RESUMO
BACKGROUND: Fatty acids (FAs) are essential cellular components and play important roles in various biological processes. Importantly, FAs produced by microorganisms from renewable sugars are considered sustainable substrates for biodiesels and oleochemicals. Their complex structures and diverse functional roles in biochemical processes necessitate the development of efficient and accurate methods for their quantitative analysis. RESULTS: Here, we developed a novel method for relative quantification of FAs by combining 12-plex isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) labeling and microchip capillary electrophoresis-mass spectrometry (CE-MS). This method enables simultaneous quantification of 12 samples in a single MS analysis. DiLeuEN labeling introduced tertiary amine center structure into FAs, which makes them compatible with the positive mode separation of commercial microchip CE systems and further improves the sensitivity. The CE separation parameters were optimized, and the quantification accuracy was assessed using FA standards. Microchip CE-MS detection exhibited high sensitivity with a femtomole level detection limit and a total analysis time within 8 min. Finally, the applicability of our method to complex biological samples was demonstrated by analyzing FAs produced by four industrially relevant yeast strains (Saccharomyces cerevisiae, Yarrowia lipolytica YB-432, Yarrowia lipolytica Po1f and Rhodotorula glutinis). The analysis time for each sample is less than 1 min. SIGNIFICANCE: This work addresses the current challenges in the field by introducing a method that combines microchip-based capillary electrophoresis separation with multiplex isobaric labeling. Our method not only offers remarkable sensitivity and rapid analysis speed but also the capability to quantify fatty acids across multiple samples simultaneously, which holds significant potential for extensive application in FA quantitative studies in diverse research areas, promising an enhanced understanding of FA functions and mechanisms.
Assuntos
Eletroforese em Microchip , Ácidos Graxos , Espectrometria de Massas , Ácidos Graxos/análise , Ácidos Graxos/química , Espectrometria de Massas/métodos , Eletroforese em Microchip/métodos , Ensaios de Triagem em Larga Escala , Yarrowia/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Eletroforese Capilar/métodosRESUMO
Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC - MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.
Assuntos
Glicômica , Ensaios de Triagem em Larga Escala , Polissacarídeos , Espectrometria de Massas em Tandem , Glicômica/métodos , Polissacarídeos/química , Polissacarídeos/análise , Espectrometria de Massas em Tandem/métodos , Ensaios de Triagem em Larga Escala/métodos , Coloração e Rotulagem/métodos , Cromatografia Líquida/métodos , Humanos , Açúcares/química , Açúcares/análiseRESUMO
Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.
Assuntos
Síndrome do Cromossomo X Frágil , Proteínas de Soja , Camundongos , Animais , Proteínas de Soja/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Síndrome do Cromossomo X Frágil/metabolismo , Proteômica , Camundongos Knockout , Modelos Animais de DoençasRESUMO
Glycans are vital biomolecules with diverse functions in biological processes. Mass spectrometry (MS) has become the most widely employed technology for glycomics studies. However, in the traditional data-dependent acquisition mode, only a subset of the abundant ions during MS1 scans are isolated and fragmented in subsequent MS2 events, which reduces reproducibility and prevents the measurement of low-abundance glycan species. Here, we reported a new method termed 6-plex mdSUGAR isobaric-labeling guide fingerprint embedding (MAGNI), to achieve multiplexed, quantitative, and targeted glycan analysis. The glycan peak signature was embedded by a triplicate-labeling strategy with a 6-plex mdSUGAR tag, and using ultrahigh-resolution mass spectrometers, the low-abundance glycans that carry the mass fingerprints can be recognized on the MS1 spectra through an in-house developed software tool, MAGNIFinder. These embedded unique fingerprints can guide the selection and fragmentation of targeted precursor ions and further provide rich information on glycan structures. Quantitative analysis of two standard glycoproteins demonstrated the accuracy and precision of MAGNI. Using this approach, we identified 304 N-glycans in two ovarian cancer cell lines. Among them, 65 unique N-glycans were found differentially expressed, which indicates a distinct glycosylation pattern for each cell line. Remarkably, 31 N-glycans can be quantified in only 1 × 103 cells, demonstrating the high sensitivity of our method. Taken together, our MAGNI method offers a useful tool for low-abundance N-glycan characterization and is capable of determining small quantitative differences in N-glycan profiling. Therefore, it will be beneficial to the field of glycobiology and will expand our understanding of glycosylation.
Assuntos
Glicômica , Espectrometria de Massas em Tandem , Feminino , Humanos , Espectrometria de Massas em Tandem/métodos , Glicômica/métodos , Reprodutibilidade dos Testes , Polissacarídeos/química , ÍonsRESUMO
High-throughput quantitative analysis of the cells' proteomes across multiple conditions such as various perturbations and different time points is essential for gaining insights into treatment-induced biological responses or disease pathological states. The advancements in mass spectrometry instrumentation and isobaric labeling methods provided useful tools to help address such demands. However, the current widely adopted isobaric labeling methods such as tandem mass tag (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) are based on low-mass reporter ions, which are indistinguishable among different peptide analytes, to achieve relative quantification. Therefore, these methods intrinsically suffer from severe ratio distortion when analyzing complex samples due to peptide coelution and cofragmentation. Here, we developed a novel set of isobaric tags named dimethylated leucine complementary ion (DiLeuC) and relied on complementary ions for relative quantification, in which the complementary ions are the remanent peptide segments after fragmentation in the high-mass range. Since those residual peptide fragments are precursor-specific, they retain the relative abundance information in an interference-free manner even in a complex matrix environment. The quantification accuracy of our method was validated in a two-proteome model where the yeast proteome was spiked with a strong background human proteome as interference. In addition, we also applied this strategy to single-cell proteome analysis, demonstrating its potential utility for sensitive high-throughput quantitative proteomics.
Assuntos
Proteoma , Proteômica , Humanos , Proteoma/análise , Proteômica/métodos , Peptídeos/análise , Espectrometria de Massas/métodos , ÍonsRESUMO
Objective: Developing a simple, rapid, reliable, sensitive, and cost-effective method for prenatal detection of fetomaternal haemorrhage by combining multi-aperture silk membrane with enzyme-linked immunosorbent assay (ELISA), which does not require any complicated instruments and can be visually colored, so as to provide a new method for clinical detection of fetomaternal haemorrhage. Methods: As a carrier, a chemically treated silk membrane was used to immobilize anti-A/anti-B antibody reagent. PBS washed slowly after vertically dropping red blood cells. After adding biotin-labeled anti-A/anti-B antibody reagent, PBS is slowly washed, enzyme-labeled avidin is added, and TMB is used for color development after washing. Results: When there were both anti-A and anti-B fetal erythrocytes in pregnant women's peripheral blood, the final color was dark brown. When there are no anti-A and anti-B fetal red blood cells in pregnant women's peripheral blood, the final color development results do not change, which corresponds to the color of chemically treated silk membrane. Conclusion: The new enzyme-linked immunosorbent assay (ELISA) based on a silk membrane can distinguish fetal red blood cells from maternal red blood cells prenatally and can be used for prenatal detection of fetomaternal haemorrhage.
RESUMO
High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimer's disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. Twenty-three proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. Seven out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R, and APOA, exhibited a potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9, and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.
Assuntos
Doença de Alzheimer , Humanos , Leucina/química , Proteólise , Proteômica/métodos , Espectrometria de Massas , Apolipoproteína A-IRESUMO
OBJECTIVE: The currently employed red blood cell reagents have a short shelf life. Some hospitals with a small number of specimens will be unable to utilize them within the validity period, resulting in a substantial increase in the purchase price. Therefore, the method of developing long-term red blood cell reagents is a problem worthy of further study. METHODS: In this experiment, the type and concentration of the red blood cell reagent treatment solution were evaluated based on the red blood cell antigen concentration 24â h after treatment. In addition, the qualified glutaraldehyde/paraformaldehyde reagent was stored for six months, and five red blood cell indices were measured every month. At the same time, the detection indices of treated red blood cell reagents and untreated red blood cell reagents were compared. RESULTS: It was discovered that treated red blood cells containing 0.005% GA and 0.05% PFA were more suitable for the preservation of red blood cells than other treated concentrations, and the preservation time could reach six months. The test tube method (n = 24) and microcolumn gel card (n = 35) were used to determine the accuracy of the treated blood cells containing 0.005% glutaraldehyde +0.05% paraformaldehyde, with an accuracy of 100%. CONCLUSION: This experiment resulted in the development of a novel reagent for treating red blood cells with glutaraldehyde/paraformaldehyde fixed solution that can effectively prolong its storage time by two to three times that of red blood cell reagents currently on the market.
Assuntos
Eritrócitos , Formaldeído , Humanos , Glutaral , Indicadores e ReagentesRESUMO
Glycans, which are widely distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as immune response and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probes greatly facilitates routine analysis of glycans. However, the requirement of high-throughput analysis still calls for advanced tools to be developed. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the subtle mass differences among different isotopologues and expanded the multiplexing capacity to 12 channels, a 3-fold throughput improvement for the original SUGAR tag design and achieved high-throughput N-glycan analysis in a single LC-MS/MS injection. We then applied 12-plex SUGAR tags to profile the N-glycans in four subtypes of human Immunoglobulin G (IgG) and to investigate the N-glycan changes in the endometrial cancer cells (ECC1) treated with Atovaquone, a quinone antimicrobial medication, and a dihydroorotate dehydrogenase (DHODH) inhibitor. Data are available via ProteomeXchange with the identifier PXD038501.
Assuntos
Glicômica , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Glicômica/métodos , Cromatografia Líquida/métodos , Indicadores e Reagentes , Polissacarídeos/químicaRESUMO
HSP90 inhibitors can target many oncoproteins simultaneously, but none have made it through clinical trials due to dose-limiting toxicity and induction of heat shock response, leading to clinical resistance. We identified diptoindonesin G (dip G) as an HSP90 modulator that can promote degradation of HSP90 clients by binding to the middle domain of HSP90 (Kd = 0.13 ± 0.02 µM) without inducing heat shock response. This is likely because dip G does not interfere with the HSP90-HSF1 interaction like N-terminal inhibitors, maintaining HSF1 in a transcriptionally silent state. We found that binding of dip G to HSP90 promotes degradation of HSP90 client protein estrogen receptor α (ER), a major oncogenic driver protein in most breast cancers. Mutations in the ER ligand-binding domain (LBD) are an established mechanism of endocrine resistance and decrease the binding affinity of mainstay endocrine therapies targeting ER, reducing their ability to promote ER degradation or transcriptionally silence ER. Because dip G binds to HSP90 and does not bind to the LBD of ER, unlike endocrine therapies, it is insensitive to ER LBD mutations that drive endocrine resistance. Additionally, we determined that dip G promoted degradation of WT and mutant ER with similar efficacy, downregulated ER- and mutant ER-regulated gene expression, and inhibited WT and mutant cell proliferation. Our data suggest that dip G is not only a molecular probe to study HSP90 biology and the HSP90 conformation cycle, but also a new therapeutic avenue for various cancers, particularly endocrine-resistant breast cancer harboring ER LBD mutations.
Assuntos
Antineoplásicos , Benzofuranos , Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Mutação , Antineoplásicos/farmacologia , Benzofuranos/farmacologiaRESUMO
Protein glycosylation, covalent attachment of carbohydrates to polypeptide chains, is a highly important post-translational modification involved in many essential physiological processes. Comprehensive site-specific and quantitative analysis is crucial for revealing the diverse functions and dynamics of glycosylation. To characterize intact glycopeptides, mass spectrometry (MS)-based glycoproteomics employs versatile fragmentation methods, among which electron-transfer/higher-energy collision dissociation (EThcD) has gained great popularity. However, the inherent limitation of EThcD in fragmenting low-charge ions has prevented its widespread applications. Furthermore, there is a need to develop a high-throughput strategy for comparative glycoproteomics with a large cohort of samples. Herein, we developed isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) tags to increase the charge states of glycopeptides, thereby improving the fragmentation efficiency and allowing for in-depth intact glycopeptide analysis, especially for sialoglycopeptides. Moreover, the unique reporter ions of DiLeuEN-labeled glycopeptides generated in tandem MS spectra enable relative quantification of up to four samples in a single analysis, which represents a new high-throughput method for quantitative glycoproteomics.
Assuntos
Elétrons , Glicopeptídeos , Etilenodiaminas , Glicopeptídeos/química , Humanos , Indicadores e Reagentes , Íons , Leucina/análogos & derivados , Peptídeos/química , Sialoglicoproteínas , Espectrometria de Massas em Tandem/métodosRESUMO
Intact glycopeptide analysis has been of great interest because it can elucidate glycosylation site information and glycan structural composition at the same time. However, mass spectrometry (MS)-based glycoproteomic analysis is hindered by the low abundance and poor ionization efficiency of glycopeptides. Relatively large amounts of starting materials are needed for the enrichment, which makes the identification and quantification of intact glycopeptides from samples with limited quantity more challenging. To overcome these limitations, we developed an improved isobaric labeling strategy with an additional boosting channel to enhance N,N-dimethyl leucine (DiLeu) tagging-based quantitative glycoproteomic analysis, termed as Boost-DiLeu. With the integration of a one-tube sample processing workflow and high-pH fractionation, 3514 quantifiable N-glycopeptides were identified from 30 µg HeLa cell tryptic digests with reliable quantification performance. Furthermore, this strategy was applied to human cerebrospinal fluid (CSF) samples to differentiate N-glycosylation profiles between Alzheimer's disease (AD) patients and non-AD donors. The results revealed processes and pathways affected by dysregulated N-glycosylation in AD, including platelet degranulation, cell adhesion, and extracellular matrix, which highlighted the involvement of N-glycosylation aberrations in AD pathogenesis. Moreover, weighted gene coexpression network analysis (WGCNA) showed nine modules of glycopeptides, two of which were associated with the AD phenotype. Our results demonstrated the feasibility of using this strategy for in-depth glycoproteomic analysis of size-limited clinical samples. Taken together, we developed and optimized a strategy for the enhanced comprehensive quantitative intact glycopeptide analysis with DiLeu labeling, showing significant promise for identifying novel therapeutic targets or biomarkers in biological systems with a limited sample quantity.
Assuntos
Glicopeptídeos , Glicopeptídeos/análise , Células HeLa , Humanos , Leucina/análogos & derivados , Leucina/química , Espectrometria de MassasRESUMO
Despite the important roles of protein sialylation in biological processes such as cellular interaction and cancer progression, simple and effective methods for the analysis of intact sialylglycopeptides (SGPs) are still limited. Analyses of low-abundance SGPs typically require efficient enrichment prior to comprehensive liquid chromatography-mass spectrometry (LC-MS)-based analysis. Here, a novel workflow combining mild periodate oxidation, hydrazide chemistry, copper-catalyzed azide/alkyne cycloaddition (CuAAC) click chemistry, and dynamic covalent exchange has been developed for selective enrichment of SGPs. The intact SGPs could be separated easily from protein tryptic digests, and the signature ions were produced during LC-MS/MS for unambiguous identification. The structure of the signature ions and corresponding dynamic covalent exchange were confirmed by using an isotopic reagent. Under the optimized condition, over 70% enrichment efficiency of SGPs was achieved using bovine fetuin digests, and the method was successfully applied to complex biological samples, such as a mouse lung tissue extract. The high enrichment efficiency, good reproducibility, and easily adopted procedure without the need to generate specialized materials make this method a promising tool for broad applications in SGP analysis.
Assuntos
Química Click , Espectrometria de Massas em Tandem , Alcinos/química , Animais , Azidas/química , Bovinos , Cromatografia Líquida/métodos , Cobre/química , Reação de Cicloadição , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
As one of the most important post-translational modifications, glycosylation plays a pivotal role in many essential physiological functions, including cell recognition, signaling, and immune response. Thus, various qualitative and quantitative analytical strategies for glycomic profiling have been developed in recent decades. However, while extensive efforts have been devoted to the analysis of N-glycans, high-throughput quantitative analysis of O-glycans is often overlooked and underexplored. This is partially due to the lack of a universal enzyme for the release of O-glycans from the protein backbone. Furthermore, the traditional chemical releasing method suffers from severe side reactions and involves tedious sample preparation procedures. Here, a multiplexed isobaric labeling method enabled by N,N-dimethyl leucine containing pyrazolone analogue (DiLeuPMP) is introduced. This method combines the release and labeling of O-glycans in a one-pot reaction and achieves accurate MS2-based relative quantification with the ability to process four samples at a time. The method has been applied to core-1 O-glycan standard and three glycoproteins first, and the results demonstrated its validity. Following this proof-of-principle demonstration, we analyzed more complex biological specimen using human serum samples. Overall, this method provides an effective and reliable approach for the profiling and high-throughput quantitative analysis of O-glycans in complex samples.
Assuntos
Polissacarídeos , Espectrometria de Massas por Ionização por Electrospray , Glicoproteínas , Glicosilação , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Long noncoding RNAs (lncRNAs) exhibit vital roles in many types of cancer, including retinoblastoma (RB), the most common primary intraocular malignancy tumor of infancy. A novel lncRNA TRPM2-AS has been demonstrated to be related to multiple cancers; however, its role in RB remains unclear. Here, we aimed to investigate the function of TRPM2-AS in RB. In this study, TRPM2-AS expression in 35 human RB tissues and RB cell lines was detected by real-time PCR. And, the relationship between its expression and clinicopathological characteristics of RB patients was analyzed. RB cells' proliferation, migration, invasion, apoptosis, and cell cycle were explored after silencing TRPM2-AS. The mechanism of TRPM2-AS in RB was focused on miR-497/WEE1 axis. Additionally, the role and mechanism of TRPM2-AS were confirmed in a xenograft mouse model. We found TRPM2-AS expression was enhanced in RB tissues and cells. And, higher TRPM2-AS expression was related to advanced clinical stage and optic nerve invasion in patients. Downregulation of TRPM2-AS significantly inhibited proliferation, migration, and invasion, elevated apoptosis, attenuated G2/M phase arrest in RB cells, and suppressed tumor growth in vivo. TRPM2-AS acted as a ceRNA for miR-497 to positively regulate WEE1 expression. miR-497 inhibitor or WEE1 overexpression dramatically reversed the effects of TRPM2-AS downregulating on the malignant phenotypes of RB cells. Therefore, TRPM2-AS is an oncogenic lncRNA in RB, and it functions largely through the miR-497/WEE1 pathway. Despite the limited sample size, this study indicates that TRPM2-AS may be a candidate target in RB therapies.
RESUMO
Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.
Assuntos
Aciltransferases/metabolismo , Anti-Infecciosos/química , Proteínas de Bactérias/metabolismo , Resorcinóis/química , Aciltransferases/antagonistas & inibidores , Aciltransferases/classificação , Aciltransferases/genética , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , COVID-19/patologia , COVID-19/virologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Avaliação Pré-Clínica de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Conformação Molecular , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Resorcinóis/isolamento & purificação , Resorcinóis/metabolismo , Resorcinóis/farmacologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Streptomyces/enzimologia , Espectrometria de Massas em TandemRESUMO
Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Formaldeído/química , Indicadores e Reagentes/química , Neoplasias Laríngeas/diagnóstico , Neoplasias Ovarianas/diagnóstico , Polissacarídeos/análise , Fixação de Tecidos , Feminino , Humanos , Hidrazinas/química , Inclusão em Parafina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The unbiased selection of peptide precursors makes data-independent acquisition (DIA) an advantageous alternative to data-dependent acquisition (DDA) for discovery proteomics, but traditional multiplexed quantification approaches employing mass difference labeling or isobaric tagging are incompatible with DIA. Here, we describe a strategy that permits multiplexed quantification by DIA using mass defect-based N,N-dimethyl leucine (mdDiLeu) tags and high-resolution tandem mass spectrometry (MS2) analysis. Millidalton mass differences between mdDiLeu isotopologues produce fragment ion multiplet peaks separated in mass by as little as 5.8 mDa, enabling up to 4-plex quantification in DIA MS2 spectra. Quantitative analysis of yeast samples displayed comparable accuracy and precision for MS2-based DIA and MS1-based DDA methods. Multiplexed DIA analysis of cerebrospinal fluid revealed the dynamic proteome changes in Alzheimer's disease, demonstrating its utility for discovery of potential clinical biomarkers. We show that the mdDiLeu tagging approach for multiplexed DIA is a viable methodology for investigating proteome changes, particularly for low-abundance proteins, in different biological matrices.
Assuntos
Leucina/análogos & derivados , Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Doença de Alzheimer/líquido cefalorraquidiano , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/química , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/química , Humanos , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Proteoma/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometria de Massas em TandemRESUMO
The proliferation and migration of Schwann cells contribute to nerve regeneration after peripheral nerve injury (PNI). In recent years, roles of long non-coding RNAs (lncRNAs) in PNI have been gradually uncovered. However, a highly conserved nuclear lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in peripheral nerve regeneration remains enigmatic. MALAT1 expression in injured sciatic nerve of mice with PNI was measured by real-time PCR. The proliferative and migrative abilities of Schwann cells were determined after upregulating or downregulating Malat1. The relationship among MALAT1, miR-129-5p, and BDNF was measured. In this study, we found elevated MALAT1 expression in injured sciatic nerve. MALAT1 upregulation in Schwann cells promoted cell proliferation and migration. However, downregulation of MALAT1 caused the suppression of Schwann cell proliferation and migration. Mechanistically, we discovered MALAT1 negatively regulated miR-129-5p through directly binding. Brain-derived neurotrophic factor (BDNF) was a target of miR-129-5p. MALAT1 positively modulated BDNF expression and secretion via decreasing miR-129-5p. Downregulation of BDNF rescued the influences of MALAT1 overexpression on Schwann cell proliferation and migration. In conclusion, MALAT1 was enhanced after PNI and it promoted the proliferation and migration of Schwann cells through sponging miR-129-5p to increase BDNF expression and secretion. This study proved that MALAT1 may be a vital regulator in peripheral nerve regeneration.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Movimento Celular , Proliferação de Células , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Células de Schwann/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Longo não Codificante/genética , Células de Schwann/fisiologiaRESUMO
Neuropathic pain caused by a peripheral nerve injury constitutes a great challenge in clinical treatments due to the unsatisfactory efficacy of the current strategy. Microglial activation-mediated neuroinflammation is a major characteristic of neuropathic pain. Oleanolic acid is a natural triterpenoid in food and medical plants, and fulfills pleiotropic functions in inflammatory diseases. Nevertheless, its role in neuropathic pain remains poorly elucidated. In the current study, oleanolic acid dose-dependently suppressed LPS-evoked IBA-1 expression (a microglial marker) without cytotoxicity to microglia, suggesting the inhibitory efficacy of oleanolic acid in microglial activation. Moreover, oleanolic acid incubation offset LPS-induced increases in the iNOS transcript and NO releases from microglia, concomitant with the decreases in pro-inflammatory cytokine transcripts and production including IL-6, IL-1ß, and TNF-α. Simultaneously, oleanolic acid shifted the microglial polarization from the M1 phenotype to the M2 phenotype upon LPS conditions by suppressing LPS-induced M1 marker CD16, CD86 transcripts, and enhancing the M2 marker Arg-1 mRNA and anti-inflammatory IL-10 levels. In addition, the LPS-induced activation of TLR4-NF-κB signaling was suppressed in the microglia after the oleanolic acid treatment. Restoring this signaling by the TLR4 plasmid transfection overturned the suppressive effects of oleanolic acid on microglial polarization-evoked inflammation. In vivo, oleanolic acid injection alleviated allodynia and hyperalgesia in SNL-induced neuropathic pain mice. Concomitantly, oleanolic acid facilitated microglial polarization to M2, accompanied by inhibition in inflammatory cytokine levels and activation of TLR4-NF-κB signaling. Collectively, these findings confirm that oleanolic acid may ameliorate neuropathic pain by promoting microglial polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype via the TLR4-NF-κB pathway, thereby indicating its usefulness as therapeutic intervention in neuropathic pain.