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Aberrant changes in site-specific core fucosylation (CF) of serum proteins contribute to cancer development and progression, which enables them as potential diagnostic markers of tumors. An optimized data-dependent acquisition (DDA) workflow involving isobaric tags for relative and absolute quantitation-labeling and enrichment of CF peptides by lens culinaris lectin was applied to identify CF of serum proteins in a test set of patients with nonalcoholic steatohepatitis (NASH)-related cirrhosis (N = 16) and hepatocellular carcinoma (HCC, N = 17), respectively. A total of 624 CF peptides from 343 proteins, with 683 CF sites, were identified in our DDA-mass spectrometry (MS) analysis. Subsequently, 19 candidate CF peptide markers were evaluated by a target parallel reaction-monitoring-MS workflow in a validation set of 58 patients, including NASH-related cirrhosis (N = 29), early-stage HCC (N = 21), and late-stage HCC (N = 8). Significant changes (p < 0.01) were observed in four CF peptides between cirrhosis and HCC, where peptide LGSFEGLVn160LTFIHLQHNR from LUM in combination with AFP showed the best diagnostic performance in discriminating HCC from cirrhosis, with an area under curve (AUC) of 0.855 compared to AFP only (AUC = 0.717). This peptide in combination with AFP also significantly improved diagnostic performance in distinguishing early HCC from cirrhosis, with an AUC of 0.839 compared to AFP only (AUC = 0.689). Validation of this novel promising biomarker panel in larger cohorts should be performed.
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Circulating tumor cells (CTCs) and exosomes, both released from the primary tumor into peripheral blood, are a promising source of cancer biomarkers. They are detectable in the blood and carry a large diversity of biological molecules, which can be used for the diagnosis and monitoring of minimally invasive cancers. However, due to their intrinsic differences in counts, size, and molecular contents, studies have focused on only one type of vesicle. Herein, we have developed an integrated system to sequentially isolate CTCs and exosomes from a single patient blood sample for further profiling and analysis. The CTCs are isolated using a commercial filtration method and then the remaining blood is processed using multiple cycles of ultracentrifugation to isolate the exosomes. The method uses two available technologies where the eluent from CTC isolation is usually discarded and interfaces them, so that the eluent can be interfaced to exosome isolation methods. The CTCs are identified based on fluorescence staining of their surface markers, while the exosomes are analyzed using transmission electron microscopy, nanosight tracking analysis, and mass spec proteomic analysis. This analysis showed CTCs detected by their surface markers for metastatic hepatocellular carcinoma (HCC), while essentially none were detected for cirrhosis. The exosome analysis resulted in the identification of â¼500-1000 exosome proteins per sample confirmed by detection of exosome surface markers CD9, CD63, CD81, and TSG101 in addition to proteins related to cancer progression. Proteins enriched in HCC exosomes were shown to be involved in the immune response, metastasis, and proliferation.
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Glycopeptide analysis by mass spectrometry may provide an important opportunity in discovery of biomarkers to aid in early detection of Alzheimer's Disease (AD). In this work, we have used a NanoLC-Stepped-HCD-DDA-MS/MS platform and a NanoLC-Stepped-HCD-PRM-MS platform for large-scale screening and quantification of novel N-glycopeptide biomarkers for early detection of AD in patient serum. N-glycopeptides were retrieved from 10 µL of serum in patients with mild cognitive impairment (MCI, a prodromal phase of AD) and normal controls, respectively, after trypsin digestion, glycopeptide enrichment, fractionation, and NanoLC-Stepped-HCD-DDA-MS/MS or NanoLC-Stepped-HCD-PRM-MS analysis. Using a combination of Byonic, Byologic and Skyline softwares, we were able to accomplish both identification and label-free quantitation of site-specific N-glycopeptides between MCI and normal controls. Differential quantitation analysis by Byologic showed that 29 N-glycopeptides derived from 16 glycoproteins were significantly changed in MCI compared to normal controls. Further, HCD-PRM-MS quantitative analysis of the selected N-glycopeptide candidates confirmed that EHEGAIYPDN138TTDFQR_HexNAc(4)Hex(5)-Fuc(2)NeuAc(1) from CERU, and VCQDCPLLAPLN156DTR_HexNAc(4)Hex(5)NeuAc(2) from AHSG can significantly discriminate MCI from normal controls. These two glycopeptides had the area under the receiver operating characteristic curve (AUC) of 0.850 (95% CI, 0.66-1.0) and 0.867 (95% CI, 0.68-1.0), respectively (p<0.05). The result demonstrates that changes in the expression level of the N-glycopeptides provide potential serum biomarkers for detection of AD at a very early stage.
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Serum-derived extracellular vesicles (EVs) are a promising source of biomarkers; however, major challenges in EV separation and proteomic profiling remain for isolating EVs from a small amount, that is, on the microliter scale, of human serum while minimizing the contamination of blood proteins and lipoprotein particles coeluting in EV preparations. Herein we have developed a column-based CD9-antibody-immobilized high-performance liquid chromatography immunoaffinity chromatography(CD9-HPLC-IAC) technology for EV isolation from a microliter scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 µL of serum in 30 min with a yield of 8.0 × 109 EVs, where EVs were further processed with a postcolumn cleaning step using the 50 kDa molecular weight cut-off filter for the buffer exchange, concentration, and reduction of potentially coeluting serum proteins. In total, 482 proteins were identified in EVs by using liquid chromatography tandem mass spectrometry, including the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101. The statistical analysis of EV protein content showed that the top 10 serum proteins in EVs were significantly decreased by using the CD9-HPLC-IAC method compared with the use of ultracentrifugation (p = 0.001) and size exclusion chromatography (p = 0.009), and apolipoproteins were significantly reduced 4.8-fold compared with the SEC method (p < 0.001). The result demonstrates the potential of the CD9-HPLC-IAC method for the efficient isolation and proteomic characterization of EVs from a microscale volume of serum.
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Vesículas Extracelulares , Proteômica , Proteínas Sanguíneas , Cromatografia Líquida de Alta Pressão , Humanos , Tecnologia , Tetraspanina 29RESUMO
Changes in N-glycosylation on specific peptide sites of serum proteins have been investigated as potential markers for diagnosis of nonalcoholic steatohepatitis (NASH)-related HCC. To accomplish this work, a novel workflow involving broad-scale marker discovery in serum followed by targeted marker evaluation of these glycopeptides were combined. The workflow involved an LC-Stepped HCD-DDA-MS/MS method coupled with offline peptide fractionation for large-scale identification of N-glycopeptides directly from pooled serum samples (each n = 10) as well as differential determination of N-glycosylation changes between disease states. We then evaluated several potentially diagnostic N-glycopeptides among 78 individual patient samples (40 cirrhosis, 28 early stage NASH HCC, and 10 late-stage NASH HCC) by LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 65 targeted glycopeptides from 7 glycoproteins. Of these targets, we found site-specific N-glycopeptides n169GSLFAFR_HexNAc(4)Hex(5)NeuAc(2) and n242ISDGFDGIPDNVDAALALPAHSYSGR_HexNAc(5)Hex(6)Fuc(1)NeuAc(3) from VTNC were significantly increased comparing samples from patients with NASH cirrhosis and NASH HCC (p < 0.05). When combining results of these 2 glycopeptides with AFP, the ROC curve analysis demonstrated the AUC value increased to 0.834 (95% CI, 0.748-0.921) and 0.847 (95% CI, 0.766-0.932), respectively, as compared to that of AFP alone (AUC = 0.791, 95% CI, 0.690-0.892). These 2 glycopeptides may serve as potential biomarkers for early HCC diagnosis in patients with NASH related cirrhosis.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Carcinoma Hepatocelular/diagnóstico , Diagnóstico Precoce , Glicopeptídeos , Humanos , Neoplasias Hepáticas/diagnóstico , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Espectrometria de Massas em TandemRESUMO
We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 µL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (â¼1 × 1011 EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.
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Proteínas Sanguíneas/análise , Vesículas Extracelulares/química , Proteoma/análise , Proteômica/métodos , Cromatografia Líquida/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , UltracentrifugaçãoRESUMO
Identifying single amino acid variants (SAAVs) in cancer is critical for precision oncology. Several advanced algorithms are now available to identify SAAVs, but attempts to combine different algorithms and optimize them on large data sets to achieve a more comprehensive coverage of SAAVs have not been implemented. Herein, we report an expanded detection of SAAVs in the PANC-1 cell line using three different strategies, which results in the identification of 540 SAAVs in the mass spectrometry data. Among the set of 540 SAAVs, 79 are evaluated as deleterious SAAVs based on analysis using the novel AssVar software in which one of the driver mutations found in each protein of KRAS, TP53, and SLC37A4 is further validated using independent selected reaction monitoring (SRM) analysis. Our study represents the most comprehensive discovery of SAAVs to date and the first large-scale detection of deleterious SAAVs in the PANC-1 cell line. This work may serve as the basis for future research in pancreatic cancer and personal immunotherapy and treatment.
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Aminoácidos , Neoplasias Pancreáticas , Antiporters , Linhagem Celular , Humanos , Proteínas de Transporte de Monossacarídeos , Neoplasias Pancreáticas/genética , Medicina de Precisão , ProteínasRESUMO
Outer membrane vesicles (OMVs) are nanosized spheres secreted by bacteria that are similar to the vesicles known as exosomes, which are secreted by most mammalian cell types. In contrast to many studies focusing on optimizing methods for enriching exosomes from biological fluid, few studies have been conducted to investigate outer membrane vesicles from fecal samples. Herein, we have developed a pipeline comprised of membrane filtration and multiple cycles of ultracentrifugation (UC) to isolate OMVs from fecal samples for proteomics analysis, where multiple cycles of UC are required for removal of contaminants. By iTRAQ labeling quantitative proteomics analysis, different filter sizes (0.22 µm and 0.45 µm) were compared in terms of their performance in enriching OMVs and eliminating background fecal material. Using the 0.45 µm filter, a slightly higher protein yield was obtained but no additional contaminating proteins from bacteria were identified compared to those from the 0.22 µm filter. The 0.45 µm filter together with the multiple cycles of UC were thus used to isolate OMVs for proteomics analysis. To our knowledge, this is the first study profiling a large number of OMV proteins from fecal samples. Such capabilities may help provide valuable information in understanding the communication between the host and microbiota, which is critical in preventing cancer and disease development.
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We have performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing, and a carrier/reference strategy. Off line fractionation of the TMT-labeled sample pooled with TMT-labeled carrier Panc-1 whole cell proteome was achieved using alkaline reversed phase spin columns. The fractionation in conjunction with the carrier/reference (C/R) proteome allowed us to detect 47â¯414 unique peptides derived from 6261 proteins, which provided a sufficient coverage to search for single amino acid variants (SAAVs) related to cancer. This high sample coverage is essential in order to detect a significant number of SAAVs. In order to verify genuine SAAVs versus false SAAVs, we used the SAVControl pipeline and found a total of 79 SAAVs from the 9-cell Panc-1 sample and 174 SAAVs from the 5000-cell Panc-1 C/R proteome. The SAAVs as sorted into high confidence and low confidence SAAVs were checked manually. All the high confidence SAAVs were found to be genuine SAAVs, while half of the low confidence SAAVs were found to be false SAAVs mainly related to PTMs. We identified several cancer-related SAAVs including KRAS, which is an important oncoprotein in pancreatic cancer. In addition, we were able to detect sites involved in loss or gain of glycosylation due to the enhanced coverage available in these experiments where we can detect both sites of loss and gain of glycosylation.
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Sequência de Aminoácidos , Variação Genética , Proteoma/análise , Tamanho da Amostra , Análise de Célula Única/métodos , Linhagem Celular , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteoma/genética , Proteômica/métodosRESUMO
A simple cloud point preconcentration method was developed and validated for the determination of gallic acid, bergenin, quercitrin, and embelin in Ardisia japonica by high-performance liquid chromatography (HPLC) using ultrasonic assisted micellar extraction. Nonionic surfactant Genapol X-080 was selected as the extraction solvent. The effects of various experimental conditions such as the type and concentration of surfactant and salt, temperature, and solution pH on the extraction of these components were studied to optimize the conditions of Ardisia japonica. The solution was incubated in a thermostatic water bath at 60°C for 10 min, and 35% NaH2PO4 (w/v) was added to the solution to promote the phase separation and increase the preconcentration factor. The intraday and interday precision (RSD) were both below 5.0% and the limits of detection (LOD) for the analytes were between 10 and 20 ng·mL-1. The proposed method provides a simple, efficient, and organic solvent-free method to analyze gallic acid, bergenin, quercitrin, and embelin for the quality control of Ardisia japonica.
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Naoxintong capsule (NXTC) was a famous patent medicine of Traditional Chinese Medicine (TCM) to treat cerebrovascular diseases in China. An LC-MS/MS method was developed for simultaneous determination of 11 major ingredients (paeoniflorin, ecdysterone, amygdalin, mulberroside A, caffeic acid, ferulic acid, salvianolic acid B, astragaloside IV, formononetin, cryptotanshinone, and tanshinone IIA) in NXTC in rat plasma. All analytes were separated on an Eclipse plus C18 column using a gradient mobile phase system of acetonitrile-0.1% formic acid aqueous solution. The lower limits of quantification of 11 ingredients were between 0.075 and 10 ng mL-1. The precision was less than 15% and the accuracies were between 85% and 115%. The results showed that caffeic acid, ferulic acid, formononetin, cryptotanshinone, and tanshinone IIA could be detected after oral administration of NXTC. The validated method was successfully applied to pharmacokinetic study of the caffeic acid, ferulic acid, formononetin, cryptotanshinone, and tanshinone IIA in rats after oral administration of NXTC at single and triple dose.
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Annexins are a multigene family of calcium and phospholipid-binding proteins that play important roles in calcium signaling, cell motility, differentiation and proliferation. Our previous mass spectrometry-based proteomics study revealed that annexin A10 (ANXA10) was uniquely overexpressed in pancreatic CD24+ adenocarcinoma cells that were dissected from clinical PDAC tissues but was absent in CD24- adjacent normal cells. The correlation between ANXA10 expression and the progression of pancreatic cancer remains unknown. In this study, we performed an immunostaining assay to evaluate ANXA10 expression in 155 primary human tissue specimens, including normal pancreas, chronic pancreatitis (CP), pancreatic adenocarcinoma (PDAC), pancreatic intraepithelial neoplasia (PanIN, the most important precursor of PDAC), and intraductal papillary mucinous neoplasm (IPMN). The immunostaining result showed that ANXA10 was significantly overexpressed in PanINs, IPMNs, and PDACs but negative in normal pancreas and the majority of chronic pancreatitis tissues. Statistical analysis revealed that ANXA10 expression was significantly associated with PDAC and its precursor lesions (p<0.0001). Abundant ANXA10 expression was predominantly present in pancreatic ductal epithelial cells of PanINs, IPMNs, and tumor cells of PDACs. Since PDAC develops through a series of PanINs which in turn arise from pancreatic ducts, the consistent overexpression of ANXA10 in ductal epithelial cells in PanINs and PDACs but negative in normal pancreatic ducts suggests that ANXA10 could serve as a potential marker indicating the presence of PDAC at its earliest precancerous stages. Double immunostaining of ANXA10 and CD24 showed that there was a large overlap between these two markers in PDAC and high-grade neoplasia lesions. The statistical analysis showed that the coexpression of ANXA10 and CD24 was significantly correlated with the progression of pancreatic precursor lesions towards PDACs.
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Adenocarcinoma/patologia , Anexinas/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno CD24/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Pancreatite Crônica/patologia , Adenocarcinoma/sangue , Adenocarcinoma/genética , Anexinas/sangue , Anexinas/genética , Biomarcadores Tumorais/sangue , Antígeno CD24/sangue , Carcinoma Ductal Pancreático/sangue , Progressão da Doença , Humanos , Família Multigênica/genética , Pâncreas/metabolismo , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Pancreatite Crônica/sangue , Pancreatite Crônica/genéticaRESUMO
A cyclodextrin-based miniaturized solid-phase extraction was developed to extract biopesticides from water and vegetable juices. The analytes were detected by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. In the solid-phase extraction (SPE) procedure, the liquid sample solution is passed through a packed column filled with 40mg of HP-ß-CD, and then the target analytes are absorbed and finally eluted with methanol-acetic acid (90:10, v/v) into a collection tube. The limits of quantification ranged from 3.73 to 16.51ng/mL for a water matrix, from 2.62 to 13.23ng/mL for an orange juice matrix and from 1.76 to 10.35ng/mL for a tomato juice matrix, respectively. The average recovery values were in the range of 88.3-95.9% for the spiked samples. The established methodology was successfully applied to analyze sanguinarine, berberine, rotenone and osthole in water, orange juice and tomato juice.
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Agentes de Controle Biológico/química , Cromatografia Líquida de Alta Pressão/métodos , Ciclodextrinas/metabolismo , Sucos de Frutas e Vegetais/microbiologia , Espectrometria de Massas/métodos , Extração em Fase Sólida/métodos , Microbiologia da Água , Agentes de Controle Biológico/análiseRESUMO
Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700-800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.
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Proteínas Sanguíneas/genética , Recidiva Local de Neoplasia/sangue , Neoplasias Pancreáticas/sangue , Proteoma/genética , Proteínas Sanguíneas/biossíntese , Exossomos/efeitos dos fármacos , Exossomos/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/radioterapiaRESUMO
Cancers are initiated and developed from a small population of stem-like cells termed cancer stem cells (CSCs). There is heterogeneity among this CSC population that leads to multiple subpopulations with their own distinct biological features and protein expression. The protein expression and function may be impacted by amino acid variants that can occur largely due to single nucleotide changes. We have thus performed proteomic analysis of breast CSC subpopulations by mass spectrometry to study the presence of single amino acid variants (SAAVs) and their relation to breast cancer. We have used CSC markers to isolate pure breast CSC subpopulation fractions (ALDH+ and CD44+/CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF-7 breast cancer cell line. By searching the Swiss-CanSAAVs database, 374 unique SAAVs were identified in total, where 27 are cancer-related SAAVs. 135 unique SAAVs were found in the CSC population compared with the mature luminal cells. The distribution of SAAVs detected in MCF-7 cells was compared with those predicted from the Swiss-CanSAAVs database, where we found distinct differences in the numbers of SAAVs detected relative to that expected from the Swiss-CanSAAVs database for several of the amino acids.
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Aldeído Desidrogenase/genética , Substituição de Aminoácidos , Biomarcadores Tumorais/genética , Antígeno CD24/genética , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/metabolismo , Aldeído Desidrogenase/metabolismo , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , Antígeno CD24/metabolismo , Separação Celular , Bases de Dados de Proteínas , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Expressão Gênica , Ontologia Genética , Humanos , Receptores de Hialuronatos/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Células MCF-7 , Células-Tronco Neoplásicas/patologia , Mapeamento de Interação de ProteínasRESUMO
A green and economical method for the extraction and preconcentration of natural pigments (curcumin, demethoxycurcumin and bisdemethoxycurcumin) was developed using ultrasound-assisted extraction combined with dispersive micro solid-phase extraction. In this work, Ionic liquids (ILs) were used for the pre-extraction of natural pigments. The pure chitosan nanoparticles (CS NPs) were then used as a sorbent for the microextraction mode. The method involves the use of ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Operating parameters influencing the performance of extraction steps such as type and concentration of ILs, concentration of CS NPs, type of elution solvent, agitation time and pH of sample-extracting solution were investigated. Under the optimum conditions, the proposed method exhibited a low detection limit in the range of 0.11-0.36ng/mL at S/N=3, and good linearities with coefficients of determination (R(2)) higher than 0.9990. The recoveries of turmeric samples were ranging from 90.45% to 105.04% for the three studied curcuminoids with SD of 3.27-6.58. The experimental results indicated that the ILs and CS NPs were the promising materials for the extraction and enrichment of target curcuminoids from complex solid samples.
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Quitosana/química , Líquidos Iônicos/química , Nanopartículas/química , Pigmentos Biológicos/isolamento & purificação , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão , Curcuma/química , Limite de Detecção , Rheum/química , Soluções , Solventes/química , Espectrometria de Massas em Tandem , Ultrassom , Raios UltravioletaRESUMO
A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.
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Fucose/química , Fucose/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , Configuração de Carboidratos , Metabolismo dos Carboidratos , Cromatografia Líquida/métodos , Fucose/análise , Interações Hidrofóbicas e Hidrofílicas , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Polissacarídeos/química , Espectrometria de Massas em Tandem/métodos , alfa 1-Antitripsina/análiseRESUMO
Thy-1 (CD90) has been shown to be a potential marker for several different types of cancer. However, reports on CD90 expression in pancreatic intraepithelial neoplasia (PanIN) lesions are still limited where PanINs are the most important precursor lesion of pancreatic ductal adenocarcinoma (PDAC). Herein, we investigate candidate markers for PanIN lesions by examining the distribution and trend of CD90 and CD24 expression as well as their co-expression in various stages of PanINs. Thirty cases of PanINs, which were confirmed histopathologically and clinically, were used to evaluate protein expression of CD90 and CD24 by immunofluoresence double staining. CD90 was found to be mainly expressed in stroma around lesion ducts while not observed in acini and islets in PanINs. CD90 also showed increased expression in PanIN III compared to PanIN III. CD24 was mainly present in the cytoplasm and membrane of pancreatic ductal epithelia, especially in the apical epithelium of the duct. CD24 had higher expression in PanIN III compared with PanIN IIIIII or PanIN III. CD90 was expressed around CD24 sites, but there was little overlap between cells that expressed each of these proteins. A correlation analysis showed that these two proteins have a moderate relationship with PanIN stages respectively. These results suggest that co-expression of CD90 and CD24 may have an important role in the development and progression of PanINs, which is also conducive to early detection and treatment of PDAC.
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Antígeno CD24/metabolismo , Carcinoma in Situ/metabolismo , Neoplasias Pancreáticas/metabolismo , Antígenos Thy-1/metabolismo , Carcinoma in Situ/patologia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Gradação de Tumores , Neoplasias Pancreáticas/patologia , Coloração e Rotulagem , Neoplasias PancreáticasRESUMO
A simple, efficient and low-cost method based on matrix solid-phase dispersion (MSPD) microextraction and ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) was developed for the determination of seven main natural phenols (gallic acid, hydroxytyrosol, methyl gallate, luteolin 7-O-ß-d-glucoside, rutin, ellagic acid and oleuropein) and other eleven compounds in olive fruits. The experimental conditions for the MSPD extraction, including the type of adsorbent, the amount of dispersing sorbent, the grinding time, and the type of elution solvent were investigated and optimized. The optimized parameters were determined to be that middle-molecular-weight chitosan was used as adsorbent, the amount of middle-molecular-weight chitosan was selected to be 25mg, the grinding time was chosen to be 60s, and methanol: water (6:4, v:v) was used as elution solvent. Compared with reported methods, the proposed method was more simple, rapid, and efficient. Moreover, this method required less extraction time and less amount of sample and solvent. The method showed good linearity (r(2)≥0.9909) for the seven analytes, with the limits of detection in the range of 69.6-358.4ng/g. And recoveries were above 80.06%. The methodology was successfully applied to the extraction and determination of seven phenolic compounds in olive fruitsï¼Canarii fructusï¼.
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Burseraceae/química , Quitosana/química , Fenóis/análise , Cromatografia Líquida de Alta Pressão , Frutas/química , Metanol , Microextração em Fase Sólida , Solventes , Espectrometria de Massas em TandemRESUMO
A rapid, simple, and strongly selective miniaturized solid phase extraction (SPE) technique, requiring only small amounts of sorbent (24 mg) and elution solvent (600 µL), coupled with ultrahigh-performance liquid chromatography and quadrupole time-of-flight mass spectrometry was developed for detecting tetracycline antibiotics. These analytes were extracted from honey and milk using chitosan-modified graphitized multiwalled carbon nanotubes (G-MWNTs) as the solid sorbent and acetonitrile/acetic acid (8:2, v/v) as the eluent in miniaturized SPE. Under the optimum experimental conditions, a satisfactory linearity (r(2) > 0.992) was obtained, and the limits of detection were in the range of 0.61-10.34 µg/kg for the analytes. The mean recoveries of the five tetracycline antibiotic residues in the real samples were between 81.5 and 101.4%. The results demonstrated that chitosan-modified G-MWNTs comprise a promising material for the enrichment of tetracycline antibiotics from complex food matrices.