Molecular weight assessment of proteins in total proteome profiles using 1D-PAGE and LC/MS/MS

BACKGROUND: The observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures. RESULTS: We have developed a set of computational tools for extracting molecular weight information of intact proteins from total proteome profiles in a high throughput manner using 1D-PAGE and LC/MS/MS. We have applied this technology to the proteome profile of a human lymphoblastoid cell line under standard culture conditions. From a total of 1 × 10(7 )cells, we identified 821 proteins by at least two tryptic peptides. Additionally, these 821 proteins are well-localized on the 1D-SDS gel. 656 proteins (80%) occur in gel slices in which the observed molecular weight of the protein is consistent with its predicted full-length sequence. A total of 165 proteins (20%) are observed to have molecular weights that differ from their predicted full-length sequence. We explore these molecular-weight differences based on existing protein annotation. CONCLUSION: We demonstrate that the determination of intact protein molecular weight can be achieved in a high-throughput manner using 1D-PAGE and LC/MS/MS. The ability to determine the molecular weight of intact proteins represents a further step in our ability to characterize gene expression at the protein level. The identification of 165 proteins whose observed molecular weight differs from the molecular weight of the predicted full-length sequence provides another entry point into the high-throughput characterization of protein modification

Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems

A novel hybrid approach of activated carbon and ultrasound cavitation for the intensification of palm oil mill effluent (POME) polishing

This investigation focuses on activated carbon (AC) adsorption and ultrasound (US) cavitation for polishing the palm oil mill effluent (POME). Both AC adsorption and US cavitation were investigated individually, in series and operating them in a combined way. The efficiency of above processes has been evaluated in terms of removal of chemical oxygen demand (COD) and total suspended solids (TSS). For the individual operation, the optimisation studies were carried out by using the following conditions: AC dosage (50–200 g/L); contact time (2, 4, 6 h); US power amplitude (50% and 80%) and US cavitation time (30–180 min). The optimisation studies utilising US power amplitude (50%) and cavitation time (15 min) followed by AC adsorption using minimum AC dosage (50 g/L) and contact time (30 min) resulted in ∼100% COD and 83.33% TSS removals which meets the discharge limits set by the Department of Environment (DoE), Malaysia. The hybrid operation was also studied by simultaneously employing AC adsorption and US cavitation and it was observed that an adsorption dosage of 50 g/L resulted into achieving 73.08% COD and 98.33% TSS removals within 15 min of US irradiation. With the possibility of continuous and feasible sonochemical reactors, this hybrid approach of US cavitation followed by AC adsorption could be an alternative processing technique for POME polishing

Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification

Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types

Background: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. Results: Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology. Conclusions: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types. Electronic supplementary material The online version of this article (10.1186/s12915-018-0527-2) contains supplementary material, which is available to authorized users

Comparison of Proteomic and Transcriptomic Profiles in the Bronchial Airway Epithelium of Current and Never Smokers

Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear.Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in <or=5% of airway samples from a previously published dataset.1D-PAGE coupled with LC-MS/MS effectively profiled the airway epithelium proteome and identified proteins expressed at different levels as a result of cigarette smoke exposure. While there was a strong correlation between protein and transcript detection within the same sample, we also identified proteins whose corresponding transcripts were not detected by microarray. This noninvasive approach to proteomic profiling of airway epithelium may provide additional insights into the field of injury induced by tobacco exposure

Can mHealth Technology Help Mitigate the Effects of the COVID-19 Pandemic?

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Transfer of Guardian’s Authority of Marriage to the Hand of the Judge: A Jurisprudential Field Study in the Marriage of Some Malaysians in Southern Thailand: انتقال ولاية النكاح إلى الحاكم: دراسة فقهية ميدانية في زواج بعض الماليزيين في جنوب تايلاند

The study aims to discuss the topic of " Transfer of guardian’s authority of marriage to the hand of the judge: a jurisprudential field study in the marriage of some Malaysians in southern Thailand." This is due to a trend among Malaysian Muslims to marry by transferring the guardianship to the Judge as they travel outside the country with the intention of transferring the right of guardianship of marriage from their guardians to the Judge and seek his services in concluding the marriage contract. This marriage, in which the guardian is purposefully absent, violates the right of the guardian of lineage to give his ward in marriage. It also violates what the majority of the scholars from the past and present held, that the guardian is a requirement for the validity of a marriage. Southern Thailand is considered one of the most desirable destinations and one of the primary options for many Malaysians who want to marry in this manner. Therefore, the authors decided to study the ruling about the marriage of Malaysians with the intention of abandoning the right of the guardian of lineage to give his ward in marriage and transferring it to the ruler in southern Thailand. The authors employed inductive and analytical approaches. The authors carried out a field study by conducting personal interviews to learn about the opinions of some scholars, judges, lawyers, muftis, and academic experts from the relevant area. The authors have divided the research topic into two primary components; the first part highlights the concept of transfer of marriage guardianship from an Islamic juristic perspective; while the second part investigates the reasons that contribute to the spread of this ugly phenomenon in Malaysian society and proposes some solutions to address this issue. One of the most important findings of this study is that this phenomenon is one of the factors causing the destruction of family relationships in particular, and the moral value system of society in general