Protein Composition Of Cells Research Articles

Advances in the application of label-free quantitative proteomics techniques in malignancy research.

Proteomics is the scientific discipline that deals with the protein composition of cells, tissues, and organisms and their patterns of change. It is considered an invaluable tool for tackling many challenges faced in medicine and biology. Among the available approaches, label-free quantitative proteomics techniques are extensively used to study malignant tumors due to their low cost, simple operation, and short cycle time. Therefore, it provides a novel approach to explore the pathogenesis, diagnostic markers, and targeted drugs of malignant tumors. Here, we summarize the research progress and potential of label-free quantitative proteomics and discuss the application of such techniques in the research on malignancies.

Data Fusion Algorithm for Myocardial Proteomics and Its Research in Sports.

Sport is a type of comprehensive activity that the human body consciously engages in to improve physical fitness. Proteomics is a comprehensive technology dedicated to the study of all protein profiles expressed by a species, individual organ, tissue, or cell under specific conditions and specific times. Proteomics is a science that studies the protein composition of cells, tissues, or organisms and their changing laws with proteomics as the research object. Related technologies are now widely used in sports and other fields. The purpose of this article is to study myocardial proteomic technology and its application in sports. During the research process, the main methods used in this study are literature survey and controlled experiment. The results achieved and the problems in this field, followed by selecting 30 SD rats into 3 groups for control experiments. The results of the study showed that among the three groups of rats, the left ventricular ejection fraction of the sham operation group was the highest, which was 7.7% and 4.6% higher than that of the operation group and the model group, respectively. The operation group had the highest left ventricular short axis shortening rate, and the left ventricle diastolic inner diameter is the longest. It can be seen that myocardial proteomics can accurately reflect the heart condition of rats. In addition, the length, diastolic velocity, and diastolic time of cardiomyocytes of the three groups of rats were different. Among them, the cardiomyocytes of the operation group had the longest time and the longest diastolic time, which were 37.1% and 8.5% higher than those of the sham operation group and the model group.

Application of Skyline for Analysis of Protein-Protein Interactions In Vivo.

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

Evolution of mechanisms controlling epithelial morphogenesis across animals: new insights from dissociation-reaggregation experiments in the sponge Oscarella lobularis

BackgroundThe ancestral presence of epithelia in Metazoa is no longer debated. Porifera seem to be one of the best candidates to be the sister group to all other Metazoa. This makes them a key taxon to explore cell-adhesion evolution on animals. For this reason, several transcriptomic, genomic, histological, physiological and biochemical studies focused on sponge epithelia. Nevertheless, the complete and precise protein composition of cell–cell junctions and mechanisms that regulate epithelial morphogenetic processes still remain at the center of attention.ResultsTo get insights into the early evolution of epithelial morphogenesis, we focused on morphogenic characteristics of the homoscleromorph sponge Oscarella lobularis. Homoscleromorpha are a sponge class with a typical basement membrane and adhaerens-like junctions unknown in other sponge classes. We took advantage of the dynamic context provided by cell dissociation-reaggregation experiments to explore morphogenetic processes in epithelial cells in a non-bilaterian lineage by combining fluorescent and electron microscopy observations and RNA sequencing approaches at key time-points of the dissociation and reaggregation processes.ConclusionsOur results show that part of the molecular toolkit involved in the loss and restoration of epithelial features such as cell–cell and cell–matrix adhesion is conserved between Homoscleromorpha and Bilateria, suggesting their common role in the last common ancestor of animals. In addition, sponge-specific genes are differently expressed during the dissociation and reaggregation processes, calling for future functional characterization of these genes.

Proteomics analysis of host cell proteins after immobilized metal affinity chromatography: Influence of ligand and metal ions

Different degrees of protein purity have been observed in immobilized metal affinity chromatography ranging from extremely high purity to moderate and low purity. It has been hypothesized that the host cell protein composition and the metal ligands are factors governing the purity of a protein obtained after immobilized metal affinity chromatography (IMAC). Ni nitrilotriacetic acid (NTA) has become the first choice for facile His-tagged protein purification, but alternative ligands such as iminodiacetic acid (IDA) with other immobilized metal ions such as Zn, Cu and Co are valuable options when the expected purity or binding capacity is not reached. Especially Cu and Zn are very attractive, due to their reduced environmental and safety concerns compared to Ni. Co and Zn are more selective than Ni and Cu. This increased selectivity comes at the cost of weaker binding. In this work, the influence of ligand choice on protein purity after IMAC was evaluated by several methods, including peptide mapping. His-tagged GFP was used as model protein. We found that host cell protein (HCP) content varies drastically between ligands, as IDA eluates generally showing higher HCP concentrations than NTA. The relative content of the key amino acids His, Cys and Trp in the sequence of the co-eluted protein does not suffice to explain co-eluting propensity. The co-elution of HCPs is mostly influenced by metal binding clusters on the protein surface and not by total content or surface concentration of metal interacting amino acids. Prediction of co-elution is not dependent on these clusters alone, due to protein-protein interactions, indicted by a relative low metal binding cluster score but high co-elution propensity and in a lot of cases these proteins are often part of complex such as ribosome and chaperones. The different co-eluting proteins were presented by a heatmap with a dendrogram. Ward's linkage method was used to calculate the distance between groups of co-eluting proteins. Clustering of co-eluting HCPs was observed according to ligand and by metal ions, with Zn and Co forming one cluster and Ni and Cu another. The co-elution of host cell proteins can be explained by clusters of metal interacting amino acids on the protein surface and by protein-protein interactions. While Ni NTA still appears to be highly advantageous, it might not be the cure-all for all applications.

The high-resolution proteomic analysis of protein composition of rat spleen lymphocytes stimulated by Concanavalin A; a comparison with morphine-treated cells

Morphine- and Concanavalin A-induced changes of protein composition of rat spleen lymphocytes were determined by high-resolution proteomic analysis, gel-free, label-free quantification, MaxLFQ. Stimulation by Con A resulted in a major reorganization of spleen cell protein composition evidenced by increased expression level of 94 proteins; 101 proteins were down-regulated (>2-fold). Interestingly, among proteins that were up-regulated to the largest extent were the prototypical brain proteins as a neuron specific enolase, synapsin-1, brain acid-soluble protein-1 and myelin basic protein. Morphine-induced change was limited to no more than 5 up-regulated and 18 down-regulated proteins (>2-fold).

THE POSSIBILITY OF USING THE BIOLOGICAL MODEL OF FRESHWATER FISH DANIO RERIO IN PRECLINICAL RESEARCH

The article presents a literary review of domestic and foreign studies that reflect the possibility of using the biological model of Danio rerio (Zebrafish) in preclinical research. Data on the use of the toxicology Biomodel, determination of toxic and teratogenic effects of embryogenic substances, including certain medications, are presented. The use of zebrafish in modeling depression and stress, hypo- and hyperthyroidism, diabetes mellitus and in reproductive technologies is described. The proven effectiveness of the use of Danio rerio in preclinical studies and in modeling a number of human diseases is noted. The data of proteomic studies of the protein composition of cells are summarized zebrafish with the ability to determine the toxic effect at the molecular level.

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation

SummaryClathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells’ limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2’s cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

Cascading effect in bioprocessing-The impact of mild hypothermia on CHO cell behavior and host cell protein composition.

A major challenge in downstream purification of monoclonal antibodies (mAb) is the removal of host cell proteins (HCPs). Previous studies have shown that cell culture conditions significantly impact the HCP content at harvest. However, it is currently unclear how process conditions affect physiological changes in the host cell population, and how these changes, in turn, cascade down to change the HCP profile. We examined how temperature downshift (TDS) to mild hypothermia affects key upstream performance indicators, that is antibody titre, HCP concentration and HCP species, across the cell culture decline phase and at harvest through the lens of changes in cellular behavior. Mild hypothermic conditions introduced on day 5 of fed-batch Chinese hamster ovary (CHO) cell bioreactors resulted in a lower cell proliferation rate but larger percentages of healthier cells across the cell culture decline phase compared to bioreactors maintained at standard physiological temperature. Moreover, the onset of apoptosis was less evident in mild hypothermic cultures. Consequently, mild hypothermic cultures took an extra 5 days to reach an integral viable cell concentration (IVCC) and antibody yield similar to that of the control at standard physiological temperature. When cell viability dropped below 80%, mild hypothermic cell cultures had a reduced variety of HCP species by 36%, including approximately 44% and 27% lower proteases and chaperones, respectively, despite having similar HCP concentration. This study suggests that TDS may be a good strategy to provide cleaner downstream feedstocks by reducing the variety of HCPs and to maintain product integrity by reducing the number of proteases and chaperones.

Quantitative telomeric chromatin isolation protocol for human cells.

The ends of eukaryotic chromosomes, known as telomeres, consist of repetitive DNA sequences, multiple proteins and noncoding RNAs. Telomeres are dynamic structures that play crucial roles as guardians of genome stability and tumor suppressors. Defects in telomere length or protein composition can accelerate aging and are seen in telomere syndromes, which affect various proliferative tissues such as the bone marrow or the lungs. One of the biggest challenges in the telomere field is to identify the molecular changes at telomeres that occur during normal development, in cancer and in telomere syndromes. To tackle this problem, our laboratory has established a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analyzed by mass spectrometry. QTIP involves stable isotope labeling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states.

Trinity of Genome on the Basis of DNA-Energy, DNA-Particle and DNA-Information: A Prokariotic Cell

The study aim is the creation of the tridimensional corpuscular-wave model of prokaryotic cell, and the justification of the trinity principle of energy, matter and information on the example of a genome of prokaryotes. Analysis was carried out at the junction of molecular biology, biochemistry, biophysics, cell biology and physics (common and quantum). It is shown that during cell cycle on the basis of DNA molecule (particle and wave) operate independently and interconnected three complex genetic systems - energetic (DNA-energy), corpuscular (DNA-particle) and informational (DNA-information). Each of them is characterized by the different nature, mechanism action and functional predestination. DNA-energy and DNA-information have the wave nature, DNA-particle – the corpuscular ones. DNA-energy associates with the work of biological harmonic oscillator, which possesses the energy and is able to lower the entropy of cell, DNA-particle forms a protein composition of cell, and DNA-information defines its species diversity. The main conclusion is that the life is realized not in one, as is accepted at the present time, and in three non-dividing and non-merging planes. Due to trinity principle a prokaryotic organisms in the thermodynamic, structural and informative relation become higher against an inanimate matter and such qualities as orderliness, particular chemical composition and species-specific organization are got.

Comparison of Protein Expression Profiles of Novel <i>Halomonas smyrnensis</i> AAD6<sup>T</sup> and <i>Halomonas salina</i> DSMZ 5928<sup>T</sup>

In this work, the protein pattern of novel Halomonas smyrnensis AAD6T was compared to that of Halomonas salina DSMZ5928T, which is the closest species on the basis of 16S rRNA sequence, to understand how AAD6T differs from type strains. Using high resolution NEPHEGE technique, the whole cell protein composition patterns of both Halomonas salina DSMZ5928T and H. smyrnensis AAD6T were mapped. The expressed proteins of the two microorganisms were mostly located at the acidic side of the gels, at molecular weight values of 60 to 17 kDa, and at isoelectric points 3.8 to 6.0, where they share a significant number of common protein spots. Identification and characterization of protein spots via whole genome sequencing data indicated that these two microorganisms used similar pathways, especially TCA cycle, for their survival; in other words, for their energy requirements. On the other hand, the protein expression differences in AAD6T and H. salina DSMZ 5928T showed that they prefer different metabolic pathways for lipid biosynthesis and in adaptation to extreme environments. Thus, we suggested that phylogenetic dissimilarities between these microorganisms could be related to the protein expression differences; in other words, metabolic flux differences in AAD6T and H. salina DSMZ 5928T. This is the first study to explain the dissimilarities of phenotypic characters and DNA-DNA hybridization between type strain and novel strain AAD6T by using protein expression differences.

A quantitative telomeric chromatin isolation protocol identifies different telomeric states

Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.

Responses of Haloarchaea to Simulated Microgravity

Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites).

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mM ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin beta(4), and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells.

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.

From genomics to proteomics

Proteomics is a modern, rapidly developing branch of biology aimed at the integral study of genome expression as the diversity of proteins. Proteomics is based on a variety of methods allowing one to isolate, detect, identify, and to analyze quantitatively thousands of proteins comprising the cellular proteome. Up to date, high-resolution two-dimensional electrophoresis is the key proteomic technology; it allows one to analyze simultaneously the protein composition of cells and tissues and to reveal quantitative and qualitative changes in the synthesis of proteins modulating multiple cell functions. The present status of proteomic technologies, i.e., of 2D electrophoresis, with accompanying methods of identification of proteins at the maps obtained, as well as a brief description of organization of human mouse 2D database suggested for global analysis of genome expression in normal and pathological conditions (http:/biobase.dk/cgi-bin/celis) are discussed.

2‐D Proteome analysis protocols, by Andrew J. Link, Ed. 1999. Totowa, New Jersey: Humana Press. 704 pp. $89.50 (hardcover)

For the past 24 years, high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been the technique of choice for analyzing the protein composition of cells, tissues, and fluids, as well as for studying changes in global patterns of gene expression elicited by a wide array of effectors. The technique, which was originally described independently by O'Farrell and Klose, offers the highest resolution of protein separation and, therefore, plays a central role in “proteomics,” an area of functional genomics that combines a plethora of techniques to resolve (2-D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting), quantify (scanners, phosphorimagers), and characterize proteins, as well as to store (image analysis and 2-D PAGE databases), communicate, and interface protein data with that emerging from the various genome mapping and sequencing projects. Proteomics, together with genomics, cDNA arrays, phage antibody libraries, and transgenics and knockouts, today provide an impressive array of technologies for studying gene expression in both health and disease.

Amino acid sequence preferences of the salt-induced peptide formation reaction in comparison to archaic cell protein composition

The salt-induced peptide formation 〈SIPF〉 reaction has been applied to a number of not yet investigated amino acids, leading to an overview of preferred peptide linkages resulting from this reaction, which is at present the simplest and most universal known possibility of a prebiotic peptide formation in chemical evolution. These relative preferences of linkages in the SIPF reaction have been compared with the relative occurrrence of amino acid pairing in the proteins of archaebacteria and prokaryotic cells. The similarity resulting from this comparison lends strong support to the assumption that salt-induced peptide formation may have supplied the basic units for the construction of protein precursors for the first primitive cells in evolution.

Cell‐surface engineering with GPI‐anchored proteins

Protein engineering of cell surfaces is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer. This technology exploits the fact that proteins that are anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, incorporate into their surface membranes and are fully functional. By substituting 3'-mRNA end sequence of naturally GPI-anchored proteins (i.e., a sequence that contains the signals that direct GPI anchoring) for endogenous 3'-mRNA end sequence, virtually any protein of interest can be expressed as a GPI-anchored derivative. The GPI-anchored product then can be purified from transfectants and the purified protein used to "paint" any target cell. Such protein engineering or "painting" of the cell surface offers several advantages over conventional gene transfer. Among these advantages are that 1) GPI-anchored proteins can be painted onto cells that are difficult to transfect, 2) cells can be altered immediately without previous culturing, 3) the amount of protein added to the surface can be precisely controlled, and 4) multiple GPI-anchored proteins can be sequentially or concurrently inserted into the same cells. Emerging applications for the technology include its use for the analysis of complex cell-surface interactions, the engineering of antigen presenting cells, the development of cancer vaccines, and possibly the protection against graft rejection.