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1.
Anal Chim Acta ; 1178: 338849, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482875

RESUMO

Various mesoporous adsorbents are of great promise for enriching small molecules from biological samples based on the size-exclusion effect. At present, the mesoporous adsorbents have adsorption sites distributed uniformly on the internal and external surfaces of mesopores. However, the adsorption sites on the external surface can adsorb proteins, interfering with the enrichment of small molecules. Herein, a novel immobilized-Ti4+ magnetic mesoporous adsorbent removing the adsorption sites on the external surface was facile prepared via the coupling chemistry of isocyanate with amine and consequent hydrolysis of urea linkage by urease. The adsorbent enables fast and selective enrichment of phosphopeptides and nucleotides from biological samples. In addition, sensitive detection methods for phosphopeptides and nucleotides in human serum are developed by coupling the magnetic solid-phase extraction with matrix-assisted laser desorption/ionization time of flight mass spectrometry and liquid chromatography-mass spectrometer, respectively. Under optimal conditions, response is linear (R2 ≥ 0.9923), limits of detection are low (0.41-9.48 ng mL-1), and reproducibility is acceptable (inter- and intra-day assay RSDs of≤15.0%) for six nucleotides. The developed strategy offers an effective method to eliminate the interference of proteins in the enrichment of small molecules from real biological samples.


Assuntos
Nucleotídeos , Fosfopeptídeos , Adsorção , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
Artigo em Russo | MEDLINE | ID: mdl-34486863

RESUMO

The article presents the results of clinical economical analysis, based on "cost-effectiveness" technology, of MALDI-TOF MS, innovative medical technology of express identification of microorganisms, calculation of incremental indicator and application of notion "willingness-to-pay-threshold". Due to the extension of sanctions against the Russian Federation, this medical equipment for national laboratories becomes difficult to access and expensive, that conditions necessity to scientifically substantiate economical effectiveness of implementation of expensive innovative MALDI-TOF MS technology as instrument to contain global antibiotic resistance increase. The understanding of importance of express identification of microorganisms as well as other positive effects that can be achieved by using modern medical equipment on the basis of mass spectrometry results in improving medical care quality, increasing reputation level of medical institution, greater commitment of physicians and patients to microbiological analysis with purpose of prescription of rational antibiotic therapy and improving population health.


Assuntos
Infecções Bacterianas , Técnicas Microbiológicas , Bactérias , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tecnologia
3.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360826

RESUMO

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Eritrócitos/metabolismo , Glicômica , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Glicosilação , Humanos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
Water Res ; 203: 117543, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34433109

RESUMO

According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment facilities. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is potentially such a method, although most of the currently available mass spectral libraries have been developed in a clinical setting and have limited environmental applicability. In this work, a MALDI-TOF MS drinking water library (DWL) was defined and developed by targeting bacteria present in water intended for human consumption. This database, made up of 319 different bacterial strains, can contribute to the routine microbiological control of either treated drinking water or mineral bottled water carried out by water treatment and distribution operators, offering a faster identification rate compared to a clinical sample-based library. The DWL, made up of 96 bacterial genera, 44 of which are not represented in the MALDI-TOF MS bacterial Bruker Daltonics (BDAL) database, was found to significantly improve the identification of bacteria present in drinking water.


Assuntos
Água Potável , Purificação da Água , Bactérias , Bases de Dados Factuais , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Fa Yi Xue Za Zhi ; 37(3): 402-401, 2021 Jun.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34379912

RESUMO

Abstract: Mass spectrometry imaging (MSI) is a new imaging technology that can simultaneously detect and record the spatial distribution information of multiple molecules on the sample surface without labeling. The main principle of MSI is to combine mass spectrometry with imaging technology and irradiate the sample slice with ion beam or laser to ionize the molecules on its surface, obtain the mass spectrometry signal through the detector, convert the obtained data into pixel points by the imaging software, and then construct the spatial distribution image of the target compound on the tissue surface. The sample preparation for MSI include: sample collection and storage, tissue section, tissue pretreatment, selection and application of matrix. At present, this technology has been widely used in the fields of biomedicine, new drug development and proteomics, and its application in the field of forensic toxicology has also gradually attracted attention. This article reviews the principles and sample preparation process of MSI, describes the application of MSI in abused substances and metabolites of various material matrices, herbal mixtures, latent fingerprints, hair and animal and plant tissues, and discusses the prospects of the application of this technology in forensic toxicology, in order to provide ideas and references for the application of MSI technology in forensic toxicology.


Assuntos
Diagnóstico por Imagem , Proteômica , Animais , Toxicologia Forense , Humanos , Espectrometria de Massas , Plantas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Klin Lab Diagn ; 66(8): 502-508, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34388322

RESUMO

Corynebacterium spp. - representatives of the normal microflora of the human body, but their role in the development of diseases in both immunocompromised and immunocompetent patients is known. Corynebacterim spp. (C. pseudodiphtheriticum, C. striatum, C. amycolatum, C. accolens, C. argentoratense, etc.) is associated with diseases of the respiratory tract: tracheitis, pharyngitis, rhinosinusitis, bronchitis, etc. They can be transmitted by airborne droplets, household contact, and possibly by hematogenic pathways. Corynebacterim spp. toxins do not produce, but are capable of adhesion and invasion, biofilm formation, production of neuraminidase, hyaluronidase, and hemolysin. It is necessary to take into account not so much the species, but the strain affiliation of isolates of Corynebacterium spp., since among the representatives of one species of non-diphtheria corynebacteria (for example, C. pseudodiphtheriticum), colonizing the respiratory tract, there may be strains that can exhibit not only pathogenic properties, but also probiotic activity. Microbiological diagnostics is based on their quantitative determination in biological material, phenotypic (culture study, test systems for biochemical identification, Vitek 2 automated systems) and genotypic (16SpRNA gene sequencing and rpoB) methods. It is possible to use mass spectrometric analysis (MALDI-ToF-MS). The greatest activity against Corynebacterium spp. in vitro studies preserve vancomycin, teicoplanin, and linezolid. Successful therapy with at least two of the following antimicrobial agents (AMP) has been reported: vancomycin, rifampicin, linezolid, and daptomycin. The sensitivity of isolates of Corynebacterium spp. to AMP is not related to the species, but is due to strain differences, and therefore it is necessary to test each isolated strain. Continuous monitoring of the sensitivity of Corynebacterium spp. strains to AMP is necessary due to the observed variability of these traits. Of particular importance is the identification of multidrug-resistant isolates that are currently considered highly pathogenic. When compiling the review, the databases Scopus, Web of Science, The Cochrane Library, CyberLeninka, RSCI were used.


Assuntos
Bronquite , Infecções por Corynebacterium , Antibacterianos/farmacologia , Corynebacterium/genética , Infecções por Corynebacterium/diagnóstico , Humanos , Testes de Sensibilidade Microbiana , Sistema Respiratório , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Molecules ; 26(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34443592

RESUMO

The bacterial infection of post-operative wounds is a common health problem. Therefore, it is important to investigate fast and accurate methods of identifying bacteria in clinical samples. The aim of the study was to analyse the use of the MALDI-TOF MS technique to identify microorganism wounds that are difficult to heal. The most common bacteria are Escherichia coli, Staphylococcus spp., and Enterococcus spp. We also demonstrate the effect of culture conditions, such as the used growth medium (solid: Brain Heart Infusion Agar, Mueller Hilton Agar, Glucose Bromocresol Purple Agar, and Vancomycin Resistance Enterococci Agar Base and liquid: Tryptic Soy Broth and BACTEC Lytic/10 Anaerobic/F), the incubation time (4, 6, and 24h), and the method of the preparation of bacterial protein extracts (the standard method based on the Bruker guideline, the Sepsityper method) to identify factors and the quality of the obtained mass spectra. By comparing the protein profiles of bacteria from patients not treated with antibiotics to those treated with antibiotics based on the presence/absence of specific signals and using the UniProt platform, it was possible to predict the probable mechanism of the action of the antibiotic used and the mechanism of drug resistance.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ferimentos e Lesões/microbiologia , Farmacorresistência Bacteriana , Humanos , Período Pós-Operatório
8.
Appl Microbiol Biotechnol ; 105(18): 6819-6833, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34432131

RESUMO

The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)-specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, "LGVING," and an adjacent conservative peptide, "TQTYNATRVGSLG." Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. KEY POINTS: • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC-MS/MS. • The specificity of mAb 2G12 is mainly determined by the "LGVING" peptide.


Assuntos
Escherichia coli O157 , Sequência de Aminoácidos , Cromatografia Líquida , Epitopos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 263: 120225, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34340052

RESUMO

In this study, highly reproducible MIR spectroscopy and highly sensitive MALDI-ToF-MS data were directly compared for the metabolomic profiling of monofloral and multifloral honey samples from three different botanical origins canola, acacia, and honeydew. Subsequently, three different classification models were applied to the data of both techniques, PCA-LDA, PCA- kNN, and soft independent modelling by class analogy (SIMCA) as class modelling technique. All monofloral external test set samples were classified correctly by PCA-LDA and SIMCA with both data sets, while multifloral test set samples could only be identified as outliers by the SIMCA technique, which is a crucial aspect in the authenticity control of honey. The comparison of the two used analytical techniques resulted in better overall classification results for the monofloral external test set samples with the MIR spectroscopic data. Additionally, clearly more multifloral external samples were identified as outliers by MIR spectroscopy (91.3%) as compared to MALDI-ToF-MS (78.3%). The results indicate that the high reproducibility of the used MIR technique leads to a generally better ability of separating monofloral honeys and in particular, identifying multifloral honeys. This demonstrates that benchtop-based techniques may operate on an eye-level with high-end laboratory-based equipment, when paired with an optimal data analysis strategy.


Assuntos
Mel , Flores , Mel/análise , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise Espectral
10.
J Agric Food Chem ; 69(35): 10358-10370, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428040

RESUMO

The advancement of mass spectrometry provides advantages for transgenic protein characterization in support of safety assessments of genetically modified crops. Here, we describe how matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS) in combination with intact mass and bottom-up analyses can be applied to achieve high confidence in the sequences of transgenic proteins expressed in plants and establish the biochemical equivalence of microbially produced protein surrogates. ISD confirmed 40-60 near terminal residues regardless of the protein size, including the improvement of the coverage of cysteine-rich proteins by the reduction/alkylation of disulfide bonds. Negative ISD significantly improved spectral quality and sequence coverage of acidic proteins. Various post-translational modifications, such as terminal truncations and N-terminal methionine excision and acetylation, were identified in plant-produced proteins by top-down MS. Finally, we demonstrated that a combination of top-down and bottom-up analyses provides high confidence in sequence equivalence of plant and microbially produced proteins.


Assuntos
Produtos Agrícolas , Proteínas , Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Curr Protoc ; 1(8): e212, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370396

RESUMO

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides a fast and easy means to identify culturable microorganisms to the species level. The sample preparation of microbial colonies for MALDI-TOF analysis requires a suitable protein extraction method. While standard MALDI-TOF sample preparation methods are well suited for the identification of and the discrimination between microorganisms belonging to different species, they are not disruptive enough to allow the discrimination between different strains of the same microorganism. More disruptive protein extraction methods lead to better discrimination power because they allow a better breakdown of bacterial cell membrane and a more efficient extraction of conserved microbial proteins that are specific to each species and strain. Here we describe how to extract proteins from single microbial colonies using formic acid and acetonitrile to disrupt cells prior to placing them on a target plate for MALDI-TOF MS analysis. Contrary to other sample preparation methods for MALDI-TOF MS, this approach allows the discrimination between different strains of microorganisms of the same species. Our approach also provides the groundwork data for building algorithms that allow the detection of specific microbial strains of interest, with a great potential for diagnostic applications in clinical settings. © 2021 Wiley Periodicals LLC. Basic Protocol: Protein extraction and MALDI-TOF bio-typing of phenotypically distinct bacterial species.


Assuntos
Bactérias , Manejo de Espécimes , Humanos , Lasers , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Front Cell Infect Microbiol ; 11: 634215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381737

RESUMO

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called "proteotyping". To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.


Assuntos
Bacteriemia , Infecções Estafilocócicas , Bacteriemia/diagnóstico , Candida albicans , Escherichia coli , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus
13.
Environ Monit Assess ; 193(8): 546, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34338921

RESUMO

The study focused on assessing drinking water quality from different sources in Gweru urban. Seventy six samples were collected from 6 different locations and analysed for physicochemical parameters and microbial quality. Bacteria isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry and antibiotic susceptibility was determined for 4 isolates that had been identified as Escherichia coli (2) and Salmonella spp. (2). Although most samples were within World Health Organisation limits for most parameters, none met coliform limits. pH ranged between 6.2 and 6.9. Salmonella prevalence was 2%. Escherichia coli and Salmonella spp. isolates were resistant to at least three antibiotics. The study showed inconsistent water quality across the city and contamination in alternative water sources.


Assuntos
Água Potável , Escherichia coli , Resistência Microbiana a Medicamentos , Monitoramento Ambiental , Salmonella , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Qualidade da Água , Zimbábue
14.
Talanta ; 234: 122640, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364449

RESUMO

Separating Yersinia pseudotuberculosis and Yersinia pestis is an important issue in plague diagnosis but can be extremely difficult because of the high similarity between the two species. MALDI-TOF MS has grown as a diagnostic tool with great potential in bacterial identification. Its application in this field is largely enhanced by multivariate analysis, especially in extracting subtle spectral differences. In this study, we built a complete MALDI-TOF MS data pipeline and found a Y. pestis-specific biomarker at 3063 Da closely related to Y. pestis plasminogen activation factor. Based on this, we achieved almost perfect separation between Y. pseudotuberculosis and Y. pestis (AUC = 0.999) using a supervised linear discriminant analysis (LDA) model. This is significantly better than the conventionally applied unsupervised spectral similarity comparison methods, such as hierarchical clustering analysis (HCA), which gave a separation accuracy of 75.0%. This new computing method paves the way for automatic differentiation between the two highly similar bacterial species with high separation accuracy.


Assuntos
Yersinia pestis , Yersinia pseudotuberculosis , Análise por Conglomerados , Análise Multivariada , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
Talanta ; 234: 122674, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364474

RESUMO

Ambient ionization of glycans is simply and efficiently achieved by spraying from an alkali metal salt-impregnated paper surface. Monosaccharides, oligosaccharides and ring glycans easily form abundant alkali metal adduct ions, and give simple and clean high-quality mass spectra. The enhancement is specific for glycans, compared to a wide variety of non-glycan compounds present in a matrix. In addition, molecular weight of unknown glycans can be further identified based on the ion mass difference of various alkali metal adduct ions from a certain compound when using a mixed salt-impregnated paper containing five cation salts. Successful determination of glycans and glycoconjugates in plant extracts, honey, blood and urine demonstrates the practicability of this approach to complicated matrices, especially biological matrices.


Assuntos
Metais Alcalinos , Polissacarídeos , Peso Molecular , Oligossacarídeos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Talanta ; 234: 122687, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364486

RESUMO

Wolfberry fruit has been attracting attention for centuries in Asian countries as a traditional herbal medicine and valuable nourishing tonic. Revealing the spatial distribution changes of important endogenous molecules during plant development is of great significance for investigating the physiological roles, nutritional and potential functional values of phytochemicals in wolfberry fruit. However, their spatial distribution information during fruit development has not been extensively explored due to the lack of efficient analytical techniques. In this work, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was performed to visualize the spatial distribution of the endogenous molecules during fruit development. From the mass spectrum imaging, the choline, betaine and citric acid were distributed evenly throughout the entire fruit at all development stages. The hexose was distributed in the endocarp and flesh tissue, while sucrose was located in the seeds. Additionally, several phenolic acids and flavonoids were accumulated in the exocarp during fruit development, which indicated that they seemingly played protective roles in wolfberry fruit growth progress against abiotic and biotic stress. From the collected data, we found that the signal intensities of citric acid were decreased, while choline, betaine, hexose and sucrose were increased with fruit development. These results indicate that MALDI-MSI may become a favorable tool for studying of the spatial distribution and effective use of endogenous molecules, which provide a simple and intuitive way for authenticity identification, classification of drug food homologous foods and further understanding the physiological roles of endogenous molecules.


Assuntos
Frutas , Lycium , Flavonoides , Lasers , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Anal Chem ; 93(31): 10982-10989, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34328720

RESUMO

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (ß-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein-protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 µM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs.


Assuntos
Lasers , Proteínas , Ligação Proteica , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Methods Mol Biol ; 2350: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331294

RESUMO

We describe a multiplexed imaging mass spectrometry approach especially suitable for fibrosis research. Fibrosis is a process characterized by excessive extracellular matrix (ECM) secretion. Buildup of ECM impairs tissue and organ function to promote further progression of disease. It is an ongoing analytical challenge to access ECM for diagnosis and therapeutic treatment of fibrosis. Recently, we reported the use of the enzyme collagenase type III to target the ECM proteome in thin histological tissue sections of fibrotic diseases including hepatocellular carcinoma, breast cancer, prostate cancer, lung cancer and aortic valve stenosis. Detection of collagenase type III peptides by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows localization of ECM peptide sequences to specific regions of fibrosis. We have further identified that the ECM proteome accessed by collagenase type III has on average 3.7 sites per protein that may be differentially N-glycosylated. N-glycosylation is a major posttranslational modification of the ECM proteome, influencing protein folding, secretion, and organization. Understanding both N-glycosylation signaling and regulation of ECM expression significantly informs on ECM signaling in fibrosis.


Assuntos
Biomarcadores , Matriz Extracelular/metabolismo , Histocitoquímica/métodos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Glicosilação , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Pesquisa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fluxo de Trabalho
19.
Environ Sci Technol ; 55(15): 10558-10568, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34286960

RESUMO

Misuse of agrochemicals has a long-lasting negative impact on aquatic systems. Mismanagement of herbicides in agri-food sectors is often linked to a simultaneous decline in the health of downstream waterways. However, monitoring the herbicide levels in these areas is a laborious task, and modern analytical approaches, such as solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) and enzyme-linked immunosorbent assay, are low-throughput and require significant sample preparation. We report here the use of microchip technology in combination with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for the assessment of the ecotoxicological effect of agrochemicals on aquatic species at the single-cell level. This approach quantifies the fluctuations in lipid content in sentinel organisms and targets the microalga, Chlamydomonas reinhardtii (C. reinhardtii), as the model system. Specifically, we investigated the cytotoxicity of three herbicides (atrazine, clomazone, and norflurazon) on C. reinhardtii by analyzing the lipid component variation upon assorted herbicide exposure. Lipidomic profiling reveals a significantly altered lipid content at >EC50 in atrazine-exposed cells. The response for norflurazon showed similar trends but diminished in magnitude, while the result for clomazone was near muted. At lower herbicide concentrations, digalactosyldiacylglycerols showed a rapid decrease in abundance, while several other lipids displayed a moderate increase. The microchip-based MALDI technique demonstrates the ability to achieve lipidomic profiling of aquatic species exposed to different stressors, proving effective for high-throughput screening and single-cell analysis in ecotoxicity studies.


Assuntos
Atrazina , Chlamydomonas reinhardtii , Herbicidas , Herbicidas/toxicidade , Lipidômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Chem Commun (Camb) ; 57(60): 7362-7365, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34196343

RESUMO

A facile strategy was introduced for room-temperature controllable synthesis of hierarchically flower-like hollow COFs (FHF-COFs). Furthermore, the universality for synthesis of the HFH-COFs was validated by altering the building units. Inspired by the unique morphology, extremely large surface area and good chemical stability, HFH-COFs could serve as an attractive adsorption probe by loading with gold nanoparticles and be applied to enrichment of brain natriuretic peptide from human serum. This work opens up a whole new approach for controllable synthesis of the HFH-COFs at room temperature and expands the application of COFs as a promising enrichment probe for complex biological samples.


Assuntos
Estruturas Metalorgânicas/química , Peptídeo Natriurético Encefálico/isolamento & purificação , Adsorção , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Estruturas Metalorgânicas/síntese química , Peptídeo Natriurético Encefálico/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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