LTQ-Orbitrap Velos Pro Research Articles

Metagenomic and metaproteomic analyses of microbial amino acid metabolism during Cantonese soy sauce fermentation.

Cantonese soy sauce is an important type of traditional Chinese brewed soy sauce that was developed in southern China, mainly in Guangdong. Due to the long fermentation period and complex microbiota in Cantonese soy sauce, there are few reports on the microbial metaproteomics of Cantonese soy sauce. In this study, integrative metagenomic and metaproteomic analyzes were used to identify the changes in the dominant microbiota and amino acid synthesis-related enzymes and metabolism during Cantonese soy sauce fermentation. Metagenomic analysis revealed that Tetragenococcus halophilus, Weissella confusa, Weissella paramesenteroides, Enterobacter hormaechei, and Aspergillus oryzae were the dominant microbiota. Using the Top 15 dominant microbiota identified by metagenomics as the database, LTQ Orbitrap Velos Pro ETD mass spectrometry was used to obtain metaproteomic information about the microbes in the soy sauce, and the results indicated that the active enzymes involved in the metabolism of amino acids were secreted by microorganisms such as A. oryzae, T. halophilus, and Zygosaccharomyces rouxii. During the Cantonese soy sauce fermentation process. Among them, early fermentation (0-15d) was dominated by A. oryzae and T. halophilus, mid-term fermentation (60-90d) was dominated by Z. rouxii, A. oryzae, and T. halophilus, and late fermentation (90-120d) was dominated by A. oryzae, Z. rouxii, and T. halophilus. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the main enzymes involved in the metabolism of umami amino acids were aspartate aminotransferase, citrate synthase, aconitase, and isocitrate dehydrogenase, which were produced by Z. rouxii and A. oryzae during early fermentation (0-15 d) and the middle fermentation stage (60-90 d). This study constructed a regulatory network of enzymes potentially involved in the metabolism of flavor amino acids, which provided a theoretical basis for studying the amino acid metabolism of Cantonese soy sauce.

Comprehensive feature-based molecular networking and metabolomics approaches to reveal the differences components in Cinnamomum cassia and Cinnamomum verum.

Cinnamon was been a widely used plant in medicinal and spices for a long time and has spread all over the world. However, the differences in the components of the bark from Cinnamomum cassia and Cinnamomum verum, the two most common types of cinnamon, have not been thoroughly investigated. In the present experiment, ultra-high-performance liquid chromatography LTQ-Orbitrap Velos Pro hybrid mass spectrometer-based metabolomics coupled with chemometrics and feature-based molecular networking were employed to dramatically distinguish and annotate Cinnamomum cassia Bark and Cinnamomum verum bark. As a consequence, principal component analysis, orthogonal projection to latent structures discriminates analysis, and heat map analysis demonstrated clear discrimination between the profiles of metabolites in cinnamon. Besides, as the known compounds, proanthocyanidins (cinnamtannin B1 and procyanidin B2) and alkaloids (norboldine, norisoboldine) with variable importance in the projection scores>6, and an unknown alkaloid (formula C24 H33 NO6 ) were selected as the best markers to discriminate cinnamon. Furthermore, large numbers of proanthocyanidins and alkaloids components were identified through feature-based molecular networking for the first time. Our investigation provides new ideas for the discovery of quality markers and identification of unknown components in natural products.

Urinary proteins detected using modern proteomics intervene in early type 2 diabetic kidney disease - a pilot study.

Aim: An advanced proteomics platform for protein biomarker discovery in diabetic chronic kidney disease (DKD) was developed, validated and implemented. Materials & methods: Three Type 2 diabetes mellitus patients and three control subjects were enrolled. Urinary peptides were extracted, samples were analyzed on a hybrid LTQ-Orbitrap Velos Pro instrument. Raw data were searched using the SEQUEST algorithm and integrated into Proteome Discoverer platform. Results & discussion: Unique peptide sequences, resulted sequence coverage, scoring of peptide spectrum matches were reported to albuminuria and databases. Five proteins that can be associated with early DKD were found: apolipoprotein AI, neutrophil gelatinase-associated lipocalin, cytidine deaminase, S100-A8 and hemoglobin subunit delta. Conclusion: Urinary proteome analysis could be used to evaluate mechanisms of pathogenesis of DKD.

Isolation, molecular identification, and characterization of a unique toxic cyanobacterium Microcystis sp. found in Hunan Province, China

ABSTRACTGlobal proliferation of cyanobacterial blooms associated with climate change and eutrophication constitutes a serious environmental threat. In Hunan Province a freshwater pond located in Changsha City was found to contain high concentrations of cyanobacteria, however, the characteristics of these cyanobacteria at present are not known. This study thus aimed to isolate, identify the most common bloom-forming cyanobacteria in this region and determine the toxigenic characteristics of the predominant cyanobacteria. The cyanobacteria were isolated by serial dilution and identified using polymerase chain reaction (PCR). The cyanotoxins generated by the cyanobacterium were detected using high-performance liquid chromatography with an ultra-high resolution LTQ Orbitrap Velos Pro ETD mass spectrometry equipped with electrospray ionization interface (HPLC-ESI-MS). One species of cyanobacterium was isolated and identified as Microcystis sp. YFM1 according to the sequence of the 16S ribosome deoxyribonucleic acid (16S rDNA). It was found that this cyanobacterium contained microcystin synthetase B gene (mcyB) and produced three types of cyanotoxins including microcystin-LR, RR and YR. Our findings indicate that the Microcystis sp. YFM1 isolated from the freshwater pond in Hunan Province exhibits unique characteristics distinguishable from other known cyanobacteria.

Investigation of the global protein content from healthy human tears

Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of 8 healthy controls (4 men and 4 women, average age: 38 ± 18) were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed on a LTQ-Orbitrap Velos Pro. Globally, 1351 proteins were identified with 2 unique peptides and 1% FDR. Gene ontology analyses showed up that 39% of the tear proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins also emerged as the main tear protein families. The glycolysis and the coagulation and complement cascades, which were already shown in tears as involved in ocular and systemic diseases, were highlighted performing pathway analyses. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

Microcystin-LR degradation utilizing a novel effective indigenous bacterial community YFMCD1 from Lake Taihu

ABSTRACTMicrocystins (MC) produced by species of cyanobacteria including Microcystis, Anabaena, and Aphanizomenon are a group of monocyclic hepatotoxins posing serious threat to public health. Microcystin-LR (MC-LR) is the most toxic and frequently encountered microcystin variant in the environment, and thus removal of this toxin using bacteria was shown to be a reliable, efficient, and cost-effective method that avoids utilization of chemicals that may produce potentially harmful by-products. The aim of this study was to determine whether a novel indigenous bacterial community designated YFMCD1 was effective in destroying MC. In addition, the influence of environmental factors such as temperature, MC concentration, and pH was examined on the effectiveness of YFMCD1 to degrade MC-LR. MC-degradation products were identified by high performance liquid chromatography coupled with an ultra-high resolution LTQ Orbitrap Velos Pro ETD mass spectrometry equipped with electrospray ionization interface (HPLC-ESI-MS). MC-LR underwent maximal degradation at rate of 0.5 µg/ml/hr with YFMCD1 containing Klebsiella sp. termed YFMCD1-1 or Stenotrophomonas sp. termed YFMCD1-2. Moreover, Adda (3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid) is a constituent within the MC-LR molecule found to be responsible for biological activity expression and critical for MC-induced toxicity, which is also degraded by YFMCD1. The results showed that YFMCD1 effectively degraded MC-LR. The degradation rate was significantly affected by temperature, pH, and MC-LR concentrations. Data indicate that this bacterial community may prove beneficial in bioremediation of lakes containing MC.

Detection of busulfan adducts on proteins.

Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin. Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides. Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides. The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John Wiley & Sons, Ltd.

Exploration of human tear proteome

PurposeThe tear film is a complex structure which constitutes an interface between our eyes and the external environment. Its protein, lipid and metabolite composition is highly regulated depending on several factors. Despite its attractive characteristics, this fluid is still poorly studied. Here we present an in‐depth human tear proteome based on a mass spectrometry approach.MethodsIn this study, tears of two healthy controls (two women aged 59 and 61 years) were collected with Schirmer's papers. After trypsin digestion and off‐gel electrophoresis fractionation, two proteomic analyses were performed by mass spectrometry (using LTQ Orbitrap Velos Pro coupled to a liquid chromatography). Resulting files were searched against the UniProt‐SwissProt/TrEMBL database (version 2014_10) and a false discovery rate of 1% was selected. The protein list was analysed using Ingenuity Pathways Analysis and Cytoscape software.ResultsGlobally 2105 and 825 proteins were identified with 1 and 2 unique peptides respectively after removing keratins and immunoglobulins. Regarding the 825 proteins, the top three pathways that we highlighted were the acute phase response signalling, the remodelling of epithelial adherens junctions and the clathrin‐mediated endocytosis signalling. Moreover, we identified 203 proteins that were not found in the previous published studies. By comparing our tears with others fluids, only 26.9% were identified in vitreous humor and 45.7% in plasma, confirming that tears have a specific composition.ConclusionsThanks to this study, we are able to propose an expanded tear proteome. Both specific proteins of the tears and correlations with other fluids give them a great potential for biomarker research.This study is kindly supported by the Provisu Foundation and the SNF_MVH (PMPDP3_158370).

Mass spectrometry profiling of non-enzymatic deamidation of articular cartilage components suggests slower protein turnover in deep regions and in hips compared with knees

Mass spectrometry profiling of non-enzymatic deamidation of articular cartilage components suggests slower protein turnover in deep regions and in hips compared with knees

Elucidation of the fragmentation pathways of a complex 3,7-O-glycosyl flavonol by CID, HCD, and PQD on an LTQ-Orbitrap Velos Pro hybrid mass spectrometer

The present study was designed to systematically investigate the ESI-MSn behavior of a complex 3, 7-O-glycosyl flavonol, kaempferol 3-O-α-L-[2,3-di-O-β-D-(6-E-p-coumaroyl)glucopyranosyl]-rhamnopyranosyl-7-O-α-L-rhamnopyranoside (KO) isolated from Epimedium wushanense, and to address the elimination priority among different glycosylation sites and different sugars/substituents. The direct-infusion ESI-MSn experiment of KO was performed on a hybrid LTQ-Orbitrap Velos Pro mass spectrometer in both negative and positive ion modes by three different fragmentation mechanisms (CID, HCD, and PQD). The CID, HCD, and PQD analyses of KO exhibited remarkable discrimination in respect of the scan range, richness, and distribution of product ions through the entire spectra. KO experienced different fragmentation pathways between two ionization modes: the negative mode CID of KO eliminated the glycosyl portions (priority: 7-sugar > 3-substituent and terminal substituents > inner sugar) and produced aglycone product ions at m/z 284.03/285.04; however, abundant sodium-adduct B32 together with subsequent i,jX30 cleavages were found characteristic for the positive mode CID-MSn. The fragmentation pathways by CID for KO were proposed by analyzing the high accuracy ESI-MSn data. Complementary structural information of KO regarding the aglycone and glycosyl portions was obtained by analyzing the ESI-MSn data in both ionization modes. In conclusion, the LTQ-Orbitrap method facilitates highly reliable qualitative analysis of bioactive flavonoids with three alternative fragmentation modes.

Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.

New triterpenic acids and alkaloids from Uncaria rhynchophylla and their CID studies by use of an LTQ-Orbitrap Velos Pro mass spectrometer

New triterpenic acids and alkaloids from Uncaria rhynchophylla and their CID studies by use of an LTQ-Orbitrap Velos Pro mass spectrometer

CID, HCD and PQD of a complex 3,7-O-glycosyl flavonol by an LTQ-Orbitrap Velos Pro hybrid mass spectrometer

CID, HCD and PQD of a complex 3,7-O-glycosyl flavonol by an LTQ-Orbitrap Velos Pro hybrid mass spectrometer

High resolution is not a strict requirement for characterization and quantification of histone post-translational modifications.

Mass spectrometry (MS) is a powerful tool to accurately identify and quantify histone post-translational modifications (PTMs). High-resolution mass analyzers have been regarded as essential for these PTM analyses because the mass accuracy afforded is sufficient to differentiate trimethylation versus acetylation (42.0470 and 42.0106 Da, respectively), whereas lower-resolution mass analyzers cannot. Noting this limitation, we sought to determine whether lower-resolution detectors are nonetheless adequate for histone PTM analysis by comparing the low-resolution LTQ Velos Pro with the high-resolution LTQ-Orbitrap Velos Pro. We first determined that the optimal scan mode on the LTQ Velos Pro is the Enhanced scan mode with respect to apparent resolution, number of MS and MS/MS scans per run, and reproducibility of label-free quantifications. We next compared the performance of the LTQ Velos Pro to the LTQ-Orbitrap Velos Pro using the same criteria for comparison, and we found that the main difference is that the LTQ-Orbitrap Velos Pro is able to resolve the difference between acetylation and trimethylation while the LTQ Velos Pro cannot. However, using heavy isotope labeled synthetic peptide standards and retention time information enables confident assignment of these modifications and comparable quantification between the instruments. Therefore, lower-resolution instruments can confidently be utilized for histone PTM analysis.

Targeted proteomics analysis of protein degradation in plant signaling on an LTQ-Orbitrap mass spectrometer.

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.

Gel-based mass spectrometric analysis of hippocampal transmembrane proteins using high resolution LTQ Orbitrap Velos Pro.

Membrane proteins (MPs) play diverse important roles for physical interactions, cell communication, molecular transport, and signal transduction. Membrane proteins comprise approximately 25∼35% of the genome in living organisms, but there are difficulties in the analysis at the protein chemical level, in particular due to low abundance and limited solubility. Sequence information on membrane proteins and their complexes would be beneficial to elucidate their function. Proteins were extracted from pooled whole mouse brains, enriched membrane fractions were prepared using either two commercially available kits or 6-aminocaproic acid under denaturing or native conditions followed by gel-based proteomic approaches using blue native (BN-) and SDS-PAGE with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin followed by nano-LC-ESI-MS/MS analysis on LTQ Orbitrap Velos Pro. By combining three different extraction methods and two separation methods, 28.39% of proteins were identified as either "integral" or "anchored/integral" MPs based on UniProtKB database searches. MPs with more than six transmembrane domains (TMDs) were identified more efficiently from BN-PAGE separation although a higher number of proteins was identified from SDS-PAGE separation. Comparative analysis of MPs containing TMDs via gel-based LC-MS/MS using BN-PAGE and SDS-PAGE may be useful to increase the number of identified membrane proteins in brain. All MS data have been deposited in the ProteomeXchange with identifier PXD000311 (http://proteomecentral.proteomexchange.org/dataset/PXD000311).

SAT0574 Peptidomic Analysis of Cartilage Secretome for the Discovery of Early OA Biomarkers

BackgroundThe low-molecular-weight subset of the cartilage proteome (also termed cartilage peptidome) may have a diagnostic potential in osteoarthritis (OA) research, not yet fully exploited.ObjectivesThe aim of this study is to...