One-dimensional Gel Research Articles

Identification of low-abundance proteins in the royal jelly using the Osborne classification method

Royal jelly (RJ) is recognized as healthy food, with a high content of proteins. These proteins play important roles in honeybee caste and human health, but the proteomic analysis of low-abundance proteins in RJ has long been a challenge. Herein, we used the Osborne classification method to separate the RJ proteins of Xinjiang black bees into various fractions. The globulin, ethanol-soluble protein, and glutelin fractions were further separated by SDS-PAGE, and proteomic analysis was carried out by LC-MS/MS and searched against the UniProt database. A total of 23 secretory proteins were identified by proteomic analysis, in which 7 proteins were identified for the first time in RJ. The Osborne classification method combining one-dimensional gel electrophoresis-based proteomic analysis allows the identification of low-abundance proteins in the RJ and greatly extends the knowledge about the components and functions of RJ proteins. The raw data are available via ProteomeXchange with the identifier PXD023315. SignificanceThis study makes an important contribution to the research of the components and functions of low-abundance royal jelly proteins for the following reasons:1.The Osborne method was applied to fractionize the royal jelly proteins, which benefits the identification of low-abundance proteins.2.23 secretory proteins were identified from the globulins, the ethanol-soluble proteins, and the glutelins of the RJ, and there were 7 proteins identified for the first time in royal jelly.3.This study extends the knowledge about the components and functions of royal jelly proteins.

Advancing drug development by leveraging microdissection for proteogenomic analysis of histological sections.

e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Development of CuO Nanoparticles from the Mucus of Garden Snail Cornu aspersum as New Antimicrobial Agents.

Several biologically active compounds involved in the green synthesis of silver and gold nanoparticles have been isolated from snail mucus and characterized. This paper presents a successful method for the application of snail mucus from Cornu aspersum as a bioreducing agent of copper sulfate and as a biostabilizer of the copper oxide nanoparticles (CuONPs-Muc) obtained. The synthesis at room temperature and neutral pH yielded nanoparticles with a spherical shape and an average diameter of 150 nm. The structure and properties of CuONPs-Muc were characterized using various methods and techniques, such as ultraviolet-visible spectroscopy (UV-vis), high-performance liquid chromatography (HPLC), one-dimensional polyacrylamide gel electrophoresis (1D-PAGE), up-conversion infrared spectroscopy Fourier transform (FTIR), scanning electron microscopy combined with energy dispersive spectroscopy (SEM/EDS), Raman spectroscopy and imaging, thermogravimetric analysis (TG-DSC), etc. Mucus proteins with molecular weights of 30.691 kDa and 26.549 kDa were identified, which are involved in the biogenic production of CuONPs-Muc. The macromolecular shell of proteins formed around the copper ions contributes to a higher efficiency of the synthesized CuONPs-Muc in inhibiting the bacterial growth of several Gram-positive (Bacillus subtilis NBIMCC2353, Bacillus spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Listeria innocua NBIMCC8755) and Gram-negative (Escherichia coli ATCC8739, Salmonella enteitidis NBIMCC8691, Salmonella typhimurium ATCC 14028, Stenotrophomonas maltophilia ATCC 17666) bacteria compared to baseline mucus. The bioorganic synthesis of snail mucus presented here provides CuONPs-Muc with a highly pronounced antimicrobial effect. These results will expand knowledge in the field of natural nanomaterials and their role in emerging dosage forms.

Abstract 730: Leveraging microdissection for proteogenomic analysis of histological sections to advance drug development

Abstract Background: Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the tumor microenvironment. This is especially important for immuno-oncology (IO) research where improvements in the predictability of patient response to IO-based drugs is sorely needed. An improved understanding of the spatial relationships involving tumor cells proper, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: LCM is used to isolate and obtain distinct histological cell types from fresh frozen tissue. Once cells have been captured, nucleic acids and proteins are extracted for in-depth multi-modality molecular profiling assays involving multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of our proteogenomics approach is focused around an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: Reported here is a proteogenomic approach that is leveraging from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume. Citation Format: Ik Jae Shin, Michael Tangrea, Michael R. Emmert-Buck, Donald J. Johann. Leveraging microdissection for proteogenomic analysis of histological sections to advance drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 730.

Identification of proteins associated with reducing regrowth and with the longevity of cane fields

Budding axillary buds are crucial for the establishment and longevity of commercial and industrial sugarcane varieties (Saccharum spp.). Increasing the longevity of sugarcane varieties beyond the fifth or sixth cutting stage without decreasing agricultural productivity is a promising economic outlook for producers. Identifying the factors that limit the longevity of cane fields can be an alternative to bypass the regrowth limits of axillary buds. This study aimed to evaluate the bud sprouting rates of axillary buds of the variety RB867515 from the first to the fifth cut and to identify the prevalent proteins in the axillary buds of sugarcanes of the first, third, and fifth cutting stages. The ultra-performance liquid chromatography in tandem with quadrupole-time-of-flight electrospray ionization from one-dimensional gel electrophoresis (1DE-UPLC-ESI-QTOF) mass spectrometry approach was used to obtain the proteome of the axillary buds in the first, third, and fifth cuts. The sprouting rate was evaluated using a caliper and considered positive when the budding of the axillary buds increased by at least 6 mm. Proteins were extracted in biological triplicate for each cutting stage, and the identification of proteins, based on acquired MS/MS spectra, was performed using the Mascot tool from Matrix Science against Viridiplantae within the SwissProt/UniProtKB and databases UP7305_Z_mays in Uniprot. The budding rate in the axillary buds of the first cut was 69.8%. In contrast, the germination rate of the axillary buds of the third cutting was 36.4%. Exclusive proteins were identified in the first, third, and fifth cutting stages, and a 49.3% reduction in the number of proteins was observed in the third cut compared to the first one. Absence of essential proteins/enzymes involved in i) carbohydrate metabolism, ii) folding and degradation of proteins (proteolytic functions), iii) 40S and 60S ribosomal subunits, and iv) proteins involved in biotic and abiotic stresses, are factors that may limit the longevity of cane fields from the third cut before the fifth or sixth cutting stage, when productivity in the field became economically unfeasible

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions(SEIs) and double-Holliday junctions(dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.

Etlingera hemisphaerica Alters One-Dimensional Profile of Serum Proteins Due to Mercury Chloride in Rats (Rattus norvegicus).

<b>Background and Objective:</b> Several previous studies have shown that leaf ethanolic extract of <i>Etlingera hemisphaerica</i> (LE3H) has the potential to reduce the toxicity and teratogenicity effects of mercury. This study aimed to describe the effects of LE3H on the protein profile of <i>Rattus norvegicus</i> serum due to treatment with HgCl<sub>2</sub>. <b>Materials and Methods:</b> Four groups of male rats, K1 was injected intraperitoneally (IP) HgCl<sub>2</sub> (5 mg kg<sup>1</sup> b.wt.), K2 was injected IP HgCl<sub>2</sub> (5 mg kg<sup>1</sup> b.wt.) and after 24 hrs it was gavage LE3H (0.27 mg g<sup>1</sup> b.wt.) every day for seven days, K3 was injected IP HgCl<sub>2</sub> (5 mg kg<sup>1</sup> b.wt.), after 24 hrs was gavage LE3H (0.55 mg g<sup>1</sup> b.wt.) every day for seven days. The K0 as control, received double-distilled water. On the ninth day, the experimental animals were killed via CD and blood was drawn from the heart to obtain serum. Serum samples were measured for protein content using the Lowry and serum was separated using the One-Dimensional Sodium dodecyl Sulfate-Polyacrylamide gel Electrophoresis (1D SDS-PAGE) technique. <b>Results:</b> The electropherograms showed four bands, 264.77, 219.53, 98.57 and 37.29 kDa, whose intensity significantly increased due to HgCl<sub>2</sub> treatment and then decreased to close to the control condition with LE3H administration. The results also revealed four bands, 31.95, 28, 06, 26, 29 and 15.09 kDa, whose intensity decreased significantly due to HgCl<sub>2</sub> treatment and then increased to close to the control condition by LE3H administration. <b>Conclusion:</b> The LE3H change profile of the eight blood serum protein bands due to HgCl<sub>2</sub> approximates the control condition in <i>R. norvegicus</i>.

Optogenetic control of migration of contractile cells predicted by an active gel model

Cell crawling on flat substrates stems from intracellular flows of the actin cytoskeleton that are driven by both actin polymerization at the front and myosin contractility at the back. Optogenetics makes it experimentally possible to spatially control contraction and possibly cell migration too. Here we theoretically analyze this situation using a one-dimensional active gel model that reflects the property of myosin II to assemble into minifilaments. Our model predicts bistability between sessile and motile solutions when cell adhesion and contractility are sufficiently large and in balance. We show that one can switch between the different states at realistic parameter values via optogenetic activation or inhibition of contractility, in agreement with recent experiments performed for neutrophils in microchannels. We predict the required activation strengths and initiation times, compare the effects of local and global increases of myosin II levels, and show that actin polymerization alone can affect a switch in direction only at high strength.

Identification of Carbohydrates Metabolic Related Enzymes for Lipid Production in Botryococcus sp., a Microalgae Isolated from Taman Negara Endau Rompin

Botryococcus is a microalgal genus known for its ability to generate and accumulate substantial amounts of lipids via carbohydrate metabolism. This work determined the metabolic pathways and enzymes involved in carbohydrate metabolism leading to increased synthesis of fat in Botryococcus sp. Relevant intracellular and extracellular metabolites were extracted and quantified using chromatographic analysis. Enzymes involved in carbohydrate metabolism leading to lipid formation in Botryococcus sp. under natural conditions were also discovered by one-dimensional gel electrophoresis followed by proteomic mass spectrometry (LC-MS/MS) and database searching. Proximate analysis demonstrated 23.0% total carbohydrate, 16.0% protein and 61.0% lipid per milligram biomass dry weight of Botryococcus sp. The extracellular metabolites constitute mostly of cyclohydrocarbons, nitrogenated hydrocarbons, siloxanes, phenols, and phenol derivatives. A glycolytic enzyme “enolase,” which can create phosphoenolpyruvate and subsequently convert it into pyruvate, was found in this study. This study revealed that enolase provided an alternate pathway to export fixed carbon to the cytoplasm, hence providing a shorter route to lipid production than the normal process via the plastid leading to the manufacture of more lipids in the cells of Botryococcus sp. than other microalgae of the same group.

Proteomics of the haemolymph of the terrestrial crustacean Porcellio scaber reveals components of its innate immunity under baseline conditions

The terrestrial crustacean Porcellio scaber is an established test organism in environmental research. We analysed the haemolymph proteome of P. scaber using a classical proteomic approach based on one-dimensional gel electrophoresis and tandem mass spectrometry. Using a publicly available protein database and our P. scaber transcriptome data, we have identified 76 proteins involved in cytoskeleton formation, protein degradation, vesicular transport, genetic information processing, detoxification, carbohydrate and lipid metabolism reflecting haemocyte metabolic activity, active intracellular transport, and intercellular communication. Compared with the data reported for other crustaceans, 28 of these P. scaber proteins have been linked to its immunity, among them hemocyanin, α-2-macroglobulin, phenoloxidase 3, superoxide dismutase, glutathione S-transferase, haemolymph clottable protein, and histones H4 and H2B. Our results thus provide a firm base for studying the innate immune response of P. scaber at the level of the haemolymph proteome. This knowledge is of particular importance in ecotoxicity studies with various environmental stressors where understanding physiological changes is important to reveal possible modes of action.

Dysregulation of metalloproteins in ischemic heart disease patients with systolic dysfunction

Ischemic heart disease (IHD) is the leading cause of mortality worldwide. Metalloproteins have been linked to human health and diseases. The molecular functions of metalloproteins in IHD is not well understood and require further exploration. The objective of this study was to find out the role of metalloproteins in the pericardial fluid of IHD patients having normal (EF > 45) and impaired (EF < 45) left ventricular ejection fraction (LVEF). IHD patients were grouped into two categories: LVEF<45 (n = 12) and LVEF >45 (n = 33). Pooled samples of pericardial fluid were fractionated by using ZOOM-isoelectric focusing (IEF) followed by further processing using one-dimensional gel electrophoresis (1D SDS-PAGE) and filter-aided sample preparation (FASP). Tryptic peptides of each fraction and differential bands were then analyzed by nano-LC-ESI-MS/MS. Protein identification was performed through a Mascot search engine using NCBI-Prot and SwissProt databases. A total of 1082 proteins including 154 metalloproteins were identified. In the differential bands, 60 metalloproteins were identified, while 115 metalloproteins were identified in all ZOOM-IEF fractions. Twelve differentially expressed metalloproteins were selected in the intense bands according to their molecular weight (MW) and isoelectric point (pI). The 12 differentially expressed metalloprotein includes ceruloplasmin, Prothrombin, Vitamin K-dependent protein, Fibulin-1, Ribosomal protein S6 kinase alpha-6, nidogen, partial, Serum albumin, Hemopexin, C-reactive protein, Serum amyloid P-component, and Intelectin-1 protein which were all up-regulated while serotransferrin is the only metalloprotein that was down-regulated in impaired (LVEF<45) group. Among the metalloproteins, Zn-binding proteins are 36.5 % followed by Ca-binging 32.2 %, and Fe-binging 12.2 %. KEGG, pathway analysis revealed the association of ceruloplasmin and serotransferrin with the ferroptosis pathway. In conclusion, 154 metalloproteins were identified of them the Zn-binding protein followed by Ca-binding and Fe-binding proteins were the most abundant metalloproteins. The two metalloproteins, the Cu-binding protein ceruloplasmin, and Fe-binding protein serotransferrin are involved in the ferroptosis pathway, an iron-dependent form of regulated cell death that has been linked to cardiac pathology, especially in IHD patients having impaired systolic (LVEF<45) dysfunction. However, further research is required to validate these findings.

Silver Nanoparticles Modified by Carbosilane Dendrons and PEG as Delivery Vectors of Small Interfering RNA.

The fact that cancer is one of the leading causes of death requires researchers to create new systems of effective treatment for malignant tumors. One promising area is genetic therapy that uses small interfering RNA (siRNA). These molecules are capable of blocking mutant proteins in cells, but require specific systems that will deliver RNA to target cells and successfully release them into the cytoplasm. Dendronized and PEGylated silver nanoparticles as potential vectors for proapoptotic siRNA (siMCL-1) were used here. Using the methods of one-dimensional gel electrophoresis, the zeta potential, dynamic light scattering, and circular dichroism, stable siRNA and AgNP complexes were obtained. Data gathered using multicolor flow cytometry showed that AgNPs are able to deliver (up to 90%) siRNAs efficiently to some types of tumor cells, depending on the degree of PEGylation. Analysis of cell death showed that complexes of some AgNP variations with siMCL-1 lead to ~70% cell death in the populations that uptake these complexes due to apoptosis.

Temperature and Light Multiphysical Fields Modulate the Propagation of Chemical Waves on Multiple Length Scales in a System

Abstract The phenomenon of multiple length scales exists in plants during growth, which is influenced not only by light but also by other physical fields such as substance concentration and temperature, and the combined effects of various physical fields form a complex multi-scale phenomenon. However, the mechanism of the multi-physical interaction of temperature and light on the formation of multiple length scales in plants has not been investigated. In this paper, we coupled a gel with a Belousov-Zhabotinsky (BZ) chemical reaction system, constructed a one-dimensional open reaction diffusion system, and investigated the changes in the multiple length scale periodic structure of chemical waves in the one-dimensional gel system under the combined effect of multiple physical fields of light and temperature. It was found that when increasing the light intensity at T = 26 °C or decreasing the temperature at the light intensity I ≤ 50 µW·cm−2, the chemical wave multiple length scale period structure of the system changed as follows: P8 → P16 → P8 → P4 → P2 → P1, and the chemical wave period structure complexity showed a non-linear change of first increasing and then decreasing. When increasing the light intensity at T ≤ 24 °C or decreasing the temperature at the light intensity I &amp;gt; 50 µW·cm−2, the chemical wave period structure complexity of the system shows a linear decrease.

Nano-LC-FTMS Analysis of the Primary Structure and Post-translational Modifications of the α1 Subunit of the GABAA Receptor

GABAAR is one of the most significant drug targets in the treatment of neuropsychiatric disorders such as epilepsy, insomnia, anxiety, as well as anesthesia in surgical operations. However, complete information on the regulation of GABAA receptor activity is lacking. Therefore, studies are needed on post-translational modifications of receptor subunits that exhibit different pharmacological and physiological properties. GABAAR has been immunopurified from rat brain membranes on protein A agarose beads immobilized with complex anti-GABAAR antibody. Primary structure and post-translational modifications of the α1 subunit of GABAAR have been characterized by nano-LC-FTMS peptide mapping by direct in situ–gel digestion on the one-dimensional gel. The primary structure of the α1 subunit of GABAAR has been identified with high sequence coverage (81%, 371 from 455 amino acids). The extracellular domains, N-terminal and the C-terminus, have been identified with extensive sequence coverage, 85 and 100 % (214 from 249 amino acids), respectively. Other soluble domains, including the M1-M2 linker, and the M3-M4 linker, have also been determined with 100 and 85 % (75 from 88 amino acids), respectively. Transmembrane domains, including M1, M2, and M4, were identified almost completely. Post-translational modifications of the α1 subunit peptides have been found, such as phosphorylation, methionine oxidation, and carbamidomethylation.

Two-dimensional Gel Analysis of Total Seed Protein of Horse Gram (Macrotyloma uniflorum) for Identification of their Proteomic Diversity

Horse gram (Macrotyloma uniflorum) is a popular pulse in the Kumaun region of Uttarakhand, locally known as Gahat dal and Kulti. An experiment was conducted with 5 horse gram cultivars having different grain colors. Total seed protein was isolated and quantified with standard protocols. Five protein samples were subjected to one-dimensional (1D) and two-dimensional (2D) gel for proteomic analysis. 300g of each sample was loaded onto immobilized pH gradient (IPG) strips (3–10 pH Linear, 18 cm) and kept for Iso-Electric Focusing (IEF) and SDS-PAGE. The gels were scanned using a scanner and analyzed for proteomic diversity. Basically, two major clusters were constructed, and samples 1, 2, and 3 are in group one and 4 and 5 are in group two, and group one has two further sub-clusters. Sample one was separately clustered based on the number of spots presented on the 2D gel and based on its color. The analysis of variance revealed that the genotypes observed had highly significant differences. Further study is needed for the identification of unique peptides presented by protein sequencing. The proteomic analysis of total seed proteins could be useful for the development of nutraceuticals and food-to-food fortification. This study’s current proteome and other proteomic information will be extremely useful to horse gram breeding programmes.

Towards the purification of IgY from egg yolk by centrifugal partition chromatography

Although their high potential as alternative biopharmaceuticals, less than 2% of the total polyclonal antibodies produced worldwide correspond to immunoglobulin Y (IgY) due to the difficulties in isolating them from egg yolk (complex biological matrix). In this work, the water-soluble proteins fraction (WSPF) of egg yolk was first obtained and the proteins present identified by one-dimensional gel electrophoresis (SDS-PAGE) and label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). The egg yolk WSPF was then applied to create aqueous biphasic systems (ABS) composed of polyethylene glycol 1000 g·mol−1 (PEG 1000) and K2HPO4/ KH2PO4 buffer, followed by centrifugal partition chromatography (CPC) to purify IgY. The characterization of the WSPF showed the presence of six major proteins: the target antibody IgY, serum albumin (α-livetin), ovalbumin, ovotransferrin, vitellogenin 1 and vitellogenin 2. The results obtained by ABS revealed a high affinity of all proteins to the polymer-rich phase. However, by changing the PEG and salt concentrations, a higher selectivity was observed for IgY, with the remaining proteins partitioning between the two phases. The best ABS were applied in CPC, finally allowing a multi-stage partition and to the technology scale-up. The CPC operating conditions were optimized, allowing to obtain IgY with 50.6% of purity.

The Identification and Characterization of Immunoreactive Fungal Proteins Recognized by Sera from Zimbabweans Sensitized to Fungi

Background: Exposure to fungal allergens poses a serious threat to human health, especially to mould-allergic individuals. The prevalence of fungal allergic disease is increasing globally but is poorly studied in Africa. Here, we aimed to identify and characterize fungal proteins that were immunoreactive against serum samples from fungal-sensitized Zimbabweans from Shamva district to inform the development of diagnostics and therapeutics. Methods: Crude protein extracts of the Ascomycota Aspergillus fumigatus, Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Penicillium chrysogenum, and Saccharomyces cerevisiae as well as mucoromycota Rhizopus nigricans were individually separated by one-dimensional gel electrophoresis for protein staining and immunoblotting. A pool of eight sera from fungi-sensitive Zimbabwean children aged 3–5 years was used to screen the crude extracts to determine their immunoreactivity. Protein bands recognized by the sera were subjected to mass spectrometry to identify the individual proteins reactive with the sera. Results: The pooled serum sample reacted with 20 bands, which resolved to 34 distinct proteins, most of which were novel immunogens. The pool was most reactive to A. alternata. The proteins identified included peptidases (8/34), hydrolases (6/34), oxidoreductases (5/34), and glucosidases (4/34), while 11/34 were unknown. Eight of the proteins were predicted to be allergens using the Structural Database of Allergenic Proteins (SDAP). Conclusions: We identified novel immunogens from fungi expanding the number of known fungal allergens. These form a potential basis for diagnostics specific for the Zimbabwean population. Validation assays will now need to be carried out to further evaluate the cross-reactivity of the identified allergen candidates as well as investigate their potential recognition in a larger cohort of patients. Furthermore, there is now a need to conduct studies relating sensitization to these immunogens and clinical diseases in the population.

Molecular markers for hemocyte subpopulations in crayfish Cherax quadricarinatus

Semigranular cells (SGCs) and granular cells (GCs) are two dominant groups of circulating hemocytes in crayfish Cherax quadricarinatus. Molecular markers are required for the clear classification of the hemocytes and the research of their function and differentiation. In this study, we compared the protein content of GCs and SGCs by using two workflows: one-dimensional gel electrophoresis followed by LC-MS/MS and in-solution digestion of cell lysate followed by LC-MS/MS. Cell type-specific proteins were identified, and their expression in SGCs and GCs was further investigated by RT-PCR, Western blotting, and immunofluorescence analysis. Three molecular markers for GCs (peroxinectin, a mannose-binding protein, and prophenoloxidase-activating enzyme 2a) and three molecular markers for SGCs (a vitelline membrane outer layer protein I-like protein, a C-type lectin, and a peptidase) were identified. The application of some of the markers in Eriocheir sinensis was also analyzed. These molecular markers are useful tools for the research of crustaceans hemocytes.

P38-MAPK recruits the proteolytic pathways in Caenorhabditis elegans during bacterial infection

In eukaryotic organisms, cell-signalling completely relies on Post Translational Modifications (PTMs) that can function as regulatory switches. Phosphorylation is a fundamental and frequently occurring PTM in almost all eukaryotes. Herein, we have studied the importance of protein phosphorylation using classical proteomic techniques in C. elegans upon bacterial infection. The differentially regulated proteins during bacterial infection were excised from SDS-PAGE (One-Dimensional) gel (TiO2 column elutes) and subjected to MALDI-TOF-MS which ended up in identifying 220 proteins kinetically. KEGG pathway analysis of those proteins suggested that MAPK pathway was part of the innate immunity. Thus, we have characterized p38-MAPK (one of the crucial downstream MAPKs) using immunoblotting, subcellular fractionation, coimmunoprecipitation, LC-MS/MS, bioinformatics studies and qPCR. Meanwhile, KU25 strain (pmk-1 mutant) exhibited an earlier mortality during infection suggesting the crucial role of p38-MAPK during host-pathogen interaction. Interestingly, Reactome pathway analysis of p38 interactors (CoIP coupled to LC-MS/MS) revealed the involvement of various proteolytic pathway players (ubiquitination, SUMOylation and Neddylation) during bacterial infection. Further, the regulation of SUMOylation and Neddylation was identified and validated using immunoblotting and qPCR analyses, respectively. Concisely, our study indicated that bacterial infection triggers the MAPK cascade to elicit innate immunity which in turn recruits proteolytic pathways to counteract the invading pathogen.