Combination of automated sample preparation and micro-flow LC-MS for high-throughput plasma proteomics.

BACKGROUND: Non-invasive detection of blood-based markers is a critical clinical need. Plasma has become the main sample type for clinical proteomics research because it is easy to obtain and contains measurable protein biomarkers that can reveal disease-related physiological and pathological changes. Many efforts have been made to improve the depth of its identification, while there is an increasing need to improve the throughput and reproducibility of plasma proteomics analysis in order to adapt to the clinical large-scale sample analysis. METHODS: We have developed and optimized a robust plasma analysis workflow that combines an automated sample preparation platform with a micro-flow LC-MS-based detection method. The stability and reproducibility of the workflow were systematically evaluated and the workflow was applied to a proof-of-concept plasma proteome study of 30 colon cancer patients from three age groups. RESULTS: This workflow can analyze dozens of samples simultaneously with high reproducibility. Without protein depletion and prefractionation, more than 300 protein groups can be identified in a single analysis with micro-flow LC-MS system on a Orbitrap Exploris 240 mass spectrometer, including quantification of 35 FDA approved disease markers. The quantitative precision of the entire workflow was acceptable with median CV of 9%. The preliminary proteomic analysis of colon cancer plasma from different age groups could be well separated with identification of potential colon cancer-related biomarkers. CONCLUSIONS: This workflow is suitable for the analysis of large-scale clinical plasma samples with its simple and time-saving operation, and the results demonstrate the feasibility of discovering significantly changed plasma proteins and distinguishing different patient groups.

A non-destructive and efficient transfer method for preparing 2D materials samples for transmission electron microscopy study.

Studying two-dimensional (2D) materials using transmission electron microscopy (TEM) is necessary and very important in many aspects. However, some 2D materials are not resistant to acids or alkalis, which are widely used in normal wet transfer techniques to transfer the exfoliated 2D nanosheets onto the TEM grids. On the other hand, dry stamping method can damage the holey carbon film on the TEM grids. In this article, we present a non-destructive, efficient, and widely applicable transfer method for preparing the TEM samples of the exfoliated 2D materials. Our method only uses the heat-release tape, PMMA, and blue Nitto tape. Neither acid nor alkali is involved in our method, therefore, impurities and damage can be avoided to the greatest extent. The method is also very efficient and can be accomplished in less than 30 min after the exfoliation of the 2D materials. This method is particularly useful for preparing the TEM samples of the 2D materials that are not resistant to acids and alkalis. The present method is also applicable to various 2D materials and various substrates.

Analytical approaches to assess metabolic changes in psoriasis.

Psoriasis is one of the most common human skin diseases, although its development is not limited to one tissue, but is associated with autoimmune reactions throughout the body. Overproduction of pro-inflammatory cytokines and growth factors systemically stimulates the proliferation of skin cells, which manifests as excessive exfoliation of the epidermis, and/or arthritis, as well as other comorbidities such as insulin resistance, metabolic syndrome, hypertension, and depression. Thus, there is a great need for a thorough analysis of the pathophysiology of psoriatic patients, including classical methods, such as spectrophotometry, chromatography, or Western blot, and also novel omics approaches such as lipidomics and proteomics. Moreover, the extensive pathophysiology forces increased research examining biological changes in both skin cells, and systemically. A wide range of techniques involved in lipidomic research based on a combination of mass spectrometry and different types of chromatography (RP-LC-QTOF-MS/MS, HILIC-QTOF-MS/MS or RP-LC-QTRAP-MS/MS), have allowed comprehensive assessment of lipid modification in psoriatic skin and provided new insight into the role of lipids and their mechanism of action in psoriasis. Moreover, proteomic analysis using gel-nanoLC-OrbiTrap-MS/MS, as well as MALDI-TOF/TOF techniques facilitates the description of panels of enzymes involved in lipidome modifications, and the response of the endocannabinoid system to metabolic changes. Psoriasis is known to alter the expression of proteins that are involved in the inflammatory and antioxidant response, as well as protein biosynthesis, degradation, as well as cell proliferation and apoptosis. Knowledge of changes in the lipidomic and proteomic profile will not only allow the understanding of psoriasis pathophysiology, but also facilitate proper and early diagnosis and effective pharmacotherapy.

Cytotoxicity assays as tools to assess water quality in the Sinos River basin / Ensaios de citotoxicidade como ferramentas para avaliar a qualidade da água da bacia do Rio do Sinos

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin.

Os ensaios de citotoxicidade utilizando culturas de células constituem uma alternativa para avaliar a toxicidade biológica de águas de superfície e podem auxiliar no controle da qualidade da água. Este estudo comparou dois métodos de preparação dos meios de cultura com amostras de água coletadas no rio Rolante, um importante afluente do Rio dos Sinos, para a exposição de células Hep-2. A toxicidade foi avaliada usando os ensaios do MTT e do vermelho neutro (VN). Dois métodos foram utilizados para preparar os meios de cultura. No método 1, a amostra foi diluída a 1:1, 1:10, 1:100, 1:1000 e 1:10.000 (v/v, amostra/ meio de cultivo) em um meio de cultura padrão; no método 2, as amostras de água foram utilizados como solventes para o meio de cultura, o qual foi preparado em concentração de 100% e nas diluições de 80, 60, 40 e 20%. Culturas semi-confluentes foram então expostas aos meios teste durante 24 horas, e a citotoxicidade foi determinada imediatamente usando os ensaios MTT e VN. A atividade mitocondrial (MTT) foi significativamente menor em todas as concentrações em ambos os métodos, exceto na diluição 1:1000 do método 1. No entanto, os resultados de viabilidade lisossomal (VN) revelaram citotoxicidade apenas no na diluição 1:1 do método 1. Ambos os métodos de preparação do meio cultura foram eficientes e sensíveis para o ensaio do MTT, mas o método 2 foi mais adequado para o ensaio do VN. O rio Rolante possui contaminantes citotóxicos para as células Hep-2, o que pode ser uma das explicações para a baixa qualidade da água da Bacia do Rio dos Sinos.

Cytotoxicity assays as tools to assess water quality in the Sinos River basin

p>Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin. /p>

p>Os ensaios de citotoxicidade utilizando culturas de células constituem uma alternativa para avaliar a toxicidade biológica de águas de superfície e podem auxiliar no controle da qualidade da água. Este estudo comparou dois métodos de preparação dos meios de cultura com amostras de água coletadas no rio Rolante, um importante afluente do Rio dos Sinos, para a exposição de células Hep-2. A toxicidade foi avaliada usando os ensaios do MTT e do vermelho neutro (VN). Dois métodos foram utilizados para preparar os meios de cultura. No método 1, a amostra foi diluída a 1:1, 1:10, 1:100, 1:1000 e 1:10.000 (v/v, amostra/ meio de cultivo) em um meio de cultura padrão; no método 2, as amostras de água foram utilizados como solventes para o meio de cultura, o qual foi preparado em concentração de 100% e nas diluições de 80, 60, 40 e 20%. Culturas semi-confluentes foram então expostas aos meios teste durante 24 horas, e a citotoxicidade foi determinada imediatamente usando os ensaios MTT e VN. A atividade mitocondrial (MTT) foi significativamente menor em todas as concentrações em ambos os métodos, exceto na diluição 1:1000 do método 1. No entanto, os resultados de viabilidade lisossomal (VN) revelaram citotoxicidade apenas no na diluição 1:1 do método 1. Ambos os métodos de preparação do meio cultura foram eficientes e sensíveis para o ensaio do MTT, mas o método 2 foi mais adequado para o ensaio do VN. O rio Rolante possui contaminantes citotóxicos para as células Hep-2, o que pode ser uma das explicações para a baixa qualidade da água da Bacia do Rio dos Sinos. /p>

Selective Extraction of Low Molecular Weight Proteins by Mesoporous Silica Particles with Phenyl-Modified Internal and Alkyl-diol-Modified External Surfaces / 分析化学

Selective extraction of low molecular weight ( LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. It is demonstrated that the unique pore structure of mesoporous material makes it efficient to enrich LMW proteins and peptides from complex matrixes. In this study, a mesoporous material, alkyl-diol@ phenyl-SiO2 , with modified exterior ( alkyl-diol group) and interior ( phenyl group) surfaces, was synthesized by co-condensation and post-grafting, and its characteristic was evaluated by FTIR and MS. The LMW proteins and peptides enriched by the alkyl-diol@phenyl-SiO2 mesoporous material could be easily eluted by organic solvents, which was compatible with the following detection by mass spectrometry ( MS ) . This new mesoporous material exhibited good selectivity for the extraction of LMW proteins and peptides ( less than 10 kDa) from complex biological samples.