RESUMEN
Alternative lengthening of telomeres (ALTs) mechanism is activated in some somatic, germ cells, and human cancer cells. However, the key regulators and mechanisms of the ALT pathway remain elusive. Here we demonstrated that ZBTB40 is a novel telomere-associated protein and binds to telomeric dsDNA through its N-terminal BTB (BR-C, ttk and bab) or POZ (Pox virus and Zinc finger) domain in ALT cells. Notably, the knockout or knockdown of ZBTB40 resulted in the telomere dysfunction-induced foci and telomere lengthening in the ALT cells. The results also show that ZBTB40 is associated with ALT-associated promyelocytic leukemia nuclear bodies, and the loss of ZBTB40 induces the accumulation of the ALT-associated promyelocytic leukemia nuclear bodies in U2OS cells. Taken together, our results implicate that ZBTB40 is a key player of telomere protection and telomere lengthening regulation in human ALT cells.
Asunto(s)
Proteínas de Unión al ADN , Telómero , Humanos , Línea Celular Tumoral , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero/genética , Unión Proteica , ADN/metabolismo , Cuerpos Nucleares/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Apoptosis/genéticaRESUMEN
OBJECTIVE: To explore the feasibility of non-invasive prenatal testing (NIPT) for detecting fetal chromosomal copy number variants (CNV). METHODS: A retrospective analysis was carried out on NIPT positive samples in Suzhou Municipal Hospital from January 1, 2019 to December 31, 2021. The effect of NIPT on fetal CNV detection was assessed by comparison with the results of karyotype analysis and/or chromosomal microarray analysis (CMA). RESULTS: Among the 525 NIPT positive samples, 146 were CNV cases, of which 84 were further verified by karyotyping and/or CMA, 29 (34.5%) were true positive. Among them, 12 cases were pathogenic variants, 2 cases were likely pathogenic variants and 15 cases were variants of uncertain significance. CONCLUSION: NIPT could detect CNV with high accuracy, and to combine CNV detection and chromosomal aneuploidy detection has great significance to improve the prenatal and postnatal care.
Asunto(s)
Variaciones en el Número de Copia de ADN , Cariotipificación , Pruebas Prenatales no Invasivas , Diagnóstico Prenatal , Humanos , Femenino , Embarazo , Estudios Retrospectivos , Pruebas Prenatales no Invasivas/métodos , Diagnóstico Prenatal/métodos , Adulto , Aneuploidia , Feto , Estudios de FactibilidadRESUMEN
A total of 15,267 pregnancies were tested by NIPT in this study. Grey zone (z score: 2.58 â¼ 4 and -4â¼-2.58) was set for screening out aneuploidy 21, 18 and 13. Cases with z score located in the grey zone were retested starting from DNA extraction. The chi-squared test and/or the Fisher's exact test were used to compare variables. One hundred and eight screening-positive samples in the first run of NIPT were common trisomies 21 (N = 83), trisomies18 (N = 13) or trisomies 13 (N = 12), with PPVs of 87.18%, 76.92%, and 30% respectively. For the cases in the grey zone, most of them (67.15%, 184/274) were reported with Z score of Chromosome 21 in the grey zone and 176 were reclassified as negative by the second run of NIPT; while 3 cases reclassified as trisomy 21 and 1 case reclassified as trisomy 13 were finally confirmed by karyotyping analysis, with PPV 25% and 20% respectively. The grey zone and the second run of NIPT in this study showed that the grey zone and second run NIPT approach was able to accurately help categorise cases as negative and positive. Invasive diagnosis is recommended to prevent false negative aneuploidies for samples located in the special z score scope of 2.58-3 for two runs of NIPT. IMPACT STATEMENTWhat is already known on this subject? Grey zone was widely used in NIPT. The performance of grey zone of clinical samples on Illumina HiSeq 2000 instrument has been reported, and the performance of grey zone on some mainstream sequencers with simulated samples has also been summarised. Reported treatments for samples located in the grey zone in NIPT usually included being classified into 'unclassified' or 'no call' followed by following up and/or karyotyping analysis.What do the results of this study add? This study investigated the performance of the grey zone on Ion Proton DA8600 with clinical samples; and it present an alternative treatment for samples in grey zone that reclassified them as negative or positive by the second run of sequencing.What are the implications of these findings for clinical practice and/or further research? Our own data for the performance of the grey zone in the cfDNA assay on the semiconductor sequencing platform might provide raw materials for other researchers' meta-analysis, cohort study, or other studies. Details of Z score distributions of chromosomes in grey zone, results of the second run of NIPT for samples in the grey zone, and false negative samples in the grey zone would help lab technicians better analyse the NIPT results and help doctors to improve genetic counselling.
Asunto(s)
Ácidos Nucleicos Libres de Células , Síndrome de Down , Aneuploidia , Estudios de Cohortes , ADN , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Femenino , Humanos , Embarazo , Diagnóstico Prenatal , Protones , Semiconductores , Trisomía/diagnóstico , Trisomía/genética , Síndrome de la Trisomía 13/diagnóstico , Síndrome de la Trisomía 13/genéticaRESUMEN
BACKGROUND: Whole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS. RESULTS: Here we provide a computational method named "BCREval" to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571-1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter. CONCLUSION: Our method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.
Asunto(s)
Biología Computacional/métodos , Sulfitos/química , Algoritmos , Citosina/química , ADN/química , ADN/genética , Metilación de ADN , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Sequencing cell-free DNA in maternal plasma is an effective noninvasive prenatal testing technique that has been used in fetal aneuploidy screening worldwide. However, its clinical application is limited by the low fetal fraction (<4%) of cell-free DNA in many singleton pregnancies, which usually results in screen failures or no calls. In addition, dizygotic twin contributions of cell-free DNA into the maternal circulation can vary by 2-fold, complicating the quantitative diagnosis of fetal aneuploidy. OBJECTIVE: We performed semiconductor sequencing of shorter fragments (107-145 bp) of circulating cell-free DNA to improve the fetal DNA fraction at lower uniquely mapped reads (1-8.5 MB) to reduce the probability of no calls. STUDY DESIGN: We identified 2903 plasma samples from pregnant women, including 86 dizygotic twin pregnancy, that were collected at a single prenatal diagnostic center between October 2015 and July 2018. Size-selection noninvasive prenatal testing for fetal aneuploidy was applied to 2817 plasma samples (1409 male and 1408 female fetuses) and 86 dizygotic twins using noninvasive prenatal testing with and without size selection. Shorter fragment size was the key factor affecting fetal fraction in multivariable linear regression models as well as to validate the accuracy of the size selection for noninvasive prenatal testing. RESULTS: Analysis of 1409 male fetuses by multivariable linear regression showed that maternal age, body mass index, number of pregnancies, average cell-free DNA size, maternal plasma cell-free DNA concentration, library concentration, and multiple gestation were negatively correlated with fetal fraction. Conversely, gestational age and uniquely mapped reads were positively correlated with fetal fraction. Compared with ≤120 bp cell-free DNA fragments, mean fetal fraction differences were -3.57% (95% confidence interval, -5.95% to -1.19%), for 121-130 bp, -9.52% (95% confidence interval, -11.89% to -7.14%) for 131-140 bp, and -14.47% (95% confidence interval, -18.37% to -10.58%) for ≥141 bp (Ptrend < .0001). These results were statistically significant after multivariable adjustments in models for fetal fraction. Meanwhile, results from 86 dizygotic twins showed that the size selection increased the fetal fraction by â¼3.2-fold, with 98.8% of samples reaching a fetal fraction >10%. Improved detection accuracy was also achieved. CONCLUSION: Sequencing shorter cell-free DNA fragments is a reasonable strategy to reduce the probability of no calls results because of low fetal fraction and should be recommended to pregnant subjects.
Asunto(s)
Aneuploidia , Ácidos Nucleicos Libres de Células/sangre , Pruebas Prenatales no Invasivas/métodos , Análisis de Secuencia de ADN/métodos , Adolescente , Adulto , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Análisis Multivariante , Embarazo , Gemelos Dicigóticos , Adulto JovenRESUMEN
Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.
Asunto(s)
Proteómica/métodos , Venenos de Araña/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cisteína/química , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada , Eliminación de Gen , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Neurotoxinas/química , Técnicas de Placa-Clamp , Péptidos/química , Filogenia , Procesamiento Proteico-Postraduccional , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Arañas , Transcripción GenéticaRESUMEN
P-21 activated kinases, or PAKs, are serine-threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.
Asunto(s)
Corazón/fisiología , Regeneración , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Activación Enzimática , Lesiones Cardíacas/metabolismo , Lesiones Cardíacas/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 µg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409.
Asunto(s)
Aminas/química , Péptidos/análisis , Péptidos/química , Escherichia coli/química , Marcaje IsotópicoRESUMEN
Glyoxalase 1 (Glo1), belonging to the glyoxalase system, participates in the detoxification of methylglyoxal (MG), a byproduct of glycolysis. Glo1 is associated with the progression of many human malignancies. However, the role of Glo1 in hepatocellular carcinoma (HCC) is unclear. We have discovered that the expression of Glo1 is up-regulated in HCC tissues compared with adjacent non-tumorous tissues, and knockdown of Glo1 expression by RNA interference significantly inhibited the proliferation of human HCC cell lines. Glo1 knockdown resulted in the accumulation of its cytotoxic substrate, MG. Overall, thus Glo1 might be essential for HCC progression and can be designated as a potential therapeutic target for HCC in the future.
Asunto(s)
Carcinoma Hepatocelular/patología , Proliferación Celular , Lactoilglutatión Liasa/metabolismo , Neoplasias Hepáticas/patología , Línea Celular Tumoral , Humanos , Regulación hacia ArribaRESUMEN
The hydrophobic nature of many membrane proteins, especially integral membrane proteins, brings great difficulties to their analysis. To improve the analysis of membrane proteins, an entirely solution-based combinative sample preparation (CSP) method was developed and its application to the shotgun analysis of rat liver membrane proteomes was evaluated in this study. This CSP method comprehensively uses the strong ability of sodium dodecyl sulfate (SDS) to lyse the membranes and solubilize hydrophobic membrane proteins, the high efficiency of the optimized acetone precipitation method in sample cleanup and protein recovery, and the advantages of sodium deoxycholate (SDC) in improving protein solubilization/digestion as well as being compatible with trypsin activity. Compared with two other representative sample preparation methods, the SDC-assisted membrane-lysing method and the tube gel method, the newly established CSP method exhibited superiority in the recovery and identification of hydrophobic peptides, larger peptides, and highly hydrophobic membrane proteins with multiple transmembrane domains. The CSP method has characteristics of easy operation, low cost, and suitability for treating protein samples in various volumes, particularly large volumes, thereby having potential in the analysis of membrane proteomes with mass spectrometry.
Asunto(s)
Proteínas de la Membrana/química , Proteómica/métodos , Acetona/química , Animales , Electroforesis en Gel de Poliacrilamida , Hígado/química , Ratas , Soluciones/químicaRESUMEN
SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.
Asunto(s)
Electroforesis en Gel de Gradiente Desnaturalizante/métodos , Proteínas de la Membrana/análisis , Mapeo Peptídico/métodos , Proteoma/análisis , Dodecil Sulfato de Sodio/aislamiento & purificación , Animales , Hígado/química , Hígado/citología , Proteínas de la Membrana/química , Proteínas de la Membrana/clasificación , Peso Molecular , Proteómica/métodos , Ratas , Dodecil Sulfato de Sodio/química , Tripsina/químicaRESUMEN
Alternative lengthening of telomeres (ALT) is a homologous recombination-based telomere maintenance mechanism activated in 10-15% of human cancers. Although significant progress has been made, the key regulators of the ALT pathway and its role in cancer development remain elusive. Bioinformatics methods were used to predict novel telomere-associated proteins (TAPs) by analysis of large-scale ChIP-Seq data. Immunostaining and fluorescence in situ hybridization experiments were applied to detect the subcellular location of target genes and telomeres. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to examine the expression of targeting genes. Overall survival (OS) analyses were used to evaluate the relationship between gene expression and survival time; immunohistochemistry was used to detect the distribution of target genes in liver cancer tissues. We found that nuclear factor related to kappaB binding protein (NFRKB), a metazoan-specific subunit of the INO80 complex, can associate with telomeres in human ALT cells. Loss of NFRKB induces dysfunction of telomeres and less PML bodies in U2OS cells. In addition, NFRKB is low/moderately expressed in cytoplasm of normal hepatocytes but heavily accumulating in the nucleus of liver cancer cells. Finally, the high expression of NFRKB is associated with short OS time and poor prognosis. NFRKB is a TAP and protects telomeres from DNA damage in ALT cells. It is highly expressed in hepatocellular carcinoma (HCC) cells and predicts a poor prognosis. NFRKB may be a promising prognostic biomarker for the treatment of HCC in the future.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/metabolismo , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Células K562 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Células MCF-7 , Unión Proteica , Telómero/metabolismoRESUMEN
Background: Whole-exome sequencing (WES) has been recommended as a first-tier clinical diagnostic test for individuals with neurodevelopmental disorders (NDDs). We aimed to identify the genetic causes of 17 children with developmental delay (DD) and/or intellectual disability (ID). Methods: WES and exome-based copy number variation (CNV) analysis were performed for 17 patients with unexplained DD/ID. Results: Single-nucleotide variant (SNV)/small insertion or deletion (Indel) analysis and exome-based CNV calling yielded an overall diagnostic rate of 58.8% (10/17), of which diagnostic SNVs/Indels accounted for 41.2% (7/17) and diagnostic CNVs accounted for 17.6% (3/17). Conclusion: Our findings expand the known mutation spectrum of genes related to DD/ID and indicate that exome-based CNV analysis could improve the diagnostic yield of patients with DD/ID.
RESUMEN
tRNAs and tRNA-derived RNA fragments (tRFs) play various roles in many cellular processes outside of protein synthesis. However, comprehensive investigations of tRNA/tRF regulation are rare. In this study, we used new algorithms to extensively analyze the publicly available data from 1332 ChIP-Seq and 42 small-RNA-Seq experiments in human cell lines and tissues to investigate the transcriptional and posttranscriptional regulatory mechanisms of tRNAs. We found that histone acetylation, cAMP, and pluripotency pathways play important roles in the regulation of the tRNA gene transcription in a cell-specific manner. Analysis of RNA-Seq data identified 950 high-confidence tRFs, and the results suggested that tRNA pools are dramatically distinct across the samples in terms of expression profiles and tRF composition. The mismatch analysis identified new potential modification sites and specific modification patterns in tRNA families. The results also show that RNA library preparation technologies have a considerable impact on tRNA profiling and need to be optimized in the future.
Asunto(s)
ARN de Transferencia/genética , Transcripción Genética/genética , Transcriptoma/genética , Algoritmos , AMP Cíclico/genética , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Genómica , Humanos , Biosíntesis de Proteínas/genética , RNA-SeqRESUMEN
Due to its compatibility with protease activity at high concentration, sodium deoxycholate (SDC) can be used to effectively improve the solubilization and enzymolysis of membrane proteins and has received increasing attention in the field of membrane proteome analysis in recent years. SDC can be removed from digests by means of acidification followed by centrifugation (i.e. acid precipitation, AP) or extraction with ethyl acetate (i.e. phase transfer, PT) so as not to interfere with the downstream analyses like LC-MS/MS. In this study, the two strategies were systematically evaluated, compared and optimized. The results of the study demonstrated that both of the AP and PT strategies led to a certain amount of tryptic peptides being lost, and in PT strategy even more peptides were lost during SDC removal process. However, the lost peptides could be mostly recovered by washing the pellet and solid content produced during AP and PT, respectively. By recovering the lost peptides, the identification efficiency of proteins, especially transmembrane and low abundance ones, was significantly improved. Comparatively, after optimization by recovering the lost peptides, AP strategy was superior to PT strategy because the former not only could achieve the comparable identification efficiency with the latter but also was more economical, safer and easier to operate than the latter.
Asunto(s)
Proteínas de la Membrana/química , Proteoma/química , Animales , Cromatografía Liquida/métodos , Biología Computacional , Ácido Desoxicólico , Hígado/química , Espectrometría de Masas , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Peso Molecular , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Conformación Proteica , Proteoma/metabolismo , Ratas , Solubilidad , TensoactivosRESUMEN
A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Proteoma/análisis , Absorción , Animales , Electroforesis en Gel de Poliacrilamida , Ratas , Ratas Sprague-Dawley , Dodecil Sulfato de Sodio/química , Tripsina/metabolismoRESUMEN
Venomous animals possess an arsenal of toxins for predation and defense. These toxins have great diversity in function and structure as well as evolution and therefore are of value in both basic and applied research. Recently, toxinomics researches using cDNA library sequencing and proteomics profiling have revealed a large number of new toxins. Although several previous groups have attempted to manage these data, most of them are restricted to certain taxonomic groups and/or lack effective systems for data query and access. In addition, the description of the function and the classification of toxins is rather inconsistent resulting in a barrier against exchanging and comparing the data. Here, we report the ATDB database and website which contains more than 3235 animal toxins from UniProtKB/Swiss-Prot and TrEMBL and related toxin databases as well as published literature. A new ontology (Toxin Ontology) was constructed to standardize the toxin annotations, which includes 745 distinct terms within four term spaces. Furthermore, more than 8423 TO terms have been manually assigned to 2132 toxins by trained biologists. Queries to the database can be conducted via a user-friendly web interface at http://protchem.hunnu.edu.cn/toxin.
Asunto(s)
Bases de Datos de Proteínas , Toxinas Biológicas/química , Animales , Internet , Péptidos/química , Señales de Clasificación de Proteína , Proteínas/química , Proteínas/genética , Toxinas Biológicas/genética , Interfaz Usuario-Computador , Vocabulario ControladoRESUMEN
In-gel digestion of gel-separated proteins is a major route to assist in proteomics-based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in-digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel-separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (> or =99.8%). Before tryptic digestion, NH(4)HCO(3) buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane-bound substances, the proteins were virtually in-solution digested in DMF-containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane-enriched sample showed that the method was superior to the reported on-membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas/aislamiento & purificación , Animales , Dimetilformamida/farmacología , Electroforesis en Gel de Poliacrilamida/métodos , Hipocampo/química , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
PM (plasma membrane) proteins play critical roles in many biological processes and are often used as molecular targets for drug discovery. In PM proteome research, fast and highly selective methods for PM preparation are highly desirable for efficient PM protein identification. In the present study, an improved PM isolation technique involving coating intact cells with synthesized cationic silica-coated magnetic nanospheres was developed and applied to the proteomic analysis of the PM from human erythroleukaemia K562 cells. Western blotting characterization and protein identification of the prepared PM indicated that the PM enrichment method using the prepared magnetic nanospheres is a fast and inexpensive strategy with high specificity. Our results demonstrate the potential of these cationic silica-coated magnetic nanospheres for high-throughput identification of PM proteins from cells.