretory cells, disintegrate releasing in the extracellular milieu its content: in the case of epididydmal principal cells, epididymosomes. These vesicles are heterogeneous with a diameter of 50-250 nm. They are characterized by a high cholesterol/phospholipids ratio and are very resistant to physico-chemical injuries such as hypertonic solution, freezing-thawing cycles and sonication. A complex electrophoretic pattern of proteins is associated with these epididymosomes. The protein composition of epididymosomes varies along the epididymis. The epididymosomes are in close relation with spermatozoa within the intraluminal epididymal compartment. Interaction between epididymosomes and spermatozoa results in the transfer of selected proteins to the maturing male gamete. The transfer of proteins from epididymosomes to spermatozoa is saturable, time and temperature dependent, and more efficient at pH 6.5. The presence of Zinc in the co-incubation increases the efficiency of protein transfer. This correlates with the relatively high concentration of Zinc in the epididymal fluid. We have characterized some proteins of epididymosomes transferred to spermatozoa. These include the enzymes of the polyol pathway, P25b proposed to be involved in zona pellucida binding and MIF (Macrophage mobility Inhibitory Factor). Whereas as P25b is GPI anchored to both epididymosomes and sperm plasma membrane, MIF is internalized and is associated to dense fibers of the flagellum. Epididymosomes contain Detergent Resistant Membrane (DRM) to which some proteins are associated while others are excluded. Once transferred, proteins associated to DRM in epididymosomes remain associated to DRM of spermatozoa. Using LC-QToF, we recently identified up to 146 different proteins associated to epididymosomes collected in the distal portion of the human excurrent duct. The function of epididymosomes is thus complex suggesting that they play a major role in sperm maturation. This work was supported by Natural Sciences and Engineering Research Council of Canada and the Canadians Institute for Health Research.