scholarly journals A Uniquely Complex Mitochondrial Proteome from Euglena gracilis

2020 ◽  
Vol 37 (8) ◽  
pp. 2173-2191 ◽  
Author(s):  
Michael J Hammond ◽  
Anna Nenarokova ◽  
Anzhelika Butenko ◽  
Martin Zoltner ◽  
Eva Lacová Dobáková ◽  
...  
Keyword(s):  
Euglena Gracilis ◽  
Translation Termination ◽  
Mitochondrial Gene ◽  
Label Free ◽  
Free Living ◽  
Ribosomal Subunits ◽  
Mitochondrial Proteome ◽  
Liquid Chromatography Tandem Mass ◽  
Emergence Of Complexity ◽  
Environmental Importance

Abstract Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

scholarly journals The Oligopeptide Transport System Is Essential for the Development of Natural Competence in Streptococcus thermophilus Strain LMD-9

2009 ◽  
Vol 191 (14) ◽  
pp. 4647-4655 ◽  
Author(s):  
Rozenn Gardan ◽  
Colette Besset ◽  
Alain Guillot ◽  
Christophe Gitton ◽  
Véronique Monnet
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Thermophilus ◽  
Defined Medium ◽  
Mass Spectrometry Analysis ◽  
Transport Systems ◽  
Label Free ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Liquid Chromatography Tandem Mass ◽  
Oligopeptide Transport

ABSTRACT In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

scholarly journals Application of Urinary Polyphenol Biomarkers Measured by Liquid Chromatography Tandem Mass Spectrometry to Assess Polyphenol Intake and Their Association with Overweight and Obesity in Free-Living Healthy Subjects

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Ruiyue Yang ◽  
Helu Xiu ◽  
Qi Zhou ◽  
Liang Sun ◽  
Hongna Mu ◽  
...  
Keyword(s):  
Regression Models ◽  
Large Scale ◽  
Epidemiologic Studies ◽  
Overweight And Obesity ◽  
Targeted Metabolomics ◽  
Free Living ◽  
Logistic Regression Models ◽  
Liquid Chromatography Tandem Mass ◽  
Polyphenol Intake ◽  
Chinese Subjects

Although some polyphenol biomarkers in serum or urine have been identified by untargeted metabolomics and proved to reflect dietary polyphenol intake, only a few of them have been validated in different studies and populations with simple and reliable targeted methods. In the present study, a targeted metabolomics method by LC/MS/MS for the measurement of twenty-two polyphenol biomarkers in urine samples was established and validated to effectively assess the habitual polyphenol intake in free-living healthy Chinese subjects. Multivariate logistic regression models were used to assess relationships of biomarkers with overweight and obesity after adjusting for potential confounders. The levels of urinary polyphenol biomarkers, especially gut microbial metabolites of polyphenols, were inversely associated with overweight and obesity, and this association was more pronounced in the inflammatory groups, suggesting that it is of great importance to maintain polyphenol biomarkers at high levels or intake-sufficient polyphenols in obesity with chronic inflammation than others. The measurement of these biomarkers may offer a valid alternative or complementary addition to self-reported survey for the evaluation of polyphenol intake and investigation into their relationships with chronic disease-related endpoints in large-scale clinical and epidemiologic studies.

scholarly journals Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins

2020 ◽  
Vol 21 (16) ◽  
pp. 5903
Author(s):  
Nicolai Bjødstrup Palstrøm ◽  
Lars Melholt Rasmussen ◽  
Hans Christian Beck
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
Plasma Proteins ◽  
Biomarker Discovery ◽  
Plasma Samples ◽  
Label Free ◽  
Affinity Capture ◽  
Liquid Chromatography Tandem Mass ◽  
Removal System ◽  
Equalization Method

In the present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the most commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MSMS) analysis. The affinity-based probes demonstrated a high reproducibility for low-abundant plasma proteins, down to picomol per mL levels, compared to the Multi Affinity Removal System (MARS) 14 and the Proteominer methods, and also demonstrated superior removal of the majority of the high-abundant plasma proteins. The ABA-based affinity probe and the Proteominer protein equalization method performed better compared to all other methods in terms of the number of analyzed proteins. All the tested methods were highly reproducible for both high-abundant plasma proteins and low-abundant proteins as measured by correlation analyses of six replicate experiments. In conclusion, our results demonstrated that small-molecule based affinity-based probes are excellent alternatives to the commonly used immune-depletion methods for proteomic biomarker discovery studies in plasma. Data are available via ProteomeXchange with identifier PXD020727.

scholarly journals A Label-Free LC/MS/MS-Based Enzymatic Activity Assay for the Detection of Genuine Caspase Inhibitors and SAR Development

2013 ◽  
Vol 18 (8) ◽  
pp. 868-878 ◽  
Author(s):  
Michel C. Maillard ◽  
Celia Dominguez ◽  
Mark J. Gemkow ◽  
Florian Krieger ◽  
Hyunsun Park ◽  
...  
Keyword(s):  
Published Data ◽  
Tandem Mass ◽  
Label Free ◽  
Activity Assay ◽  
Fluorogenic Substrates ◽  
Enzymatic Assays ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Enzymatic Activity Assay ◽  
Assay Interference

The resurgence of interest in caspases (Csp) as therapeutic targets for the treatment of neurodegenerative diseases prompted us to examine the suitability of published nonpeptidic Csp-3 and Csp-6 inhibitors for our medicinal chemistry programs. To support this effort, fluorescence-based Csp-2, Csp-3, and Csp-6 enzymatic assays were optimized for robustness against apparent enzyme inhibition caused by redox-cycling or aggregating compounds. The data obtained under these improved conditions challenge the validity of previously published data on Csp-3 and Csp-6 inhibitors for all but one series, namely, the isatins. Furthermore, in this series, it was observed that the nature of the rhodamine-labeled substrate, typically used to measure caspase activity, interfered with the pharmacological sensitivity of the Csp-2 assay. As a result, a liquid chromatography/tandem mass spectrometry–based assay that eliminates label-dependent assay interference was developed for Csp-2 and Csp-3. In these label-free assays, the activity values of the Csp-2 and Csp-3 reference inhibitors were in agreement with those obtained with the fluorogenic substrates. However, isatin 10a was 50-fold less potent in the label-free Csp-2 assay compared with the rhodamine-based fluorescence format, thus proving the need for an orthogonal readout to validate inhibitors in this class of targets highly susceptible to artifactual inhibition.

scholarly journals Exploring the Bile Stress Response of Lactobacillus mucosae LM1 through Exoproteome Analysis

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5695
Author(s):  
Bernadette B. Bagon ◽  
Ju Kyoung Oh ◽  
Valerie Diane V. Valeriano ◽  
Edward Alain B. Pajarillo ◽  
Dae-Kyung Kang
Keyword(s):  
Stress Response ◽  
Ribosomal Proteins ◽  
High Performance ◽  
Phosphoglycerate Kinase ◽  
Label Free ◽  
Beneficial Effects ◽  
Extracellular Proteome ◽  
Liquid Chromatography Tandem Mass ◽  
Lactobacillus Mucosae

Lactobacillus sp. have long been studied for their great potential in probiotic applications. Recently, proteomics analysis has become a useful tool for studies on potential lactobacilli probiotics. Specifically, proteomics has helped determine and describe the physiological changes that lactic acid bacteria undergo in specific conditions, especially in the host gut. In particular, the extracellular proteome, or exoproteome, of lactobacilli contains proteins specific to host– or environment–microbe interactions. Using gel-free, label-free ultra-high performance liquid chromatography tandem mass spectrometry, we explored the exoproteome of the probiotic candidate Lactobacillus mucosae LM1 subjected to bile treatment, to determine the proteins it may use against bile stress in the gut. Bile stress increased the size of the LM1 exoproteome, secreting ribosomal proteins (50S ribosomal protein L27 and L16) and metabolic proteins (lactate dehydrogenase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenases, among others) that might have moonlighting functions in the LM1 bile stress response. Interestingly, membrane-associated proteins (transporters, peptidase, ligase and cell division protein ftsH) were among the key proteins whose secretion were induced by the LM1 bile stress response. These specific proteins from LM1 exoproteome will be useful in observing the proposed bile response mechanisms via in vitro experiments. Our data also reveal the possible beneficial effects of LM1 to the host gut.

scholarly journals Metabolic Responses of a Model Green Microalga Euglena gracilis to Different Environmental Stresses

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiayi He ◽  
ChenChen Liu ◽  
Mengzhe Du ◽  
Xiyi Zhou ◽  
Zhangli Hu ◽  
...  
Keyword(s):  
Euglena Gracilis ◽  
High Performance ◽  
Unsaturated Fatty Acids ◽  
Metabolic Response ◽  
Environmental Stresses ◽  
Metabolic Responses ◽  
Potential Candidate ◽  
Cell Biomass ◽  
Green Microalga ◽  
Liquid Chromatography Tandem Mass

Euglena gracilis, a green microalga known as a potential candidate for jet fuel producers and new functional food resources, is highly tolerant to antibiotics, heavy metals, and other environmental stresses. Its cells contain many high-value products, including vitamins, amino acids, pigments, unsaturated fatty acids, and carbohydrate paramylon as metabolites, which change contents in response to various extracellular environments. However, mechanism insights into the cellular metabolic response of Euglena to different toxic chemicals and adverse environmental stresses were very limited. We extensively investigated the changes of cell biomass, pigments, lipids, and paramylon of E. gracilis under several environmental stresses, such as heavy metal CdCl2, antibiotics paromomycin, and nutrient deprivation. In addition, global metabolomics by Ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was applied to study other metabolites and potential regulatory mechanisms behind the differential accumulation of major high-valued metabolites. This study collects a comprehensive update on the biology of E. gracilis for various metabolic responses to stress conditions, and it will be of great value for Euglena cultivation and high-value [154mm][10mm]Q7metabolite production.

Analysis of the uterine flush fluid proteome of healthy mares and mares with endometritis or fibrotic endometrial degeneration

2020 ◽  
Vol 32 (6) ◽  
pp. 572 ◽  
Author(s):  
Mariana Diel de Amorim ◽  
Firdous A. Khan ◽  
Tracey S. Chenier ◽  
Elizabeth L. Scholtz ◽  
M. Anthony Hayes
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass ◽  
Label Free ◽  
Chromatography Tandem Mass Spectrometry ◽  
Uterine Health ◽  
Liquid Chromatography Tandem Mass ◽  
Specific Proteins ◽  
Health And Disease ◽  
Flush Fluid

The objective of this study was to evaluate the differences in the uterine flush fluid proteome between healthy mares and mares with endometritis or fibrotic endometrial degeneration (FED). Uterine flush fluid samples were collected from healthy mares (n=8; oestrus n=5 and dioestrus n=3) and mares with endometritis (n=23; oestrus n=14 and dioestrus n=9) or FED (n=7; oestrus n=6 and dioestrus n=1). Proteomic analysis was performed using label-free liquid chromatography–tandem mass spectrometry. Of 216 proteins identified during oestrus, 127 were common to all three groups, one protein was exclusively detected in healthy mares, 47 proteins were exclusively detected in mares with endometritis and four proteins were exclusively detected in mares with FED. Of 188 proteins identified during dioestrus, 113 proteins were common between healthy mares and mares with endometritis, eight proteins were exclusively detected in healthy mares and 67 proteins were exclusively detected in mares with endometritis. Quantitative analysis revealed a subset of proteins differing in abundance between the three groups during oestrus and between healthy mares and mares with endometritis during dioestrus. These results provide a springboard for evaluation of specific proteins as biomarkers of uterine health and disease and for investigation of their roles in the establishment and maintenance of pregnancy.

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