Mobilization of seed storage proteins is crucial to high vigor in common bean seeds / Mobilização das proteínas de reserva é crucial para o alto vigor em sementes de feijão

Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.

A germinação de sementes é um processo complexo controlado por muitos fatores, nos quais mecanismos físicos e bioquímicos estão envolvidos e a mobilização de reservas é decisiva para que esse processo ocorra. Embora a mobilização de reservas de sementes seja considerada um processo pós-germinativo, a mobilização das proteínas de reserva de sementes ocorre durante a germinação. Este estudo teve como objetivo quantificar as proteínas de sementes de genótipos de feijão durante os diferentes tempos de hidratação, a fim de compreender o processo de mobilização proteica e se há relação desse componente bioquímico com o vigor das sementes. Este estudo foi realizado utilizando sementes com diferentes níveis de vigor, genótipos com maior (13, 42, 55 e 81) e menor (07, 23, 44, 50, IPR-88-Uirapurú e Iapar 81) qualidade fisiológica. Os genótipos de alto vigor apresentaram maior eficiência na hidrólise e mobilização do componente proteico, pois apresentaram baixo teor de globulinas nos cotilédones na protrusão radicular em relação aos genótipos de baixo vigor (07, 23 e 50). A proteína inibidora da alfa-amilase, observada em todos os genótipos, está envolvida com o maior tempo necessário para a protrusão da radícula, de acordo com a diferença de intensidade da banda nos genótipos 07, 44 e Iapar 81.

Toxicogenomic Analysis.

The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.

Data on proteomic profiling of extracellular vesicles of Acholeplasma laidlawii strains with increased resistance to antibiotics of different classes - ciprofloxacin and tetracycline.

To elucidate the regularities of adaptation of the representatives of class Mollicutes to antimicrobials and to identify the promising targets for eradication of mycoplasma infections and contaminations the comparative analysis of the molecular basis of bacterial resistance to antibiotics of different classes is needed. Previously, we presented the data on the whole-genome sequences of Acholeplasma laidlawii strains with different susceptibility to ciprofloxacin (GenBank: LXYB00000000.1), tetracycline (GenBank: NELO00000000.2) and melittin (GenBank: NELN00000000.2) as well as the data on cell and extracellular vesicle proteomes of melittin-resistant A. laidlawii strain [1]. The lists of extracellular vesicle proteins secreted by A. laidlawii strains with the increased resistance to ciprofloxacin (PG8R10) and tetracycline (PG8RTet) are presented here. The vesicle proteome profiles were obtained by 1D SDS-PAGE and liquid chromatography-mass spectrometry.

Protocol for the Analysis of Laser Capture Microdissected Fresh-Frozen Tissue Homogenates by Silver-Stained 1D SDS-PAGE.

The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements. Collectively, the stroma forms an essential microenvironment, which is indispensable for the survival and growth of the malignant neoplasm. This cellular heterogeneity makes molecular profiling of solid tumors via mass spectrometry (MS)-based proteomics a daunting task. Laser capture microdissection (LCM) is commonly used to obtain distinct histological cell types (e.g., tumor parenchymal cells, stromal cells) from tumor tissue and attempt to address the tumor heterogeneity interference with downstream liquid chromatography (LC) MS analysis. To provide optimal LC-MS analysis of micro-scale and/or nano-scale tissue sections, we modified and optimized a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) protocol for the LC-MS analysis of LCM-procured fresh-frozen tissue specimens. Presented is a detailed in-gel digestion protocol adjusted specifically to maximize the proteome coverage of amount-limited LCM samples, and facilitate in-depth molecular profiling. Following LCM, targeted tissue sections are further fractionated using silver-stained 1D-SDS-PAGE to resolve and visualize tissue proteins prior to in-gel digestion and subsequent LC-MS analysis.

Preparation of wool follicles for proteomic studies.

A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs.

Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry.

Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC-ESI mass spectrometry. We identified 222 membrane-associated proteins, which represent approximately 24% of the predicted membrane-associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane-associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment.

Proteomic Analysis of the Protein Expression Profile in the Mature Nigella sativa (Black Seed).

Nigella sativa (N. sativa) seed has been used as an important nutritional flavoring agent and in traditional medicine for treating many illnesses since ancient times. Understanding the proteomic component of the seed may lead to enhance the understanding of its structural and biological functional complexity. In this study, we have analyzed its proteome profile based on gel-based proteome mapping technique that includes one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy. We have not come across any such studies that have been performed in N. sativa seeds up to date. A total of 277 proteins were identified, and their functional, metabolic, and location-wise annotations were carried out using the UniProt database. The majority of proteins identified in the proteome dataset based on their function were those involved in enzyme catalytic activity, nucleotide binding, and protein binding while the major cellular processes included regulation of biological process followed by regulation of secondary biological process, cell organization and biogenesis, protein metabolism, and transport. The identified proteome was localized mainly to the nucleus then to the cytoplasm, plasma membrane, mitochondria, plastid, and others. A majority of the proteins were involved in biochemical pathways involving carbohydrate metabolism, amino acid and shikimate pathway, lipid metabolism, nucleotide, cell organization and biogenesis, transport, and defense processes. The identified proteins in the dataset help to improve our understanding of the pathways involved in N. sativa seed metabolism and its biochemical features and detail out useful information that may help to utilize these proteins. This study could thus pave a way for future further high-throughput studies using a more targeted proteomic approach.

Statistical analysis of the individual variability of 1D protein profiles as a tool in ecology: an application to parasitoid venom.

Understanding the forces that shape eco-evolutionary patterns often requires linking phenotypes to genotypes, allowing characterization of these patterns at the molecular level. DNA-based markers are less informative in this aim compared to markers associated with gene expression and, more specifically, with protein quantities. The characterization of eco-evolutionary patterns also usually requires the analysis of large sample sizes to accurately estimate interindividual variability. However, the methods used to characterize and compare protein samples are generally expensive and time-consuming, which constrains the size of the produced data sets to few individuals. We present here a method that estimates the interindividual variability of protein quantities based on a global, semi-automatic analysis of 1D electrophoretic profiles, opening the way to rapid analysis and comparison of hundreds of individuals. The main original features of the method are the in silico normalization of sample protein quantities using pictures of electrophoresis gels at different staining levels, as well as a new method of analysis of electrophoretic profiles based on a median profile. We demonstrate that this method can accurately discriminate between species and between geographically distant or close populations, based on interindividual variation in venom protein profiles from three endoparasitoid wasps of two different genera (Psyttalia concolor, Psyttalia lounsburyi and Leptopilina boulardi). Finally, we discuss the experimental designs that would benefit from the use of this method.

One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).

This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins.

Silver staining of SDS-polyacrylamide gel.

To detect nanogram quantities of protein and nucleic acids on SDS-PAGE gels.