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1.
Nucleic Acids Res ; 52(15): 9076-9091, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39188014

RESUMO

The MUT-7 family of 3'-5' exoribonucleases is evolutionarily conserved across the animal kingdom and plays essential roles in small RNA production in the germline. Most MUT-7 homologues carry a C-terminal domain of unknown function named MUT7-C appended to the exoribonuclease domain. Our analysis shows that the MUT7-C is evolutionary ancient, as a minimal version of the domain exists as an individual protein in prokaryotes. In animals, MUT7-C has acquired an insertion that diverged during evolution, expanding its functions. Caenorhabditis elegans MUT-7 contains a specific insertion within MUT7-C, which allows binding to MUT-8 and, consequently, MUT-7 recruitment to germ granules. In addition, in C. elegans and human MUT-7, the MUT7-C domain contributes to RNA binding and is thereby crucial for ribonuclease activity. This RNA-binding function most likely represents the ancestral function of the MUT7-C domain. Overall, this study sheds light on MUT7-C and assigns two functions to this previously uncharacterized domain.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Exorribonucleases , Domínios Proteicos , Animais , Exorribonucleases/metabolismo , Exorribonucleases/química , Exorribonucleases/genética , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Humanos , Evolução Molecular , RNA/metabolismo , RNA/química , Sequência de Aminoácidos , Ligação Proteica
2.
Proc Natl Acad Sci U S A ; 119(47): e2211827119, 2022 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-36383601

RESUMO

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses.


Assuntos
Engenharia Metabólica , Pichia , Humanos , Pichia/metabolismo , Engenharia Metabólica/métodos , Dióxido de Carbono/metabolismo , Processos Autotróficos
3.
Anal Chem ; 96(41): 16215-16226, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39353203

RESUMO

Nontargeted screening (NTS) utilizing liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI/HRMS) is increasingly used to identify environmental contaminants. Major differences in the ionization efficiency of compounds in ESI/HRMS result in widely varying responses and complicate quantitative analysis. Despite an increasing number of methods for quantification without authentic standards in NTS, the approaches are evaluated on limited and diverse data sets with varying chemical coverage collected on different instruments, complicating an unbiased comparison. In this interlaboratory comparison, organized by the NORMAN Network, we evaluated the accuracy and performance variability of five quantification approaches across 41 NTS methods from 37 laboratories. Three approaches are based on surrogate standard quantification (parent-transformation product, structurally similar or close eluting) and two on predicted ionization efficiencies (RandFor-IE and MLR-IE). Shortly, HPLC grade water, tap water, and surface water spiked with 45 compounds at 2 concentration levels were analyzed together with 41 calibrants at 6 known concentrations by the laboratories using in-house NTS workflows. The accuracy of the approaches was evaluated by comparing the estimated and spiked concentrations across quantification approaches, instrumentation, and laboratories. The RandFor-IE approach performed best with a reported mean prediction error of 15× and over 83% of compounds quantified within 10× error. Despite different instrumentation and workflows, the performance was stable across laboratories and did not depend on the complexity of water matrices.

4.
Microb Cell Fact ; 23(1): 43, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38331812

RESUMO

BACKGROUND: Specific productivity (qP) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (µ). Understanding the factors limiting productivity at extremely low µ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions. RESULTS: By the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h-1. Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average qP at the end of the retentostat was estimated at 0.019 mg g-1 h-1. Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival. CONCLUSIONS: In conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.


Assuntos
Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomyces cerevisiae/metabolismo , Perfilação da Expressão Gênica , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Lipídeos
5.
Anal Bioanal Chem ; 415(21): 5151-5163, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37347300

RESUMO

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO2 and CO2-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for 13C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using 13C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.


Assuntos
Dióxido de Carbono , Metanol , Carbono/metabolismo , Tetra-Hidrofolatos/metabolismo , Espectrometria de Massas , Formiatos
6.
Anal Bioanal Chem ; 415(5): 823-840, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36547703

RESUMO

Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value < 0.05). All approaches revealed the same trend between genotypes, namely up-regulation of most metabolites in the root hair defective mutant (rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20-79 features, 13-31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula.


Assuntos
Metaboloma , Metabolômica , Metabolômica/métodos , Espectrometria de Massas/métodos , Exsudatos e Transudatos , Carbono/análise , Raízes de Plantas/química
7.
Anal Bioanal Chem ; 414(15): 4359-4368, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34642781

RESUMO

We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.


Assuntos
Saccharomyces cerevisiae , Espectrometria de Massas em Tandem , Cromatografia Gasosa-Espectrometria de Massas , Isótopos/metabolismo , Metaboloma , Metabolômica/métodos , Pichia/química , Espectrometria de Massas em Tandem/métodos
8.
Anal Bioanal Chem ; 414(25): 7483-7493, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35960317

RESUMO

The major benefits of integrating ion mobility (IM) into LC-MS methods for small molecules are the additional separation dimension and especially the use of IM-derived collision cross sections (CCS) as an additional ion-specific identification parameter. Several large CCS databases are now available, but outliers in experimental interplatform IM-MS comparisons are identified as a critical issue for routine use of CCS databases for identity confirmation. We postulate that different routine external calibration strategies applied for traveling wave (TWIM-MS) in comparison to drift tube (DTIM-MS) and trapped ion mobility (TIM-MS) instruments is a critical factor affecting interplatform comparability. In this study, different external calibration approaches for IM-MS were experimentally evaluated for 87 steroids, for which TWCCSN2, DTCCSN2 and TIMCCSN2 are available. New reference CCSN2 values for commercially available and class-specific calibrant sets were established using DTIM-MS and the benefit of using consolidated reference values on comparability of CCSN2 values assessed. Furthermore, use of a new internal correction strategy based on stable isotope labelled (SIL) internal standards was shown to have potential for reducing systematic error in routine methods. After reducing bias for CCSN2 between different platforms using new reference values (95% of TWCCSN2 values fell within 1.29% of DTCCSN2 and 1.12% of TIMCCSN2 values, respectively), remaining outliers could be confidently classified and further studied using DFT calculations and CCSN2 predictions. Despite large uncertainties for in silico CCSN2 predictions, discrepancies in observed CCSN2 values across different IM-MS platforms as well as non-uniform arrival time distributions could be partly rationalized.


Assuntos
Calibragem , Cromatografia Líquida , Espectrometria de Massas/métodos , Padrões de Referência
9.
Plant Mol Biol ; 105(4-5): 435-447, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33296063

RESUMO

KEY MESSAGE: LC-MS based metabolomics approach revealed that putative metabolites other than flavonoids may significantly contribute to the sexual compatibility reactions in Prunus armeniaca. Possible mechanisms on related microtubule-stabilizing effects are provided. Identification of metabolites playing crucial roles in sexual incompatibility reactions in apricot (Prunus armeniaca L.) was the aim of the study. Metabolic fingerprints of self-compatible and self-incompatible apricot pistils were created using liquid chromatography coupled to time-of-flight mass spectrometry followed by untargeted compound search. Multivariate statistical analysis revealed 15 significant differential compounds among the total of 4006 and 1005 aligned metabolites in positive and negative ion modes, respectively. Total explained variance of 89.55% in principal component analysis (PCA) indicated high quality of differential expression analysis. The statistical analysis showed significant differences between genotypes and pollination time as well, which demonstrated high performance of the metabolic fingerprinting and revealed the presence of metabolites with significant influence on the self-incompatibility reactions. Finally, polyketide-based macrolides similar to peloruside A and a hydroxy sphingosine derivative are suggested to be significant differential metabolites in the experiment. These results indicate a strategy of pollen tubes to protect microtubules and avoid growth arrest involved in sexual incompatibility reactions of apricot.


Assuntos
Flores/genética , Metabolômica/métodos , Polinização/genética , Prunus armeniaca/genética , Autoincompatibilidade em Angiospermas/genética , Cromatografia Líquida/métodos , Flores/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Genótipo , Espectrometria de Massas/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Componente Principal , Prunus armeniaca/metabolismo
10.
Electrophoresis ; 42(4): 490-500, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33332608

RESUMO

Nontargeted analysis of water samples using liquid chromatography combined with high-resolution mass spectrometers is an emerging approach for surface water monitoring and evaluation of water treatment processes. In this study, sample preconcentration via direct, large volume injection with 500 µL and 1000 µL injection volumes was compared to SPE regarding analytical performance parameters in targeted and nontargeted workflows. In targeted analysis, the methods were evaluated in terms of LOD and intrabatch precision of the selected compounds, whereas in nontargeted analysis, the number of detected unknown compounds, the method's intra-batch precision, and the retention time versus molecular mass pattern of the detected unknowns were evaluated. In addition, a novel intensity drift correction method was developed that is not based on quality control samples and makes use of the signals obtained for continuously infused reference compounds, which are conventionally utilized for online mass drift correction. It could be demonstrated that the new correction method significantly reduced the bias introduced by instrumental drift and is important for the reliable intercomparison of different nontargeted methods. Intercomparison of results showed that the 1000 µL large volume injection method revealed the best performance in terms of precision under repeatability conditions of measurement as well as lower LODs for targeted compound analysis. In nontargeted analysis, the SPE method detected a higher number of unknown compounds but exhibited also a higher uncertainty of measurement caused by matrix effects.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Extração em Fase Sólida/métodos , Poluentes Químicos da Água/isolamento & purificação , Água Doce/química , Limite de Detecção , Reprodutibilidade dos Testes , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química
11.
Electrophoresis ; 42(4): 473-481, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33188545

RESUMO

Rugged analytical methods for the screening and identity confirmation of anthocyanins require a dedicated sample preparation, chromatographic setup, and the reliable generation of multiple identification points to confirm identity against the wide range of phenolic compounds typically present in food, beverage, and plant material samples. To this end, combinations of spectroscopic and mass spectrometric detection are frequently employed for this application to provide higher confidence in the absence of authentic standards. In the present work, low-field drift tube ion mobility (DTIM) separation is evaluated for this task using a LC-DAD-DTIM-QTOFMS method. DTIM-MS allows accurate determination of collision cross sections (DT CCS) for all analysed compounds as well as a precise alignment tool for reconciling fragment and precursor ions in data independent acquisition mode. The presented approach thereby allows for an anthocyanin screening method taking true advantage of all dimensions of the analytical platform: relative retention (RPLC), UV/VIS absorption spectrum, accurate mass, DT CCSN2 , and confirmed high-resolution fragment ions. From the analysis of authentic standards and several berry samples primarily from the Vaccinium genus, Level 1 confirmation data for six anthocyanins from the cyanidin family, and Level 2 confirmation for a further 29 anthocyanins confirmed in berry samples is provided. The method and accompanying dataset provided as part of this work provides a means to develop anthocyanin screening methods using the ion mobility dimension as an additional alignment and filtering parameter in data independent analysis acquisition across any LC-IM-MS platform.


Assuntos
Antocianinas/análise , Frutas/química , Vaccinium/química , Cromatografia Líquida , Espectrometria de Massas/métodos , Ribes/química , Rubus/química
12.
FEMS Yeast Res ; 21(2)2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33599728

RESUMO

Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut- phenotype. The Mut- phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut- strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut- increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.


Assuntos
Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/metabolismo , Regulação Fúngica da Expressão Gênica , Metanol/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Álcool Desidrogenase/análise , Álcool Desidrogenase/genética , Proteínas Fúngicas/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes , Saccharomycetales/enzimologia
13.
J Anal At Spectrom ; 36(11): 2306-2311, 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34764530

RESUMO

Turbulent flow chromatography is an online solid phase extraction mode that achieves the extraordinary effect of proxying an upper molecular weight cutoff for the retained molecules, based on loading the sample at high linear velocities. Despite the potential of being a universal sample preparation technique prior to inductively coupled plasma mass spectrometry and liquid chromatography mass spectrometry, it employs specific hardware and expensive consumables. In the present work we apply this technique using off-the-shelf fluidic components and the niche "bead injection" methodology. For the first time, this procedure has been executed with a pressure of approximately 20 bar, compared to the low pressure of the classic setup, achieving a sample throughput >285 h-1 for the SPE/TFC procedure, or 20 h-1 if the procedure involves renewing the sorbent, using no more than 4 mg of sorbent for every µ-SPE. Another novelty is that sorbent packing and unpacking has been controlled with a smart method using real-time pressure feedback as quality control for truly unattended operation. Finally, the turbulent flow chromatography principle has been comprehensively characterized, providing similar performance to that demonstrated in earlier literature, and the ancillary sample preparation capabilities, e.g., in-valve acidification, have been demonstrated by the fractionation of gadolinium in surface waters prior to ICP-MS, an element of increasing surface water concern due to its use as a magnetic resonance contrast agent.

14.
Molecules ; 26(23)2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34885835

RESUMO

Though not regulated in directives such as the Water Framework Directive of the European Union, the investigation of geogenic background concentrations of certain elements such as precious metals is of increasing interest, in particular for the early detection of a potential environmental pollution due to the increased use in various industrial and technological applications and in medicine. However, the precise and accurate quantification of precious metals in natural waters is challenging due to the complex matrices and the ultra-low concentrations in the (sub-) ng L-1 range. A methodological approach, based on matrix separation and pre-concentration on the strong anion exchange resin TEVA® Resin in an online mode directly coupled to ICP-SFMS, has been developed for the determination of Ag, Pt, Pd and Au in ground water. Membrane desolvation sample introduction was used to reduce oxide-based spectral interferences, which complicate the quantification of these metals with high accuracy. To overcome errors arising from matrix effects-in particular, the highly varying major ion composition of the investigated ground water samples-an isotope dilution analysis and quantification based on standard additions, respectively, were performed. The method allowed to process four samples per hour in a fully automated mode. With a sample volume of only 8 mL, enrichment factors of 6-9 could be achieved, yielding detection limits <1 ng L-1. Validation of the trueness was performed based on the reference samples. This method has been used for the analysis of the total concentrations of Ag, Pt, Pd and Au in highly mineralized ground waters collected from springs located in important geological fault zones of Austria's territory. Concentrations ranges of 0.21-64.2 ng L-1 for Ag, 0.65-6.26 ng L-1 for Pd, 0.07-1.55 ng L-1 for Pt and 0.26-1.95 ng L-1 for Au were found.

15.
Anal Chem ; 92(7): 4875-4883, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32096989

RESUMO

N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. It plays a variety of roles at the cell surface structure and is under discussion to be involved in signaling pathways. The presence of a number of N-acetylhexosamine stereoisomers in samples of biological or biotechnological origin demands for dedicated high efficiency separation methods, due to identical exact mass and similar fragmentation patterns of the stereoisomers. Gas chromatography offers high sample capacity, separation efficiency, and precision under repeatability conditions of measurement, which is a necessity for the analysis of low abundant stereoisomers in biological samples. Automated online derivatization facilitates to overcome the main obstacle for the use of gas chromatography in metabolomics, namely, the derivatization of polar metabolites prior to analysis. Using alkoximation and subsequent trimethylsilylation, carbohydrates and their derivatives are known to show several derivatives, since derivatization is incomplete as well as highly matrix dependent inherent to the high number of functional groups present in carbohydrates. A method based on efficient separation of ethoximated and trimethylsilylated N-acetylglucosamines was developed. Accurate absolute quantification is enabled using biologically derived 13C labeled internal standards eliminating systematic errors related to sample pretreatment and analysis. Due to the lack of certified reference materials, a methodological comparison between tandem and time-of-flight mass spectrometric instrumentation was performed for mass spectrometric assessment of trueness. Both methods showed limits of detection in the lower femtomol range. The methods were applied to biological samples of Penicillium chrysogenum cultivations with different matrices revealing excellent agreement of both mass spectrometric techniques.


Assuntos
Acetilglucosamina/análise , Penicillium chrysogenum/química , Automação , Configuração de Carboidratos , Células Cultivadas , Cromatografia Gasosa , Espectrometria de Massas , Penicillium chrysogenum/citologia
16.
Metab Eng ; 61: 288-300, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32619503

RESUMO

BACKGROUND: Cell line-specific, genome-scale metabolic models enable rigorous and systematic in silico investigation of cellular metabolism. Such models have recently become available for Chinese hamster ovary (CHO) cells. However, a key ingredient, namely an experimentally validated biomass function that summarizes the cellular composition, was so far missing. Here, we close this gap by providing extensive experimental data on the biomass composition of 13 parental and producer CHO cell lines under various conditions. RESULTS: We report total protein, lipid, DNA, RNA and carbohydrate content, cell dry mass, and detailed protein and lipid composition. Furthermore, we present meticulous data on exchange rates between cells and environment and provide detailed experimental protocols on how to determine all of the above. The biomass composition is converted into cell line- and condition-specific biomass functions for use in cell line-specific, genome-scale metabolic models of CHO. Finally, flux balance analysis (FBA) is used to demonstrate consistency between in silico predictions and experimental analysis. CONCLUSIONS: Our study reveals a strong variability of the total protein content and cell dry mass across cell lines. However, the relative amino acid composition is independent of the cell line and condition and thus needs not be explicitly measured for each new cell line. In contrast, the lipid composition is strongly influenced by the growth media and thus will have to be determined in each case. These cell line-specific variations in biomass composition have a small impact on growth rate predictions with FBA, as inaccuracies in the predictions are rather dominated by inaccuracies in the exchange rate spectra. Cell-specific biomass variations only become important if the experimental errors in the exchange rate spectra drop below twenty percent.


Assuntos
Biomassa , Simulação por Computador , Modelos Biológicos , Animais , Células CHO , Cricetulus , Meios de Cultura/análise , Meios de Cultura/química
17.
Mass Spectrom Rev ; 38(3): 291-320, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30707468

RESUMO

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

18.
Environ Exp Bot ; 177: 104122, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34103771

RESUMO

Pteris vittata (PV) and Pteris quadriaurita (PQ) are reported to hyperaccumulate arsenic (As) when grown in Asrich soil. Yet, little is known about the impact of their unique As accumulation mechanisms on As transformations and cycling at the soil-root interface. Using a combined approach of two-dimensional (2D), sub-mm scale solute imaging of arsenite (AsIII), arsenate (AsV), phosphorus (P), manganese (Mn), iron (Fe) and oxygen (O2), we found localized patterns of AsIII/AsV redox transformations in the PV rhizosphere (AsIII/AsV ratio of 0.57) compared to bulk soil (AsIII/AsV ratio of ≤0.04). Our data indicate that the high As root uptake, translocation and accumulation from the As-rich experimental soil (2080 mg kg-1) to PV fronds (6986 mg kg-1) induced As detoxification via AsV reduction and AsIII root efflux, leading to AsIII accumulation and re-oxidation to AsV in the rhizosphere porewater. This As cycling mechanism is linked to the reduction of O2 and MnIII/IV (oxyhydr)oxides resulting in decreased O2 levels and increased Mn solubilization along roots. Compared to PV, we found 4-fold lower As translocation to PQ fronds (1611 mg kg-1), 2-fold lower AsV depletion in the PQ rhizosphere, and no AsIII efflux from PQ roots, suggesting that PQ efficiently controls As uptake to avoid toxic As levels in roots. Analysis of root exudates obtained from soil-grown PV showed that As acquisition by PV roots was not associated with phytic acid release. Our study demonstrates that two closely-related As-accumulating ferns have distinct mechanisms for As uptake modulating As cycling in As-rich environments.

19.
Rapid Commun Mass Spectrom ; 33 Suppl 2: 66-74, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30801790

RESUMO

RATIONALE: The wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front-end separation techniques combined with high-resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high-throughput analyses. METHODS: Prior to low-field drift tube ion mobility (IM) separation and quadrupole time-of-flight mass spectrometry (QTOFMS) detection, rapid ultrahigh-performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data-independent all-ions acquisition and a quadrupole broad band isolation (Q-BBI) directed by IM drift separation. RESULTS: For Q-BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision-induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 µM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM-TOFMS-based study. CONCLUSIONS: Chimeric mass spectra, inherent to data-independent analysis approaches, are reduced when using a drift time directed Q-BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front-end separation, a powerful approach for metabolite screening.


Assuntos
Espectrometria de Massas/métodos , Metabolômica/métodos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Metaboloma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Analyst ; 144(15): 4653-4660, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31257367

RESUMO

In this work, we introduce a high-throughput quantitative multi-element method for biological fluids enabled by omitting sample preparation and an analysis time of a few seconds per sample. For the first time, flow injection of an undiluted cerebrospinal fluid (CSF) was combined to state-of-the-art ICP-TOFMS detection for multi-element analysis. Owing to the low sample volume and trace element concentrations of the CSF, flow injection methods with only 5 µL sample intake were used in combination with an icpTOF 2R TOF-based ICP-MS instrument. Due to the lack of certified reference materials for CSF analysis, a validated method employing open vessel digestion of the CSF material in combination with ICP-sectorfield-MS analysis was carried out and used as a reference. Additionally, the performance of the flow injection ICP-TOFMS was cross-validated by flow injection quadrupole-based ICP-MS/MS analysis using both external calibration and isotope dilution strategies. In the latter case, the sample had to be injected several times because of the need for tailored gas conditions for different elements. Overall, flow injection of biological fluids delivered quantitative values, which were in excellent agreement with the gold standard established by ICP-SFMS demonstrating the capability of ICP-TOFMS analysis in terms of resolution and sensitivity for the accurate quantification of trace elements in biological samples.


Assuntos
Espectrometria de Massas em Tandem/métodos , Oligoelementos/líquido cefalorraquidiano , Animais , Calibragem , Análise de Injeção de Fluxo/métodos , Humanos , Limite de Detecção , Oligoelementos/sangue
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