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1.
Niger J Clin Pract ; 22(8): 1083-1090, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31417051

RESUMO

Aims: The aim of this study was to provide epidemiological data about the presence of Salmonella spp. and Shigella spp. in raw milk samples collected from different animals. Methods: A total of 231 raw milk samples from 48 cows, 65 goats, 65 sheep, and 53 donkeys were studied. The ISO 6579:2002 and ISO 21567:2004 methods, antimicrobial susceptibility tests, and serotyping were performed. Species and subspecies discriminations were made via matrix-assisted laser desorption/ionization-time of flight mass spectrometry. After DNA isolation from all samples, Salmonella spp. and Shigella spp. were detected using real-time polymerase chain reaction (PCR) kits. Results: Five samples (2.16%) showed positivity out of 231 raw milk samples for Salmonella spp., and 2 (0.87%) samples were detected to be positive by multiplex real-time PCR design. Conclusion: We found that raw milk samples were not free of Salmonella spp. and Shigella spp. and need to be tested routinely to avoid public health problems. Rapid and reliable real-time PCR method can be developed and used for this purposes instead of slow bacterial culture processes.


Assuntos
DNA Bacteriano/análise , Contaminação de Alimentos/análise , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , Salmonella/genética , Salmonella/isolamento & purificação , Shigella/genética , Shigella/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Equidae , Feminino , Cabras , Humanos , Salmonella/classificação , Sensibilidade e Especificidade , Ovinos , Shigella/classificação
2.
Chem Commun (Camb) ; 55(67): 9979-9982, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31367719

RESUMO

A chemical tag enhances peptide detection by multiple orders in mass spectrometry. The substantial improvement in the peptide mapping along with simplified and enhanced fragmentation pattern enables the unambiguous sequencing of a protein and antibody. The chemoselective sensitivity booster provides a tool for remarkably improved analysis of protein bioconjugates.


Assuntos
Peptídeos/análise , Proteínas/análise , Citocromos c/análise , Lisina/química , Mapeamento de Peptídeos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
3.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
4.
Klin Lab Diagn ; 64(7): 430-434, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31408596

RESUMO

Corynebacteria non-diphtheria and, in particular, C. pseudodiphtheriticum species that are closely related to C. propinquum and C. striatum form a group of new respiratory pathogens leading to the development of bronchitis, tracheitis, exacerbation of chronic obstructive pulmonary diseases, nosocomial pneumonia and other pathology. The goal is to analyze the frequency of the release of Сorynebacteria non-diphtheria from the upper respiratory tract of patients with various inflammatory diseases of the respiratory tract. Strains of Сorynebacteria non-diphtheria (C. pseudodiphtheriticum, C. propinquum, C. accolens, et al.), isolated from patients with inflammatory diseases of the respiratory tract (60 pcs.) and practically healthy individuals (31 pcs.) were studied. Identification of Сorynebacteria was performed using the method of mass spectrometry (MALDI-ToFMS). Сorynebacteria nondiphtheria in the amount of 105 and higher were more frequently detected with the development of chronic tonsillitis (60.0%) and nasopharyngitis (30%). The strains of C. pseudodiphtheriticum (40.0±6.4%) and the closely related species C. propinquum (21.7±5.3%) were mainly found; much less often - C. accolens (8.3±3.6%), C. afermentans (6.7±3.3%), et al. In 86.7% of cases, Corynebacteria non-diphtheria were isolated from children. In chronic tonsillitis, C. pseudodiphtheriticum and the closely related species of C. propinquum were isolated more often; in nasopharyngitis and bronchitis - С. pseudodiphtheriticum. Isolation of Corynebacteria non-diphtheria and, especially, C. pseudodiphtheriticum, C. propinquum, C. accolens species from patients with inflammatory diseases of the respiratory tract in the amount of 105 and above, if there are no other pathogenic microorganisms in the role of microbial associates, of clinical importance.


Assuntos
Infecções por Corynebacterium/diagnóstico , Corynebacterium/isolamento & purificação , Infecções Respiratórias/microbiologia , Criança , Corynebacterium/classificação , Humanos , Sistema Respiratório/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Toxicol Lett ; 313: 150-158, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276768

RESUMO

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen ß). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity.


Assuntos
Anexina A3/metabolismo , Carcinógenos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Ocratoxinas/toxicidade , Neoplasias Gástricas/induzido quimicamente , Anexina A3/genética , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Biologia Computacional , Eletroforese em Gel Bidimensional , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Invasividade Neoplásica , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
6.
J Agric Food Chem ; 67(32): 8958-8966, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31334644

RESUMO

The functional role of human milk oligosaccharides (HMOs) is closely associated with their type, composition, and structure. However, a detailed analysis of HMOs is difficult because neutral oligosaccharides (NHMOs) are mixed with sialylated oligosaccharides (SHMOs) in milk. Here, NHMOs were separated from SHMOs by DEAE-52 anion chromatography, and lactose was removed by graphite carbon solid-phase extraction. Lactose-free NHMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) based on Girard's reagent P on-target derivatization (GPOD), and SHMOs were analyzed by MALDI-TOF-MS following selective sialic acid derivatization and GPOD. Sixty-four oligosaccharides were detected: 36 NHMOs, of which 28 were fucosylated, and 28 SHMOs, of which 8 with α-2,3-linked monosialic acid, 2 with α-2,3-linked disialic acid, 10 with α-2,6-linked monosialic acid, 2 with α-2,6-linked disialic acid, and 5 with both α-2,3- and α-2,6-linked disialic acid. These findings provide the groundwork for further characterization of the structure and activity of HMOs.


Assuntos
Betaína/análogos & derivados , Leite Humano/química , Oligossacarídeos/química , Betaína/química , Feminino , Humanos , Ácido N-Acetilneuramínico/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Plant Foods Hum Nutr ; 74(3): 414-420, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278561

RESUMO

The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.


Assuntos
Nigella sativa/metabolismo , Peptídeos/análise , Proteínas de Plantas/análise , Proteoma , Eletroforese em Gel Bidimensional , Peso Molecular , Proteômica , Sementes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo
8.
Adv Exp Med Biol ; 1140: 27-43, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347040

RESUMO

Matrix assisted laser desorption ionization (MALDI) mass spectrometry allows for the rapid profiling of different biomolecular species from both biofluids and tissues. Whilst originally focused upon the analysis of intact proteins and peptides, MALDI mass spectrometry has found further applications in lipidomic analysis, genotyping, microorganism identification, biomarker discovery and metabolomics. The combining of multiple profiles data from differing locations across a sample furthermore, allows for spatial distribution of biomolecules to be established utilizing imaging MALDI analysis. This chapter discusses the MALDI process, its usual applications in the field of protein identification via peptide mass fingerprinting before focusing upon advances in the application of the profiling potential of MALDI mass spectrometry and its' various applications in biomedicine.


Assuntos
Peptídeos/análise , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Humanos , Mapeamento de Peptídeos
9.
Adv Exp Med Biol ; 1140: 45-54, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347041

RESUMO

Matrix-Assisted Laser Desorption Ionization In-Source Decay (MALDI-ISD) Mass Spectrometry is a very powerful tool for providing terminal sequence information of biomolecules with minimal sample preparations. Fragmentation is induced at the position where hydrogen radical transfers from matrix to analyte in the MALDI-ISD process by proposed mechanism. Uniform fragmentation in MALDI-ISD generates relative simple ion spectra of readable sequence ladders with labile modifications retained, which is advantageous over other fragmentation methods such as collision-induced dissociation (CID) for characterizing modifications. MALDI-ISD has been applied to de novo sequencing of a 13.6 kDa protein and fully validate sequences of therapeutic antibodies, showing its promising potential in examining reference sequences of biotherapeutics unambiguously. It has also been successfully applied to the analysis of modifications such as post-translational modifications (PTMs) and PEGylation. Here we discuss the applications of MALDI-ISD in protein sequencing and modification analysis by featuring representative studies in details.


Assuntos
Proteínas , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Hidrogênio
10.
Adv Exp Med Biol ; 1140: 55-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347042

RESUMO

In order to overcome the limitations of classic imaging in Histology during the actually era of multiomics, the multi-color "molecular microscope" by its emerging "molecular pictures" offers quantitative and spatial information about thousands of molecular profiles without labeling of potential targets. Healthy and diseased human tissues, as well as those of diverse invertebrate and vertebrate animal models, including genetically engineered species and cultured cells, can be easily analyzed by histology-directed MALDI imaging mass spectrometry. The aims of this review are to discuss a range of proteomic information emerging from MALDI mass spectrometry imaging comparative to classic histology, histochemistry and immunohistochemistry, with applications in biology and medicine, concerning the detection and distribution of structural proteins and biological active molecules, such as antimicrobial peptides and proteins, allergens, neurotransmitters and hormones, enzymes, growth factors, toxins and others. The molecular imaging is very well suited for discovery and validation of candidate protein biomarkers in neuroproteomics, oncoproteomics, aging and age-related diseases, parasitoproteomics, forensic, and ecotoxicology. Additionally, in situ proteome imaging may help to elucidate the physiological and pathological mechanisms involved in developmental biology, reproductive research, amyloidogenesis, tumorigenesis, wound healing, neural network regeneration, matrix mineralization, apoptosis and oxidative stress, pain tolerance, cell cycle and transformation under oncogenic stress, tumor heterogeneity, behavior and aggressiveness, drugs bioaccumulation and biotransformation, organism's reaction against environmental penetrating xenobiotics, immune signaling, assessment of integrity and functionality of tissue barriers, behavioral biology, and molecular origins of diseases. MALDI MSI is certainly a valuable tool for personalized medicine and "Eco-Evo-Devo" integrative biology in the current context of global environmental challenges.


Assuntos
Imagem Molecular , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Humanos , Proteoma
11.
Adv Exp Med Biol ; 1140: 251-263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347052

RESUMO

ADCs are empowered monoclonal antibodies that are designed to harness their targeting ability by linking them to cell-killing agents. They are made up of three main components, the antibody, linker and the cytotoxic drug. The specificity of the antibody with the antigen on the tumor cell surface helps with its internalization into the cell after which the active drug is released causing cell death. The investigation of ADCs can be done using a variety of MS methods. Here, we talk about the bottom-up approach, the top-down approaches such as ECD and ETD, the ESI/MS method and IM-MS. Further, we also focus on the applications of MALDI/MS such as UV-MALDI, IR-MALDI and IMS-MALDI and provide examples of the mass spectra that provide tremendous amount of information on ADC structures.


Assuntos
Anticorpos Monoclonais/análise , Antineoplásicos/análise , Imunoconjugados/análise , Neoplasias , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Adv Exp Med Biol ; 1140: 377-388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347059

RESUMO

Identifying antigen-antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen-antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen-antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.


Assuntos
Complexo Antígeno-Anticorpo/análise , Mapeamento de Epitopos , Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Antígenos de Neoplasias/análise , Epitopos , Humanos
13.
Adv Exp Med Biol ; 1140: 401-415, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347061

RESUMO

Mass spectrometry is a powerful analytical technique becoming increasingly important in different biomedical research area. Mass spectrometric based methods were developed and applied to detect and identify multiple metal ion complexes of peptides and proteins with high sensitivity and high mass accuracy. Aggregation of amyloid-ß (Aß) peptides is one of the main pathological features of Alzheimer's disease (AD), and some metal ions seem to play a key role in AD pathogenesis. Consequently, mass spectrometry was used to investigate heavy metal binding to AD-related peptides. Therefore, the purpose of this chapter is to review the methodology and application of identifying coordination chemistry and binding properties of several metal ion-binding sites to synthetic ß-amyloid (Aß) and anti-amyloid model peptides. The selective metal-amyloid-ß peptide interaction studies using (a) Matrix-assisted laser desorption/ionization mass spectrometry (MALDI); (b) Electrospray ionization mass spectrometry (ESI-MS), and (c) Tandem mass spectrometry (MS/MSn) will be reported.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Metais Pesados/metabolismo , Humanos , Modelos Moleculares , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
14.
Exp Parasitol ; 204: 107723, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299265

RESUMO

Toxoplasmosis, caused by apicomplexan parasite Toxoplasma gondii, is a common food-borne disease in humans. Undercooked meat is a potential source of T. gondii infection. As meat of chicken or rabbit is consumed worldwide, tools such as ELISA for the detection of infection of this parasite in rabbits and chickens are much-needed. To search diagnostic antigens of T. gondii special for rabbits and chickens, we conducted two dimensional electrophoresis (2-DE), Western blotting and mass spectrometry (MS) with T. gondii tachyzoite proteins. When probed with rabbit or chicken anti-T. gondii sera, about 60 positive spots among over 500 visible protein spots were detected. In subsequent mass spectrometric analysis, microneme 4 (MIC4) and a putative rhoptry protein are of diagnositic value among the 13 spots selectively picked from the equivalent gel. This study encourages further validation of these candidate antigens for the development of immunologic tools for the detection of T. gondii infection in chickens and rabbits.


Assuntos
Antígenos de Protozoários/análise , Parasitologia de Alimentos , Carne/parasitologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/diagnóstico , Animais , Western Blotting , Galinhas , Biologia Computacional , Soros Imunes/imunologia , Immunoblotting , Proteínas de Membrana/imunologia , Distribuição Normal , Coelhos , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Toxoplasmose Animal/imunologia , Eletroforese em Gel Diferencial Bidimensional
15.
Medicine (Baltimore) ; 98(25): e16117, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31232959

RESUMO

The study aimed to find novel effect biomarkers for occupational benzene exposure and chronic benzene poisoning (CBP), which might also provide clues to the mechanism of benzene toxicity.We performed a comparative serological proteome analysis between healthy control workers with no benzene exposure, workers with short-term benzene exposure, workers with long-term benzene exposure, and CBP patients using 2D-DIGE and MALDI-TOF-MS. Two of the differentially expressed proteins were then selected to be validated by immune turbidimetric analysis.A total of 10 proteins were found to be significantly altered between different groups. The identified deferentially expressed proteins were classified according to their molecular functions, biological processes, and protein classes. The alteration of 2 important serum proteins among them, apolipoprotein A-I and transthyretin, were further confirmed.Our findings suggest that the identified differential proteins could be used as biomarkers for occupational benzene exposure and CBP, and they may also help elucidate the mechanisms of benzene toxicity.


Assuntos
Benzeno/toxicidade , Proteômica/métodos , Adulto , Análise de Variância , Benzeno/efeitos adversos , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos
16.
Adv Exp Med Biol ; 1073: 77-123, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236840

RESUMO

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science.This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies.In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution.With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies.Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest.


Assuntos
Medicina Legal , Oncologia , Proteômica , Manejo de Espécimes/métodos , Toxicologia , Cromatografia Líquida , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
17.
Microbiol Res ; 223-225: 129-136, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178045

RESUMO

Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.


Assuntos
Antifúngicos/farmacologia , Bacillus subtilis/metabolismo , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , Agentes de Controle Biológico/metabolismo , Lipopeptídeos/antagonistas & inibidores , Lipopeptídeos/metabolismo , Antifúngicos/isolamento & purificação , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Basidiomycota/patogenicidade , Agentes de Controle Biológico/isolamento & purificação , Técnicas de Cocultura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Glucose , Lipopeptídeos/isolamento & purificação , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/genética , Metabolismo Secundário , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Anal Bioanal Chem ; 411(20): 5007-5012, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31147760

RESUMO

MALDI-MSI represents an ideal tool to explore the spatial distribution of proteins directly in situ, integrating molecular and cytomorphological information, enabling the discovery of potential diagnostic markers in thyroid cytopathology. However, red cells present in the fine needle aspiration biopsy (FNAB) specimens caused ion suppression of other proteins during the MALDI-MSI analysis due to large amount of haemoglobin. Aim of this study was to set up a sample preparation workflow able to manage this haemoglobin interference. Three protocols were compared using ex vivo cytological samples collected from fresh thyroid nodules of 9 patients who underwent thyroidectomy: (A) conventional air-dried smears, (B) cytological smears immediately fixed in ethanol, and (C) ThinPrep liquid-based preparation. Protocols C and A were also evaluated using real FNABs. Results show that protocol C markedly decreased the amount of haemoglobin, with respect to protocols A and B. Protein profiles obtained with protocols A and B were characterised by high inter-patient variability, probably related to the abundance of the haemoglobin, whereas similar spectra were observed for protocol C, where haemoglobin contents were lower. Our findings suggest protocol C as the sample preparation method for MALDI-MSI analysis. Graphical abstract.


Assuntos
Biópsia por Agulha/métodos , Hemoglobinas/análise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glândula Tireoide/patologia , Artefatos , Humanos , Tireoidectomia
19.
Anal Bioanal Chem ; 411(19): 4509-4522, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31165185

RESUMO

Metal-organic frameworks (MOFs) have drawn great interest in recent decades due to their fascinating structures, unusual physical properties, versatile modification strategies, and biological compatibilities. In this review, we describe recent progress in the application of MOFs to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Owing to their high porosities, specific affinities, and photon absorption capacities, MOFs can be used as solid adsorbents to selectively capture peptides (including endogenous peptides, phosphopeptides, and glycopeptides) and as matrices in MALDI MS. Current developments in and future prospects for this field of research are also discussed.


Assuntos
Estruturas Metalorgânicas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Glicopeptídeos/química , Humanos
20.
BMC Bioinformatics ; 20(1): 303, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164082

RESUMO

BACKGROUND: The spatial distribution and colocalization of functionally related metabolites is analysed in order to investigate the spatial (and functional) aspects of molecular networks. We propose to consider community detection for the analysis of m/z-images to group molecules with correlative spatial distribution into communities so they hint at functional networks or pathway activity. To detect communities, we investigate a spectral approach by optimizing the modularity measure. We present an analysis pipeline and an online interactive visualization tool to facilitate explorative analysis of the results. The approach is illustrated with synthetical benchmark data and two real world data sets (barley seed and glioblastoma section). RESULTS: For the barley sample data set, our approach is able to reproduce the findings of a previous work that identified groups of molecules with distributions that correlate with anatomical structures of the barley seed. The analysis of glioblastoma section data revealed that some molecular compositions are locally focused, indicating the existence of a meaningful separation in at least two areas. This result is in line with the prior histological knowledge. In addition to confirming prior findings, the resulting graph structures revealed new subcommunities of m/z-images (i.e. metabolites) with more detailed distribution patterns. Another result of our work is the development of an interactive webtool called GRINE (Analysis of GRaph mapped Image Data NEtworks). CONCLUSIONS: The proposed method was successfully applied to identify molecular communities of laterally co-localized molecules. For both application examples, the detected communities showed inherent substructures that could easily be investigated with the proposed visualization tool. This shows the potential of this approach as a complementary addition to pixel clustering methods.


Assuntos
Visualização de Dados , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias Encefálicas/patologia , Análise por Conglomerados , Glioblastoma/patologia , Hordeum , Humanos , Análise de Componente Principal , Sementes/anatomia & histologia , Sementes/química
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