Mass Spectrometry Profiling Research Articles

Combined Study of Transcriptome and Metabolome Reveals Involvement of Metabolites and Candidate Genes in Flavonoid Biosynthesis in Prunus avium L.

Sweet cherry (Prunus avium L.) is a popular fruit tree grown for its juicy fruit and pleasing appearance. The fruit pf the sweet cherry contains active antioxidants and other chemical compounds essential for human health. For this study, we performed the transcriptomics and metabolomics analysis using young Green Peel (GP) and mature Red Peel (RP) from sweet cherries to understand the underlying genetic mechanism regulating fruit development and ripening. Using high-throughput RNA sequencing and ultra-performance liquid chromatography, with quadrupole time-of-flight tandem mass spectrometry, respectively, metabolic and transcript profiling was obtained. Relative to GP, there were equal quantities of pronouncedly varied metabolites in RP (n = 3564). Differentially expressed genes (DEGs, n = 3564), containing 45 transcription factor (TF) families, were recorded in RP. Meanwhile, 182 differentially expressed TF (DETF) members of 37 TF families, were displayed in abundance in RP compared to GP sweet cherries. The largest quantities of DETFs were members of the ERF (25) and basic helix–loop–helix (bHLH) (19) families, followed by the MYB (18), WRKY (18), and C2H2 (12) families. Interestingly, most ERF genes were down-regulated, whereas CCCH genes were mainly up-regulated in RP. Other DETFs exhibited significant variations. In addition, RT-QPCR results and metabolomics data together with transcriptomic data revealed that the abundance of catechin, epicatechin, rhoifolin, myricetin, keracyanin, and the other six glycosyltransferase genes was highly increased in RP when compared to GP sweet cherries. The relatively higher expression of DETFs, metabolite, and flavonoid biosynthesis in RP sweet cherries suggests the accumulation of distinct metabolites that cause red coloring during fruit development and ripening. Thus, the metabolomics and transcriptomic analysis of the current study are powerful tools for providing more valuable information for the metabolic engineering of flavonoids biosynthesis in sweet cherries. They are also helpful in understanding the relationship between genotype and phenotype.

Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms.

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Effects of Silane Monolayers on Lysophosphatidylcholine (LysoPC) Detection by Desorption Ionization on Silicon Mass Spectrometry (DIOS-MS) in Solution and Plasma

Desorption ionization on silicon mass spectrometry (DIOS-MS) enables high throughput analysis of low-molecular-weight biomolecules. However, detection of metabolite biomarkers in complex fluids such as plasma requires sample pretreatment, limiting clinical application. Here, we show that porous silicon, chemically modified using monolayers of n-propyldimethylmethoxysilane molecules, is a good candidate for fingerprinting lysophosphatidylcholine (lysoPC) in plasma, without sample pretreatment, for DIOS-MS-based diagnosis (e.g., sepsis). Results were correlated to lysoPC molecule location inside/outside the pores, determined by time-of-flight secondary ion mass spectrometry profiling, and to physicochemical properties.

Hydroxysafflor yellow A protects against colitis in mice by suppressing pyroptosis via inhibiting HK1/NLRP3/GSDMD and modulating gut microbiota

Hydroxysafflor yellow A (HSYA), a chalcone glycoside, is a component of Carthamus tinctorius L. and exerts anti-inflammatory and antioxidative effects. However, the therapeutic effect and the underlying mechanism of HSYA on ulcerative colitis is unclear. This study aimed to investigate the unexplored protective effects and underlying mechanisms of HSYA on UC. In vitro analyses showed that HSYA reduced the secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 and inhibited nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3)/gasdermin D (GSDMD)-mediated pyroptosis in lipopolysaccharide/ adenosine-5′-triphosphate (LPS/ATP)-stimulated macrophages. Gas chromatography–mass spectrometry (GC–MS) profiling of intracellular metabolites showed that HSYA reduced the increased levels of glucose, glucose 6-phosphate, and lactic acid, and inhibited the increased hexokinase 1 (HK1) expression caused by LPS/ATP stimulation. HK1 shRNA transfection further confirmed that HSYA inhibited the NLRP3/GSDMD-mediated pyroptosis via HK1 downregulation. In vivo analyses showed that HSYA drastically attenuated UC symptoms by relieving body weight loss, a decline in colon length, and inflammatory infiltration in colonic tissues induced by dextran sulfate sodium (DSS). HSYA also reduced the secretion of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and IL-18. Moreover, HSYA inhibited HK1/NLRP3/GSDMD-mediated pyroptosis in DSS-induced colitis mice. Finally, 16S rRNA sequencing analyses of gut microbiota revealed that HSYA reversed gut microbiota dysbiosis by reducing the abundance of Proteobacteria and increasing that of Bacteroidetes. This study demonstrated that HSYA not only exerted anti-inflammatory effects by inhibiting HK1/NLRP3/GSDMD and suppressing pyroptosis but also regulated gut microbiota in mice with DSS-induced colitis. Our findings provide new experimental evidence that HSYA might be a potential candidate for treating inflammatory bowel diseases.

Growth, yield productivity, and oil composition of two Amaranthus species grown under two different environmental conditions in Egypt

AbstractAmaranthus plants are very rich in nutritional benefits with significant traditional uses especially against inflammations. Herein, the growth productivity, oil quality, and compositions of the two promising novel food resources, Amaranthus caudatus and Amaranthus tricolor, were evaluated in two different locations in Egypt (El‐Sharkia and El‐Minia) which have different geographical regions. Analyses of geographic positions, climatic data, and soil characteristics of the two locations were evaluated. Plant heights, number of branches/plant, seed yield, and inflorescence length of the two plant species were studied in both locations. Additionally, the seed oil yields along with their gas chromatography–mass spectroscopy (GC–MS) profiles were characterized. The findings revealed a significant difference between the two locations, with a strong correlation between the yields and the climate conditions. The two plant species cultivated in El‐Minia showed significant records of overall growth indicators than El‐Sharkia. A worthy variability in the oil yields and composition of El‐Minia samples exhibited higher contents of dry matter in both plants (89.01 and 89.03 g/100 g dry tissues, respectively) than those of El‐Sharkia. Amaranthus caudatus cultivated in El‐Sharkia showed higher lipids components (7.02 g/100 g dry seeds) and saturated (19.13 g/100 g dry seeds) and unsaturated fatty acids (61.32 g/100 g) than those of El‐Minia site. In addition, A. tricolor cultivated in El‐Sharkia contained higher lipids contents (6.87 g/100 g) and unsaturated fatty acids (61.75 g/100 g dry seeds) than El‐Minia with the supremacy of El‐Minia samples in saturated fatty acids (19.03 g/100 g dry seeds). A strong correlation between the lipids and fatty acids of A. caudatus and A. tricolor was determined, and the cultivation locations and conditions were found based upon the GC–MS analysis as well as statistical significance.

Psoralea corylifolia (babchi) seeds enhance proliferation of normal human cultured melanocytes: GC–MS profiling and biological investigation

Abstract Plants are considered as a source of medicine to treat skin-related disorders since decades. Seeds of Psoralea corylifolia were used thousands of years ago for re-pigmentation in vitiligo patients especially in South Asia. It is a well-known fact that melanocytes, which are the specialized cells in the skin causes vitiligo through a series of chemical and enzymatic reactions, mainly due to the copper containing enzyme tyrosinase. Conditions associated with overproduction of melanin are termed hyperpigmentation. The treatment options are generally limited due to the complicated and slow pathogenesis of the disease. Therefore, therapeutic repigmentation modalities for the treatment of vitiligo are highly desirable. The aim of this study was to investigate the effects of various doses of ethanol and water extracts of P. corylifolia seeds on proliferation of normal human cultured melanocytes and its gas chromatography–mass spectrometry profiling.

Discrimination of Olive Oil and Extra-Virgin Olive Oil from Other Vegetable Oils by Targeted and Untargeted HRMS Profiling of Phenolic and Triterpenic Compounds Combined with Chemometrics.

Extra-virgin olive oil (EVOO) and virgin olive oil (VOO) are valuable natural products of great economic interest for their producing countries, and therefore, it is necessary to establish methods capable of proving the authenticity of these oils on the market. This work presents a methodology for the discrimination of olive oil and extra-virgin olive oil from other vegetable oils based on targeted and untargeted high-resolution mass spectrometry (HRMS) profiling of phenolic and triterpenic compounds coupled with multivariate statistical analysis of the data. Some phenolic compounds (cinnamic acid, coumaric acids, apigenin, pinocembrin, hydroxytyrosol and maslinic acid), secoiridoids (elenolic acid, ligstroside and oleocanthal) and lignans (pinoresinol and hydroxy and acetoxy derivatives) could be olive oil biomarkers, whereby these compounds are quantified in higher amounts in EVOO compared to other vegetable oils. The principal component analysis (PCA) performed based on the targeted compounds from the oil samples confirmed that cinnamic acid, coumaric acids, apigenin, pinocembrin, hydroxytyrosol and maslinic acid could be considered as tracers for olive oils authentication. The heat map profiles based on the untargeted HRMS data indicate a clear discrimination of the olive oils from the other vegetable oils. The proposed methodology could be extended to the authentication and classification of EVOOs depending on the variety, geographical origin, or adulteration practices.

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a multistage activation mechanism. Activation is instigated by conformational rearrangements relieving autoinhibitory FERM domain interactions. The kinase autophosphorylates a central linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loops to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. We employ hydrogen/deuterium exchange mass spectrometry and kinase activity profiling to map the conformational dynamics associated with substrate binding and Src-mediated activation loop phosphorylation. Nucleotide engagement stabilizes the autoinhibitory interface, while phosphorylation deprotects both FERM and kinase regulatory surfaces. Phosphorylation organizes active site motifs linking catalytic loop with activation segment. Dynamics of the activation segment anchor propagate to EF/G helices to prevent reversion of the autoinhibitory FERM interaction. We employ targeted mutagenesis to dissect how phosphorylation-induced conformational rearrangements elevate kinase activity above the basal autophosphorylation rate.

Cutaneous protothecosis in a dog successfully treated with oral itraconazole in pulse dosing

BackgroundProtothecosis is a rare infectious disease caused by unicellular, achlorophyllous, microalgae of the genus Prototheca, ubiquitously distributed in nature. The algae are emerging pathogens, whose incidence is increasing in both human and animal populations and serious systemic infections related to this pathogen have been increasingly described in humans in recent years. After mastitis in dairy cows, canine protothecosis is the second most prevalent form of the protothecal disease in animals. Here, we report the first case of chronic cutaneous protothecosis due to P. wickerhamii in a dog in Brazil, successfully treated with a long-term therapy with itraconazole in pulse.Case presentationUpon clinical examination, exudative nasolabial plaque, ulcered, and painful lesions in central and digital pads and lymphadenitis were observed in a 2-year-old mixed-breed dog, with a 4-month history of cutaneous lesions and contact with sewage water. Histopathological examination revealed intense inflammatory reaction, with numerous spherical to oval, encapsulated structures stained with Periodic Acid Schiff, compatible with Prototheca morphology. Tissue culture on Sabouraud agar revealed yeast-like, greyish-white colonies after 48 h of incubation. The isolate was subjected to mass spectrometry profiling and PCR-sequencing of the mitochondrial cytochrome b (CYTB) gene marker, leading to identification of the pathogen as P. wickerhamii. The dog was initially treated with oral itraconazole at a dosage of 10 mg/kg once daily. After six months, the lesions resolved completely, yet recurred shortly after cessation of therapy. The dog was then treated with terbinafine at a dose of 30 mg/kg, once daily for 3 months, with no success. The resolution of clinical signs, with no recurrence over a 36-months follow-up period, was achieved after 3 months of treatment with itraconazole (20 mg/kg) in pulse intermittently on two consecutive days a week.ConclusionsThis report highlights the refractoriness of skin infections by Prototheca wickerhamii with therapies proposed in the literature and suggests a new treatment option with oral itraconazole in pulse dosing for long-term disease control successfully performed in a dog with skin lesions.

Unraveling the Steric Link to Copper Precursor Decomposition: A Multi-Faceted Study for the Printing of Flexible Electronics.

The field of printed electronics strives for lower processing temperatures to move toward flexible substrates that have vast potential: from wearable medical devices to animal tagging. Typically, ink formulations are optimized using mass screening and elimination of failures; as such, there are no comprehensive studies on the fundamental chemistry at play. Herein, findings which describe the steric link to decomposition profile: combining densityfunctionaltheory, crystallography, thermal decomposition, mass spectrometry, and inkjet printing, are reported. Through the reaction of copper(II) formate with excess alkanolamines of varying steric bulk, tris-co-ordinated copper precursor ions: "[CuL3 ]," each with a formate counter-ion (1-3) are isolated and their thermal decomposition mass spectrometry profiles are collected to assess their suitability for use in inks (I1-3 ). Spin coating and inkjet printing of I1,2 provides an easily up-scalable method toward the deposition of highly conductive copper device interconnects (ρ = 4.7-5.3 × 10-7 Ω m; ≈30% bulk) onto paper and polyimide substrates and forms functioning circuits that can power light-emitting diodes. The connection among ligand bulk, coordination number, and improved decomposition profile supports fundamental understanding which will direct future design.

Solid-State Fermentation of Trichoderma spp.: A New Way to Valorize the Agricultural Digestate and Produce Value-Added Bioproducts.

In this study, the agricultural digestate from anaerobic biogas production mixed with food wastes was used as a substrate to grow Trichoderma reesei RUT-C30 and Trichoderma atroviride Ta13 in solid-state fermentation (SSF) and produce high-value bioproducts, such as bioactive molecules to be used as ingredients for biostimulants. The Trichoderma spp. reached their maximum growth after 6 and 3 SSF days, respectively. Both Trichoderma species were able to produce cellulase, esterase, and citric and malic acids, while T. atroviride also produced gibberellins and oxylipins as shown by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) profiling. Experimental evaluation of germination parameters highlighted a significant promotion of tomato seed germination and root elongation induced by T. atroviride crude extracts from SSF. This study suggests an innovative sustainable use of the whole digestate mixed with agro-food waste as a valuable substrate in fungal biorefineries. Here, it has been applied to produce plant growth-promoting fungi and bioactive molecules for sustainable agriculture.

Phytochemical analysis and biological activities of Artemisia vulgaris grown in different altitudes of Nepal

ABSTRACT Herbal medication developed from natural products has antibacterial, antioxidant, and antidiabetic effects on human health. This research aimed at the investigation of the biological activities, estimation of total phenolic and flavonoid content, and Gas Chromatography and Mass Spectrometry (GC-MS) profiling of chemical compounds in Artemisia vulgaris root and leaf grown at three different altitudes of Nepal. The radical scavenging property of the extracts was studied by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, antidiabetic activity was performed by α-amylase inhibition activity, and the total phenolic and flavonoid content was estimated by Folin–Ciocalteu phenol reagent and aluminium chloride colorimetric methods. The IC50 against the DPPH activity shown by the root extract of A. vulgaris grown in Kathmandu was evaluated to be 67.31 ± 2.80 µg/mL, whereas the lowest activity was shown by the leaf extract of A. vulgaris grown in Chitwan of IC50 149.62 ± 2.40 µg/mL. The α-amylase enzyme inhibition activity shown by the root extract grown in Gorkha was found to be 39.43% as compared to the leaf extract grown in Gorkha of 8.69%. The total phenolic content was found to be maximum in root grown in Kathmandu of 96.30 ± 0.52 mg.GAE/g, whereas the least was found in leaf grown in Chitwan of 26.04 ± 1.66 mg.GAE/g. The flavonoid content was found to be maximum in leaf extracts of A. vulgaris grown in Kathmandu of 71.15 ± 2.07 mg.QE/g, whereas the least was found in leaf grown in Gorkha of 31.54 ± 0.70 mg.QE/g. The essential oils extracted from the A. vulgaris growing in Gorkha (862 m altitude) showed the effective antibacterial property against Klebsiella pneumonia and Acinetobacter baumannii with zone of inhibition (ZOI) 12 and 15 mm, respectively. Similarly, the essential oils of A. vulgaris growing in Chitwan (208 m altitude) showed the ZOI 12 and 11 mm against Klebsiella pneumonia and Acinetobacter baumannii, respectively. The positive control used was ampicillin and polymyxin with ZOI of 22 mm. However, the extracts from plants growing in all these altitudes are found to be inactive against these organisms. From the results of this study, it can be concluded that the biological activities of leaf and root extracts of A. vulgaris increased with an increase in altitudes, and the plants grown in high altitudes are the rich sources of secondary metabolites that play a significant role against infectious diseases and diabetes in human beings.

Volatiles from Pseudomonas palleroniana Strain B-BH16-1 Suppress Aflatoxin Production and Growth of Aspergillus flavus on Coix lacryma-jobi during Storage

Semen coicis is not only a traditional Chinese medicine (TCM), but also a typical food in China, with significant medical and healthcare value. Because semen coicis is rich in starch and oil, it can be easily contaminated with Aspergillus flavus and its aflatoxins (AFs). Preventing and controlling the contamination of semen coicis with Aspergillus flavus and its aflatoxins is vital to ensuring its safety as a drug and as a food. In this study, the endosphere bacteria Pseudomonas palleroniana strain B-BH16-1 produced volatiles that strongly inhibited the mycelial growth and spore formation activity of A. flavus. Gas chromatography-mass spectrometry profiling revealed three volatiles emitted from B-BH16-1, of which 1-undecene was the most abundant. We obtained authentic reference standards for these three volatiles; these significantly reduced mycelial growth and sporulation in Aspergillus, with dimethyl disulfide showing the most robust inhibitory activity. Strain B-BH16-1 was able to completely inhibit the biosynthesis of aflatoxins in semen coicis samples during storage by emitting volatile bioactive components. The microscope revealed severely damaged mycelia and a complete lack of sporulation. This newly identified plant endophyte bacterium was able to strongly inhibit the sporulation and growth of Aspergillus and the synthesis of associated mycotoxins, thus not only providing valuable information regarding an efficient potential strategy for the prevention of A. flavus contamination in TCM and food, but potentially also serving as a reference in the control of toxic fungi.

Classic and 11-oxygenated androgens in serum and saliva across adulthood: a cross-sectional study analyzing the impact of age, body mass index, and diurnal and menstrual cycle variation.

11-oxygenated androgens significantly contribute to the circulating androgen pool. Understanding the physiological variation of 11-oxygenated androgens and their determinants is essential for clinical interpretation, for example, in androgen excess conditions. We quantified classic and 11-oxygenated androgens in serum and saliva across the adult age and body mass index (BMI) range, also analyzing diurnal and menstrual cycle-dependent variation. Cross-sectional. Morning serum samples were collected from 290 healthy volunteers (125 men, 22-95 years; 165 women, 21-91 years). Morning saliva samples were collected by a sub-group (51 women and 32 men). Diurnal saliva profiles were collected by 13 men. Twelve women collected diurnal saliva profiles and morning saliva samples on 7 consecutive days during both follicular and luteal menstrual cycle phases. Serum and salivary steroids were quantified by liquid chromatography-tandem mass spectrometry profiling assays. Serum classic androgens decreased with age-adjusted BMI, for example, %change kg/m2 for 5α-dihydrotestosterone: men -5.54% (95% confidence interval (CI) -8.10 to -2.98) and women -1.62% (95%CI -3.16 to -0.08). By contrast, 11-oxygenated androgens increased with BMI, for example, %change kg/m2 for 11-ketotestosterone: men 3.05% (95%CI 0.08-6.03) and women 1.68% (95%CI -0.44 to 3.79). Conversely, classic androgens decreased with age in both men and women, while 11-oxygenated androgens did not. Salivary androgens showed a diurnal pattern in men and in the follicular phase in women; in the luteal phase, only 11-oxygenated androgens showed diurnal variation. Classic androgens decrease while active 11-oxygenated androgens increase with increasing BMI, pointing toward the importance of adipose tissue mass for the activation of 11-oxygenated androgens. Classic but not 11-oxygenated androgens decline with age.

Targeted and explorative profiling of kallikrein proteases and global proteome biology of pancreatic ductal adenocarcinoma, chronic pancreatitis, and normal pancreas highlights disease-specific proteome remodelling

Pancreatic ductal adenocarcinoma (PDAC) represents one of the most aggressive and lethal malignancies worldwide with an urgent need for new diagnostic and therapeutic strategies. One major risk factor for PDAC is the pre-indication of chronic pancreatitis (CP), which represents highly inflammatory pancreatic tissue. Kallikreins (KLKs) are secreted serine proteases that play an important role in various cancers as components of the tumor microenvironment. Previous studies of KLKs in solid tumors largely relied on either transcriptomics or immunodetection. We present one of the first targeted mass spectrometry profiling of kallikrein proteases in PDAC, CP, and normal pancreas. We show that KLK6 and KLK10 are significantly upregulated in PDAC (n=14) but not in CP (n=7) when compared to normal pancreas (n=16), highlighting their specific intertwining with malignancy. Additional explorative proteome profiling identified 5936 proteins in our pancreatic cohort and observed disease-specific proteome rearrangements in PDAC and CP. As such, PDAC features an enriched proteome motif for extracellular matrix (ECM) and cell adhesion while there is depletion of mitochondrial energy metabolism proteins, reminiscent of the Warburg effect. Although often regarded as a PDAC hallmark, the ECM fingerprint was also observed in CP, alongside with a prototypical inflammatory proteome motif as well as with an increased wound healing process and proteolytic activity, thereby possibly illustrating tissue autolysis. Proteogenomic analysis based on publicly accessible data sources identified 112 PDAC-specific and 32 CP-specific single amino acid variants, which among others affect KRAS and ANKHD1. Our study emphasizes the diagnostic potential of kallikreins and provides novel insights into proteomic characteristics of PDAC and CP.

Revealing the small proteome of Haloferax volcanii by combining ribosome profiling and small-protein optimized mass spectrometry.

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon Haloferax volcanii. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence in vivo for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in Haloferax species and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.

Chemical Composition of the Volatile Oil from the Leaves of Kaempferia champasakensis Picheans. & Koonterm. (Zingiberaceae)

The genus Kaempferia (Zingiberaceae) is well-known for its diverse phytochemical constituents and characteristic essential oil. Kaempferia champasakensis was firstly described in 2008 by Chayan Picheansoonthon and Supachai Koonterm in Champasak province of Southern Laos. To date, little is known about phytochemical components and pharmacological activities of the plant. In the present study, the extraction of essential oil from K. champasakensis leaves as well as Gas Chromatography - Mass Spectrometry (GC/MS) profile were investigated. The major components in essential oil extracted from K. champasakensis leaves were determined as β-caryophyllene (16.07%), β-elemenone (14.53%), β-pinene (13.80%), germacrone (9.85%), myrcene (9.46%) and diisooctyl phthalate (8.62%). This is the first report on chemical constituents of essential oil extracted from the leaves of K. champasakensis.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.

RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of Genome Engineering and Stable Transfection.

To study the function of RNA-binding proteins (RBPs), an overexpression or knockout approach is generally used. However, as many RBPs are essential to cellular functions, the complete knockout of these proteins may be lethal to the cell. Overexpression of RBPs, on the other hand, may create an altered transcriptome and aberrant phenotypes that can mask their physiological function. Additionally, biochemical characterization of RBP often requires highly specific antibodies for efficient immunoprecipitation for downstream mass spectrometry or RNA footprinting profiling. To overcome these hurdles, we have developed a strategy to generate cellular systems either using a CRISPR-Cas9-mediated epitope tag knock-in approach or a two-step workflow to first stably express an exogenous Flag-tagged RBP and subsequently knockout the endogenous RBP using CRISPR-Cas9 gene editing. The generation of these cell lines will be beneficial for downstream RNA footprinting studies and mass spectrometry-mediated interactome studies.

Mass tag-encoded nanointerfaces for multiplexed mass spectrometric analysis and imaging of biomolecules.

Revealing multiple biomolecules in the physiopathological environment simultaneously is crucial in biological and biomedical research. Mass spectrometry (MS) features unique technical advantages in multiplexed and label-free analyses. However, owing to comparably low abundance and poor ionization efficiency of target biomolecules, direct MS profiling of these biological species in vitro or in situ remains a challenge. An emerging route to solve this issue is to devise mass tag (MT)-encoded nanointerfaces which specifically convert the abundance or activity of biomolecules into amplified ion signals of mass tags, offering an ideal strategy for synchronous MS assaying and mapping of multiple targets in biofluids, cells and tissues. This review provides a thorough and organized overview of recent advances in MT-encoded nanointerfaces elaborately tailored for several practical applications in multiplexed MS bioanalysis and biomedical research. First, we start with elucidation of the structural characteristics and working principle of MT-encoded nanointerfaces in specific labeling and sensing of multiple biological targets. In addition, we further discuss the application scenarios of MT-encoded nanointerfaces particularly in multiplexed biomarker assays, cell analysis, and tissue imaging. Finally, the current challenges are pointed out and future prospects of these nanointerfaces in MS analysis are forecast.