Shotgun Approach Research Articles

Data- and diversity-driven development of a Shotgun crystallization screen using the Protein Data Bank.

Protein crystallization has for decades been a critical and restrictive step in macromolecular structure determination via X-ray diffraction. Crystallization typically involves a multi-stage exploration of the available chemical space, beginning with an initial sampling (screening) followed by iterative refinement (optimization). Effective screening is important for reducing the number of optimization rounds required, reducing the cost and time required to determine a structure. Here, an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) is proposed and compared with the previously derived (2014) Shotgun I screen. In an update to that analysis, it is clarified that the Shotgun approach entails finding the crystallization conditions that cover the most diverse space of proteins by sequence found in the PDB, which can be mapped to the well known maximum coverage problem in computer science. With this realization, it was possible to apply a more effective algorithm for selecting conditions. In-house data demonstrate that compared with alternatives, the Shotgun I screen has been remarkably successful over the seven years that it has been in use, indicating that Shotgun II is also likely to be a highly effective screen.

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The crystallomics pipeline, a shotgun approach on native proteomes to (re)discover the unsuspected

The crystallomics pipeline, a shotgun approach on native proteomes to (re)discover the unsuspected

Aquaculture industry prospective from gut microbiome of fish and shellfish: An overview.

The microbiome actually deals with micro-organisms that are associated with indigenous body parts and the entire gut system in all animals, including human beings. These microbes are linked with roles involving hereditary traits, defence against diseases and strengthening overall immunity, which determines the health status of an organism. Considerable efforts have been made to find out the microbiome diversity and their taxonomic identification in finfish and shellfish and its importance has been correlated with various physiological functions and activities. In recent past due to the availability of advanced molecular tools, some efforts have also been made on DNA sequencing of these microbes to understand the environmental impact and other stress factors on their genomic structural profile. There are reports on the use of next-generation sequencing (NGS) technology, including amplicon and shot-gun approaches, and associated bioinformatics tools to count and classify commensal microbiome at the species level. The microbiome present in the whole body, particularly in the gut systems of finfish and shellfish, not only contributes to digestion but also has an impact on nutrition, growth, reproduction, immune system and vulnerability of the host fish to diseases. Therefore, the study of such microbial communities is highly relevant for the development of new and innovative bio-products which will be a vital source to build bio and pharmaceutical industries, including aquaculture. In recent years, attempts have been made to discover the chemical ingredients present in these microbes in the form of biomolecules/bioactive compounds with their functions and usefulness for various health benefits, particularly for the treatment of different types of disorders in animals. Therefore, it has been speculated that microbiomes hold great promise not only as a cure for ailments but also as a preventive measure for the number of infectious diseases. This kind of exploration of new breeds of microbes with their miraculous ingredients will definitely help to accelerate the development of the drugs, pharmaceutical and other biological related industries. Probiotic research and bioinformatics skills will further escalate these opportunities in the sector. In the present review, efforts have been made to collect comprehensive information on the finfish and shellfish microbiome, their diversity and functional properties, relationship with diseases, health status, data on species-specific metagenomics, probiotic research and bioinformatics skills. Further, emphasis has also been made to carry out microbiome research on priority basis not only to keep healthy environment of the fish farming sector but also for the sustainable growth of biological related industries, including aquaculture.

Data- and diversity-driven development of a Shotgun crystallisation screen using the Protein Data Bank

Protein crystallisation has for decades been a critical and restrictive step in macro-molecular structure determination via X-ray diffraction. Crystallisation typically involves a multi-stage exploration of the available chemical space, beginning with an initial sampling (screening) followed by iterative refinement (optimisation). Effective screening is important for reducing the number of optimisation rounds required, reducing the cost and time required to determine a structure. Here, we propose an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) and compare it with the previously derived (2014) Shotgun I screen. In an update to that analysis, we clarify that the Shotgun approach entails finding the crystallisation conditions which cover the most diverse space of proteins by sequence found in the PDB - which can be mapped to the well known Maximum Coverage problem in computer science. With this realisation we are able to apply a more effective algorithm for selecting conditions, such that the Shotgun II screen outperforms the Shotgun I screen both in protein coverage and quantity of data input. Our data demonstrates that the Shotgun I screen, compared with alternatives, has been remarkably successful over the seven years it has been in use, indicating that Shotgun II is likely to be a highly effective screen.

Novel Techniques to Improve Precise Cell Injection.

We noted recently that the injection of cells with a needle through a cystoscope in the urethral sphincter muscle of pigs failed to deposit them nearby or at the intended target position in about 50% of all animals investigated (n > 100). Increasing the chance for precise cell injection by shotgun approaches employing several circumferential injections into the sphincter muscle bears the risk of tissue injury. In this study, we developed and tested a novel needle-free technique to precisely inject cells in the urethral sphincter tissue, or other tissues, using a water-jet system. This system was designed to fit in the working channels of endoscopes and cystoscopes, allowing a wide range of minimally invasive applications. We analyze key features, including the physical parameters of the injector design, pressure ranges applicable for tissue penetration and cell injections and biochemical parameters, such as different compositions of injection media. Our results present settings that enable the high viability of cells post-injection. Lastly, the method is suitable to inject cells in the superficial tissue layer and in deeper layers, required when the submucosa or the sphincter muscle of the urethra is targeted.

Impact of cropping systems on the functional diversity of rhizosphere microbial communities associated with maize plant: a shotgun approach.

Understanding the functions carried out by rhizosphere microbiomes will further explore their importance in biotechnological improvement and agricultural sustainability. This study presents one of the foremost attempts to understand the functional diversity of the rhizosphere microbiome in mono-cropping and crop rotation farming sites using shotgun metagenomic techniques. We hypothesized that the functional diversity would vary in the cropping sites and more abundant in the rotational cropping site. Hence, we carried out complete DNA extraction from the bulk and rhizospheric soils associated with maize plant cultivated on the mono-cropping farm (LT and LTc) and the crop rotation farm (VD and VDc), respectively, and sequenced employing shotgun approach. Using the SEED subsystem, our result revealed that a total of 24 functional categories dominated the rotational cropping site, while four functional categories dominated the mono-cropping sites. Alpha diversity assessment showed that no significant difference (p > 0.05) was observed across the cropping sites, while beta diversity assessment revealed a significant difference. Going by the high abundance of functional groups observed in the samples from the crop rotational site, it is evident that cropping systems influenced the functions of soil microbiomes. Worthy of note is the high abundance of unknown functions associated with these maize rhizosphere microbiomes. This is an indication that there are still some under-investigated functional genes associated with the maize rhizosphere microbiome. It is, therefore, imperative that further studies explore these functional genes for their agricultural and biotechnological potentials.

Lipin Expression Correlates to Differences in Phospholipid Profiles in Prostate Cancer Progression

PURPOSE The association between clinical outcomes of prostate cancer progression and lipid remodeling is not well understood. Although it is known that increases in select circulating lipids correlate to decreased patient survival, the mechanisms mediating alterations in these lipids are not fully explained. We addressed this gap-in-knowledge using in vitro models of non-cancerous, hormone-sensitive, and castration-resistant prostate cancer (CRPC) cell lines combined with quantitative HPLC-ESI-Orbitrap-MS lipidomic analysis. HYPOTHESIS We hypothesized that the expression of lipin (phosphatidic acid phosphatase), an enzyme mediating the conversion of phosphatidic acid to diacylglycerol, correlates to changes in lipidomic profiles in prostate cancer cells. We further hypothesized that changes in this enzyme influences the overall survivability of prostate cancer patients. METHODS Lipids were isolated using the Bligh-Dyer extraction from non-cancerous, hormone-sensitive, and CRPC cell lines. Lipids present in both cells and media were initially analyzed using an untargeted shotgun approach (ESI-MS), followed by a targeted-based quantitative HPLC-ESI-Orbitrap-MS approach. Post data acquisition for both shotgun and HPLC-ESI/MS/MS lipidomics were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency-based corrections. Online resources and software's utilized included MetaboAnalyst, LIPIDMATCH, LIPID MAPS, XCMS, and MZmine. Gene alteration frequency and overall survival data were generated using cBioPortal for Cancer Genomics and GEPIA 2. Lipin expression in cells was determined using immunoblot analysis. RESULTS Our data showed significant changes in the abundance of lipid species between the various cell lines and a correlation between select lipid species and the progression of prostate cancer. Prominent among these were the levels of phosphatidic acid (PA) and diacylglycerol (DAG), a process controlled by lipin. Most notably, PA levels in cells tended to inversely correlate with DAG levels. These levels correlated to the decreased expression on lipin, specifically lipin-1, suggesting that decreased lipin-1 expression correlates to more aggressive prostate cancer. This hypothesis was supported by analysis of alteration in lipin genes expressed in numerous cohorts of prostate cancer patients, demonstrating that lipin mutations indicated a decrease in the overall survival of prostate cancer patients. CONCLUSIONS Distinct phospholipid classes were observed to have increased levels in prostate cancer cells, as compared to non-cancer cells. Preliminary data shows a correlation between the abundance of PA and DAG with lipin expression in cells representing more aggressive prostate cancers, which is supported by data from patient populations, suggesting that altered lipin expression correlates to decreased survival.

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach.

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more “natural” and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010–2011 and 2011–2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier <PXD024204>.

Grape Seeds Proanthocyanidins: Advanced Technological Preparation and Analytical Characterization.

A “green” solvent-free industrial process (patent pending) is here described for a grape seed extract (GSE) preparation (Ecovitis™) obtained from selected seeds of Veneto region wineries, in the northeast of Italy, by water and selective tangential flow filtration at different porosity. Since a comprehensive, non-ambiguous characterization of GSE is still a difficult task, we resorted to using an integrated combination of gel permeation chromatography (GPC) and electrospray ionization high resolution mass spectrometry (ESI-HRMS). By calibration of retention time and spectroscopic quantification of catechin as chromophore, we succeeded in quantifying GPC polymers up to traces at n = 30. The MS analysis carried out by the ESI-HRMS method by direct-infusion allows the detection of more than 70 species, at different polymerization and galloylation, up to n = 13. This sensitivity took advantage of the nanoscale shotgun approach, although paying the limit of missed separation of stereoisomers. GPC and MS approaches were remarkably well cross-validated by overlapping results. This simple integrated analytical approach has been used for quality control of the production of Ecovitis™. The emerging feature of Ecovitis™ vs. a popular benchmark in the market, produced by a different technology, is the much lower content of species at low n and the corresponding increase of species at high n.

Unveiling the putative functional genes present in root-associated endophytic microbiome from maize plant using the shotgun approach.

To ensure food security for the ever-increasing world's population, it is important to explore other alternatives for enhancing plant productivity. This study is aimed at identifying the putative plant growth-promoting (PGP) and endophytic gene clusters in root-associated endophytic microbes from maize root and to also verify if their abundance is affected by different farming practices. To achieve this, we characterize endophytic microbiome genes involved in PGP and endophytic lifestyle inside maize root using the shotgun metagenomic approach. Our results revealed the presence of genes involved in PGP activities such as nitrogen fixation, HCN biosynthesis, siderophore, 4-hydroxybenzoate, ACC deaminase, phenazine, phosphate solubilization, butanediol, methanol utilization, acetoin, nitrogen metabolism, and IAA biosynthesis. We also identify genes involved in stress resistance such as glutathione, catalase, and peroxidase. Our results further revealed the presence of putative genes involved in endophytic behaviors such as aerotaxis, regulator proteins, motility mechanisms, flagellum biosynthesis, nitrogen regulation, regulation of carbon storage, formation of biofilm, reduction of nitric oxide, regulation of beta-lactamase resistance, type III secretion, type IV conjugal DNA, type I pilus assembly, phosphotransferase system (PTS), and ATP-binding cassette (ABC). Our study suggests a high possibility in the utilization of endophytic microbial community for plant growth promotion, biocontrol activities, and stress mitigation. Further studies in ascertaining this claim through culturing of the beneficial isolates as well as pot and field experiments are necessary.

The development of mature gait patterns in children during walking and running

PurposeWe sought to identify the developing maturity of walking and running in young children. We assessed gait patterns for the presence of flight and double support phases complemented by mechanical energetics. The corresponding classification outcomes were contrasted via a shotgun approach involving several potentially informative gait characteristics. A subsequent clustering turned out very effective to classify the degree of gait maturity.MethodsParticipants (22 typically developing children aged 2–9 years and 7 young, healthy adults) walked/ran on a treadmill at comfortable speeds. We determined double support and flight phases and the relationship between potential and kinetic energy oscillations of the center-of-mass. Based on the literature, we further incorporated a total of 93 gait characteristics (including the above-mentioned ones) and employed multivariate statistics comprising principal component analysis for data compression and hierarchical clustering for classification.ResultsWhile the ability to run including a flight phase increased with age, the flight phase did not reach 20% of the gait cycle. It seems that children use a walk-run-strategy when learning to run. Yet, the correlation strength between potential and kinetic energies saturated and so did the amount of recovered mechanical energy. Clustering the set of gait characteristics allowed for classifying gait in more detail. This defines a metric for maturity in terms of deviations from adult gait, which disagrees with chronological age.ConclusionsThe degree of gait maturity estimated statistically using various gait characteristics does not always relate directly to the chronological age of the child.

Application and optimization of label-free shotgun approaches in the study of Quercus ilex

Advances in proteomic equipment, algorithms and wet protocols are being increasingly reported. Each step in the experimental workflow must be adapted and optimized to the target experimental system and objectives. The influence of the amount of peptides loaded onto the column in shotgun platforms has rarely been considered to date even though it dictates the confidence with which proteins can be identified and quantified. An experiment using variable dilutions of protein equivalent mixtures of root, leaf and seed tissue extracts of Quercus ilex was performed by subjecting BSA protein equivalent amounts of 1–100 μg to SDS-PAGE, the resulting bands being trypsin digested and peptides (10–1000 ng protein equivalents) loaded onto an LC column. Mass spectra were used to identify proteins against the in-house Q. ilex transcriptome database. Determinations included SEQUEST quantification (average of the three most abundant distinct peptides for each protein) and proteotypic peptides. The number of proteins identified was found to depend on peptide load and to peak at 2054 with 600 ng. Smaller loads led to linearly decreasing identifications from 1859 with 400 ng to 495 with 10 ng. Both quantification strategies provided similar results. The linear dynamic range was from 100 to 600 ng.

GeLC-Orbitrap/MS and 2-DE-MALDI-TOF/TOF comparative proteomics analysis of seed cotyledons from the non-orthodox Quercus ilex tree species

Gel electrophoresis-based and shotgun approaches are the most employed proteomic platforms in plant biology research, with the latter replacing the former in the last years. We have compared 2-DE-MALDI-TOF/TOF and GeLC-Orbitrap/MS analyses using the same protein extracts from Quercus ilex cotyledons at different development stages. The results obtained (ProteomeXchange available data, PXD020603) showed that both platforms were complementary, showing common and specific proteins identified in each case, but leading to similar biological conclusions. Protein analysis identified 562 spots in gel-based (292 variables) and 2409 proteins in shotgun (560 variables), that were detected with both platforms and represent common key pathways related to maturation and germination. The main differences concern hormone metabolism, storage and late embryogenesis abundant proteins. Deeper proteome coverage was obtained with the shotgun approach, with a greater number of metabolic pathways represented, as gibberellin biosynthesis, not observed in the gel-based analysis. Nevertheless, several storage proteins, highly abundant in cotyledons and well represented in gel-based platform were not identified using the shotgun platform. These results support that when analyzing any plant biological process, the use of both platforms is complementary rather than redundant, that favors an in-depth proteomic analysis and a more confident biological interpretation of the data obtained.

PROTEIN IDENTIFICATION USING SEQUENCE DATABASES

The bottom-up proteomics approach (also known as the shotgun approach), based on the digestion of proteins in peptides and their sequencing using tandem mass spectrometry (MS/MS), has become widespread. The identification of peptides from the obtained MS/MS data is most often done using available sequence databases. In this paper, we present a detailed overview of the peptide identification workflow and description of the main protein bioinformatics databases. Choosing the correct search parameters and the sequence database is essential to the success of this method, and we pay special attention to the practical aspects of searching for efficient analysis of MS/MS spectra. We also consider possible reasons why database search tools cannot find the correct sequence for some MS/MS spectra, and highlight the issues of misidentification that can significantly reduce the value of published data. To help assess the assignment of peptides to MS/MS spectra, we will look at the scoring algorithms that are used in the most popular database search tools. We also analyze statistical methods and computational tools for validating peptide compliance with MS/MS data. The final part describes the process of determining the identity of protein samples from a list of peptide identifications and discusses the limitations of bottom-up proteomics

Taxonomic Binning Approaches and Functional Characteristics of the Microbial Community during the Anaerobic Digestion of Hydrolyzed Corncob

Maize forms the basis of Mexican food. As a result, approximately six million tons of corncob are produced each year, which represents an environmental issue, as well as a potential feedstock for biogas production. This research aimed to analyze the taxonomic and functional shift in the microbiome of the fermenters using a whole metagenome shotgun approach. Two strategies were used to understand the microbial community at the beginning and the end of anaerobic digestion: (i) phylogenetic analysis to infer the presence and coverage of clade-specific markers to assign taxonomy and (ii) the recovery of the individual genomes from the samples using the binning of the assembled scaffolds. The results showed that anaerobic digestion brought some noticeable changes and the main microbial community was composed of Corynebacterium variable, Desulfovibrio desulfuricans, Vibrio furnissii, Shewanella spp., Actinoplanes spp., Pseudoxanthomonas spp., Saccharomonospora azurea, Agromyces spp., Serinicoccus spp., Cellulomonas spp., Pseudonocardia spp., Rhodococcus rhodochrous, Sphingobacterium spp. Methanosarcina mazei, Methanoculleus hydrogenitrophicus, Methanosphaerula spp., Methanoregula spp., Methanosaeta spp. and Methanospirillum spp. This study provides evidence of the drastic change in the microbial community structure in a short time and the functional strategy that the most representative microorganisms of the consortia used to carry out the process.

Shotgun Proteomics and Mass Spectrometry as a Tool for Protein Identification and Profiling of Bio-Carrier-Based Therapeutics on Human Cancer Cells.

Shotgun proteomics has been widely applied to study proteins in complex biological samples. Combination of high-performance liquid chromatography with mass spectrometry has allowed for comprehensive protein analysis with high resolution, sensitivity, and mass accuracy. Prior to mass spectrometry analysis, proteins are extracted from biological samples and subjected to in-solution trypsin digestion. The digested proteins are subjected for clean-up and injected into the liquid chromatography-mass spectrometry system for peptide mass identification. Protein identification is performed by analyzing the mass spectrometry data on a protein search engine software such as PEAKS studio loaded with protein database for the species of interest. Results such as protein score, protein coverage, number of peptides, and unique peptides identified will be obtained and can be used to determine proteins identified with high confidence. This method can be applied to understand the proteomic changes or profile brought by bio-carrier-based therapeutics in vitro. In this chapter, we describe methods in which proteins can be extracted for proteomic analysis using a shotgun approach. The chapter outlines important in vitro techniques and data analysis that can be applied to investigate the proteome dynamics.

Epipelagic microbiome of the Small Aral Sea: Metagenomic structure and ecological diversity.

Microbial diversity studies regarding the aquatic communities that experienced or are experiencing environmental problems are essential for the comprehension of the remediation dynamics. In this pilot study, we present data on the phylogenetic and ecological structure of microorganisms from epipelagic water samples collected in the Small Aral Sea (SAS). The raw data were generated by massive parallel sequencing using the shotgun approach. As expected, most of the identified DNA sequences belonged to Terrabacteria and Actinobacteria (40% and 37% of the total reads, respectively). The occurrence of Deinococcus‐Thermus, Armatimonadetes, Chloroflexi in the epipelagic SAS waters was less anticipated. Surprising was also the detection of sequences, which are characteristic for strict anaerobes—Ignavibacteria, hydrogen‐oxidizing bacteria, and archaeal methanogenic species. We suppose that the observed very broad range of phylogenetic and ecological features displayed by the SAS reads demonstrates a more intensive mixing of water masses originating from diverse ecological niches of the Aral‐Syr Darya River basin than presumed before.

Metagenomic Approach to Bacterial Diversity and Lipolytic Enzymes’ Genes from a Steam Soil of Los Humeros Geothermal Field (Puebla, México)

Los Humeros volcanic complex, located at the eastern end of the Trans Mexican Volcanic Belt, possess superficial thermal manifestations (fumaroles, heat steam-soils and alteration zones), with temperatures between 50 °C and 90 °C. This is the first work to explore the bacterial diversity of a steam soil of Los Humeros geothermal field using a metagenomic 16S RNA gene analysis, and the presence of lipolytic enzymes’ genes after an enrichment culture by a whole-genome shotgun approach. High diversity indexes were found in soil samples, with Firmicutes, Chloroflexi, Proteobacteria, Actinobacteria and Acidobacteria as the most prevalent phyla. Alicyclobacillus was the most prevalent genus at 15 cm and Anoxybacillus at 50 cm depth; anaerobic and obligate thermophilic and hyperthermophilic members were found at 50 cm depth. Firmicutes was favored by the oil-rich medium, and the dominant genera were Geobacillus and Caldibacillus. 165 genes related to lipolytic enzymes were found; carboxilesterases and phospholipases C were the most abundant, and 43% were assigned to uncharacterized lipolytic enzymes. The diversity as well as the high percentage of non-characterized lipolytic enzymes found after soil enrichment in an oil-rich medium suggest the presence of biotechnologically promising organisms and enzymes that deserve to be investigated.

Sequenced application of glutathione as an antioxidant with an organic biostimulant improves physiological and metabolic adaptation to salinity in wheat

Globally, salinity threatens the agricultural crops productivity by inhibiting plant growth and development through osmotic stress and ionic cytotoxicity. The polygenic nature of salinity offers several pragmatic shotgun approaches to improve salinity tolerance. The present study investigated the potential of glutathione (GSH; 1 mM) as an antioxidant and moringa leaf extract (MLE; 3%) as an organic biostimulant applied in sequence as seed priming and foliar spray on wheat growth, physiology and metabolic adaptation under saline conditions (9.16 dS m−1). Plants without any treatment and water spray (H2O) were considered controls. Salinity induced osmotic stress reduced the plant tissue water status and photosynthetic performance, and perturbed ionic (K+/Na+, Ca2+/Na+, K++Ca2+/Na+) and hormonal (IAA, GA3, zeatin, ABA) homeostasis, consequently affected growth and yield in wheat. Sequenced applied MLE and/or GSH improved osmotic stress tolerance by stabilizing membrane integrity and decreasing electrolyte leakage. These positive results were owed to enhanced endogenous GSH and ascorbate levels. Improved tissue water status was attributed to increased osmotic adjustment, better ionic and hormonal homeostasis contributed to improving photosynthetic efficiency and growth under salinity. Exogenously applied MLE and GSH sequences improved grain yield, which was attributed to the maintenance of green leaf area and delayed senescence associated with an increase in photosynthetic pigments and chlorophyll fluorescence traits. In crux, exogenous applied MLE and/or GSH can be the best physiological strategy to reduce the deleterious effects of salinity and improve physiological and metabolic adaptation in wheat under saline field conditions.