RESUMEN
Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias/genética , ADN de Neoplasias/genética , Humanos , Mutación/genética , Proteínas de Neoplasias/genética , Neoplasias/patología , ARN Neoplásico/genéticaRESUMEN
Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purificationmass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.
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Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Espectrometría de Masas , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Purificación por Afinidad en TándemRESUMEN
Extracellular vesicles (EVs) are valued candidates for the development of new tools for medical applications. Vesicles carrying melanoma-associated antigen A (MAGEA) proteins, a subfamily of cancer-testis antigens, are particularly promising tools in the fight against cancer. Here, we have studied the biophysical and chemical properties of MAGEA4-EVs and show that they are stable under common storage conditions such as keeping at +4 °C and -80 °C for at least 3 weeks after purification. The MAGEA4-EVs can be freeze-thawed two times without losing MAGEA4 in detectable quantities. The attachment of MAGEA4 to the surface of EVs cannot be disrupted by high salt concentrations or chelators, but the vesicles are sensitive to high pH. The MAGEA4 protein can bind to the surface of EVs in vitro, using robust passive incubation. In addition, EVs can be loaded with recombinant proteins fused to the MAGEA4 open reading frame within the cells and also in vitro. The high stability of MAGEA4-EVs ensures their potential for the development of EV-based anti-cancer applications.
Asunto(s)
Antígenos de Neoplasias/química , Vesículas Extracelulares/química , Proteínas de Neoplasias/química , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/aislamiento & purificación , Antígenos de Neoplasias/metabolismo , Almacenaje de Medicamentos , Vesículas Extracelulares/metabolismo , Congelación , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Concentración de Iones de Hidrógeno , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Octoxinol/química , Proteínas Recombinantes/química , Sales (Química)/químicaRESUMEN
Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and ß-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.
Asunto(s)
Quinasas de Receptores Acoplados a Proteína-G/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Antineoplásicos/síntesis química , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatografía/métodos , Clonación Molecular , Diseño de Fármacos , Quinasas de Receptores Acoplados a Proteína-G/química , Quinasas de Receptores Acoplados a Proteína-G/genética , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/enzimología , Mieloma Múltiple/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Sf9 , SpodopteraRESUMEN
The early diagnosis and monitoring of cancers are key factors in effective cancer treatment. Particularly, the separation of biomolecules is an essential step for both diagnostic and analytical purposes. However, the current techniques used to isolate biomolecules are intensive, laborious, and require multiple instruments as well as repeated sample preparations to separate each biomolecule. Thus, an efficient separation system that can simultaneously separate biomolecules from scarce samples is highly desirable. Hence, in this study, we developed a biosilica-based syringe filtration system for the efficient separation of biomolecules from cancer samples using amine-modified diatomaceous earth (AD) with dimethyl 3,3'-dithiobispropionimidate (DTBP). The syringe filter can be an efficient and rapid tool for use in various procedures without complex instruments. The DTBP-based AD system was combined with the syringe filter system for nucleic acid and protein separation from various cancer cells. We demonstrated the efficacy of the DTBP-based AD in a single-filter system for the efficient separation of DNA and proteins within 40 min. This DTBP-based AD syringe filter system showed good rapidity, efficiency, and affordability in the separation of biomolecules from single samples for the early diagnosis and clinical analysis of cancers.
Asunto(s)
Técnicas Biosensibles/métodos , ADN de Neoplasias/aislamiento & purificación , Tierra de Diatomeas/química , Imidoésteres/química , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias/metabolismo , ADN de Neoplasias/análisis , Humanos , Proteínas de Neoplasias/análisis , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
Prostate cancer most frequently metastasizes to bone, resulting in abnormal bone metabolism and the release of components into the blood stream. Here, we evaluated the capacity of convolutional neural networks (CNNs) to use Raman data for screening of prostate cancer bone metastases. We used label-free surface-enhanced Raman spectroscopy (SERS) to collect 1281 serum Raman spectra from 427 patients with prostate cancer, and then we constructed a CNN based on LetNet-5 to recognize prostate cancer patients with bone metastases. We then used 5-fold cross-validation method to train and test the CNN model and evaluated its actual performance. Our CNN model for bone metastases detection revealed a mean training accuracy of 99.51% ± 0.23%, mean testing accuracy of 81.70% ± 2.83%, mean testing sensitivity of 80.63% ± 5.07%, and mean testing specificity of 82.82% ± 2.94%.
Asunto(s)
Neoplasias Óseas/sangre , Detección Precoz del Cáncer , Proteínas de Neoplasias/sangre , Neoplasias de la Próstata/sangre , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Humanos , Masculino , Nanopartículas/química , Proteínas de Neoplasias/aislamiento & purificación , Redes Neurales de la Computación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Espectrometría RamanRESUMEN
Introduction: Despite being rare cancers, gliomas account for a significant number of cancer-related deaths. Identification and treatment of these tumors at an early stage would greatly improve the therapeutic outcomes. There is an urgent need for diagnostic and prognostic markers, which can identify disease early and discriminate the subtypes of these tumors thereby improving the existing treatment modalities.Areas covered: In this article, we have reviewed published literature on proteomics biomarkers for gliomas and their importance in diagnosis or prognosis. Proteomic studies for the discovery of protein, autoantibody biomarkers, and biological pathway alterations in serum, CSF and tumor biopsies have been discussed in this review.Expert opinion: The rapid development in the field of mass spectrometry and increased sensitivity and reproducibility in assays has led to the identification and quantification of large number of proteins very precisely. Though genomic markers are the prime focus in the classification of gliomas, incorporating protein markers would further improve the existing classification. In this regard, data mining and studies on large cohorts of glioma patients would help in the identification of diagnostic and prognostic markers ultimately translating to the clinics.
Asunto(s)
Biomarcadores de Tumor/genética , Glioma/genética , Proteínas de Neoplasias/genética , Proteómica , Glioma/patología , Humanos , Espectrometría de Masas , Proteínas de Neoplasias/aislamiento & purificación , PronósticoRESUMEN
The protein ALL1 fused from chromosome 1q (AF1q) is overexpressed in a variety of cancers and acts to activate several signaling pathways that lead to oncogenesis. For example, AF1q has been shown to interact with T-cell Factor 7 (TCF7; also known as TCF1) from the Wnt/ß-catenin pathway resulting in the transcriptional activation of the CD44 and the enhancement of breast cancer metastasis. Despite the importance of AF1q in facilitating oncogenesis and metastasis, the structural and biophysical properties of AF1q remain largely unexplored due to the absence of a viable method for producing recombinant protein. Here, we report the overexpression of AF1q in E. coli as a fusion to a N-terminal His6-tag, which forms inclusion bodies (IBs) during expression. The AF1q protein was purified from IBs under denaturing conditions by immobilized metal affinity chromatography followed by a successful one-step dialysis refolding. Refolded AF1q was further purified to homogeneity by gel filtration chromatography resulting in an overall yield of 35â¯mg/L culture. Our nuclear magnetic resonance (NMR) and analytical ultracentrifugation (AUC) measurements reveal AF1q interacts with TCF7, specifically with TCF7's high-mobility group (HMG) domain (residues 154-237), which is, to our knowledge, the first biophysical characterization of the AF1q and TCF7 interaction.
Asunto(s)
Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Clonación Molecular , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , UltracentrifugaciónRESUMEN
The genomic landscape of breast cancer (BC) is complex. The purpose of this study was to decipher the mutational profiles of Taiwanese patients with BC using next-generation sequencing. We performed whole-exome sequencing on DNA from 24 tumor tissue specimens from BC patients. Sanger sequencing was used to validate the identified variants. Sanger sequencing was also performed on paired adjacent nontumor tissues. After genotype calling and algorithmic annotations, we identified 49 deleterious variants in canonical cancer-related genes in our BC cohort. The most frequently mutated genes were PIK3CA (16.67%), FKBP9 (12.5%), TP53 (12.5%), ATM (8.33%), CHEK2 (8.33%), FOXO3 (8.33%), NTRK1 (8.33%), and NUTM2B (8.33%). Seven mutated variants (ATR p.V1581fs, CSF1R p.R579Q, GATA3 p.T356delinsTMKS, LRP5 p.W389*, MAP3K1 p.T918fs, MET p.K1161fs, and MTR p.P1178S) were novel variants that are not present in any gene mutation database. After grouping the samples according to molecular subtype, we found that the cell cycle, MAPK, and chemokine signaling pathways in the luminal A subtype of BC; the focal adhesion, axon guidance, and endocytosis pathways in the luminal B subtype; and amyotrophic lateral sclerosis in the basal-like subtype were exclusively altered. Survival curve analysis showed that the presence of the MAPK signaling pathway and endocytosis mutations were correlated with a poor prognosis. These survival data were consistent with cBioPortal analyses of 2,051 BC cases. We discovered novel mutations in patients with BC. These results have implications for developing strategic, adjuvant, and gene-targeted therapies.
Asunto(s)
Neoplasias de la Mama/genética , Mutación/genética , Proteínas de Neoplasias/genética , Pronóstico , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Femenino , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Proteínas de Neoplasias/aislamiento & purificación , Transducción de Señal/genética , Secuenciación del ExomaRESUMEN
Acute respiratory distress syndrome is a severe form of respiratory failure, occurring in up to 20% of patients admitted to the intensive care unit with sepsis. Dysregulated leukocyte diapedesis is a major contributor to acute respiratory distress syndrome. Endocan is a circulating proteoglycan that binds to the leukocyte integrin leukocyte functional antigen-1 and blocks its interaction with its endothelial ligand, ICAM-1. The objective of this study was to evaluate the role of endocan in the control of acute lung inflammation. In vitro, endocan inhibited human leukocyte transendothelial migration as well as ICAM-1-dependent migration but had a very mild effect on ICAM-1-dependent adhesion. Endocan also acted as an inhibitor of transendothelial migration of mouse leukocytes. The effect of systemic administration of recombinant human endocan was assessed in a model of acute lung inflammation in BALB/c mice. Treatment with endocan 1 h after intratracheal LPS challenge reduced the alveolar inflammatory response, diminished histological features of acute lung injury, and improved respiratory function. These results highlight the anti-inflammatory role of human endocan and its protective effect against acute lung injury.NEW & NOTEWORTHY We show here that endocan inhibits ICAM-1-dependent human leukocyte transendothelial migration and ICAM-1-dependent adhesion. We also found that in BALB/c mice with tracheal LPS-induced acute lung injury treatment with recombinant human endocan reduces lung inflammation, notably through reduction of neutrophilic recruitment, and restores normal lung function. These results confirm the hypothesis that human endocan may have a protective effect against acute lung inflammation.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Leucocitos/efectos de los fármacos , Proteínas de Neoplasias/uso terapéutico , Proteoglicanos/uso terapéutico , Migración Transendotelial y Transepitelial/efectos de los fármacos , Animales , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Lipopolisacáridos , Masculino , Ratones Endogámicos BALB C , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/farmacología , Proteoglicanos/aislamiento & purificación , Proteoglicanos/farmacología , Frecuencia Respiratoria/efectos de los fármacosRESUMEN
The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage. We demonstrate the incorporation of Evosep One technology into our multiplexing workflow for analysis of tandem mass tag (TMT)-labeled nonsmall cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). By the use of a 30 samples per day Evosep workflow, >12â¯000 proteins were identified in 48 h of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 60 h. Shorter Evosep gradient lengths reduced the number of protein identifications by 10% while decreasing the mass analysis time by 50%. This Evosep workflow will enable quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/química , Cromatografía Liquida/métodos , Neoplasias Pulmonares/química , Proteínas de Neoplasias/aislamiento & purificación , Péptidos/aislamiento & purificación , Proteoma/aislamiento & purificación , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Cromatografía Liquida/instrumentación , Expresión Génica , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones SCID , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Péptidos/química , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Coloración y Etiquetado/métodos , Espectrometría de Masas en Tándem , Factores de Tiempo , Flujo de TrabajoRESUMEN
The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas de Neoplasias/metabolismo , Multimerización de Proteína/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias de Aminoácidos/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Línea Celular Tumoral , Cristalografía por Rayos X , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Fluorescente , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Conformación Proteica en Hélice alfa , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Imagen de Lapso de TiempoRESUMEN
Programmed cell death 5 (PDCD5) is a vital signaling protein in the apoptosis pathway in eukaryotes. It is known that there are two dissociated N-terminal regions and a triple-helix core in eukaryotic PDCD5. Structural and functional studies of PDCD5 from hyperthermophilic archaea have been limited to date. Here, the PDCD5 homolog Sso0352 (SsoPDCD5) was identified in Sulfolobus solfataricus, the SsoPDCD5 protein was expressed and crystallized, and the phase was identified by single-wavelength anomalous diffraction. The native SsoPDCD5 crystal belonged to space group C2 and diffracted to 1.49â Å resolution. This is the first crystal structure of a PDCD5 homolog to be solved. SsoPDCD5 shares a similar triple-helix bundle with eukaryotic PDCD5 but has a long α-helix in the N-terminus. A structural search and biochemical data suggest that SsoPDCD5 may function as a DNA-binding protein.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas de Neoplasias/química , Sulfolobus solfataricus/enzimología , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis/aislamiento & purificación , Proteínas Reguladoras de la Apoptosis/metabolismo , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Unión Proteica , Conformación Proteica , Homología de SecuenciaRESUMEN
PURPOSE: We evaluated whether multiplex protein quantification using antibody bar-coding with photocleavable oligonucleotides (NanoString) can be applied to evaluate protein expression in breast cancer FFPE specimens. We also assessed whether diagnostic core-cuts fixed immediately at time of procedures and surgical excision sections from routinely fixed breast cancers are affected by the same fixation related differences noted using immunohistochemistry (IHC). METHODS: The expression of 26 proteins was analysed using NanoString technology in 16 pairs of FFPE breast cancer core-cuts and surgical excisions. The measurements yielded were compared with those by IHC on Ki67, PgR and HER2 biomarkers and pAKT and pERK1/2 phosphorylated proteins. RESULTS: When considered irrespective of sample type, expression measured by the two methods was strongly correlated for all markers (p < 0.001; ρ = 0.69-0.88). When core-cuts and excisions were evaluated separately, the correlations between NanoString and IHC were weaker but significant except for pAKT in excisions. Surgical excisions showed lower levels of 8/12 phosphoproteins and higher levels of 4/13 non-phosphorylated proteins in comparison to core-cuts (p < 0.01). Reduced p4EBP1, pAMPKa, pRPS6 and pRAF1 immunogenicity in excisions was correlated with tumour size and mastectomy specimens showed lower p4EBP1 and pRPS6 expression than lumpectomy (p < 0.05). CONCLUSIONS: Our study supports the validity of the new multiplex approach to protein analysis but indicates that, as with IHC, caution is necessary for the analysis in excisions particularly of phosphoproteins. The specimen type, tumour size and surgery type may lead to biases in the quantitative analysis of many proteins of biologic and clinical interest in excision specimens.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Proteínas de Neoplasias/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Biopsia con Aguja Gruesa , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Femenino , Formaldehído , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Antígeno Ki-67/genética , Mastectomía , Persona de Mediana Edad , Proteínas de Neoplasias/aislamiento & purificación , Adhesión en Parafina , Proteínas Quinasas/genética , Proteínas Proto-Oncogénicas c-raf/genética , Receptor ErbB-2/genética , Receptores de Progesterona/genéticaRESUMEN
The main objective of this study was to develop an effective in-gel trypsin digestion protocol using aqueous-acetonitrile solvent system to facilitate MS analysis and maximize the number of identified proteins from biological samples. The procedure, where 80% acetonitrile was present in the trypsin reaction mixture, increased the number of matched peptides, and allowed the identification of more proteins with higher coverage than the common digestion protocol. Vimentin, annexins, tubulin, actin, peptidyl-prolyl cis-trans isomerase or alpha-enolase are examples of important proteins that change during the progress cancer. These were isolated from human breast cancer cells and were used for this study.
Asunto(s)
Acetonitrilos/química , Proteínas de Neoplasias/análisis , Proteómica/métodos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Humanos , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
The efficient extraction of proteins of interest from cells and tissues can be challenging. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 and the transcriptional repressor Snail from choriocarcinoma cells using NP-40 and RIPA lysis buffer. We also show the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness with the often utilized RIPA lysis buffer for solubilization of heat shock proteins (HSP) B1 and B5 and the cytoplasmic adapter protein integrin-linked kinase (ILK) from smooth muscle. Overall, the results demonstrate the importance of optimizing lysis buffers for specific protein solubilization prior to finalizing the experimental workflow.
Asunto(s)
Electroforesis/métodos , Proteínas de la Membrana/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Polietilenglicoles/química , Factores de Transcripción de la Familia Snail/aislamiento & purificación , Western Blotting/métodos , Tampones (Química) , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Octoxinol , SolubilidadRESUMEN
Efficient extraction of proteins is a great challenge for numerous downstream proteomic analyses. During the protein extraction procedure, it is critical to maintain the conformational stability, integrity, as well as higher yield of the protein. To do so, 5-different lysis buffers of Tris and HEPES have been used as the primary buffering reagents with variable compositions at different concentrations and pH using human cancer cells. In this study, different protein lysates of human breast cancer cells T47D and MDA-MB-231 and ovarian cancer cell PA-1 were subjected to run SDS-PAGE for separation of proteins based on their molecular size, followed by Coomassie blue, silver staining, and immunoblot assays to compare the extraction yield of total cytoplasmic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the integral membrane protein, integrin ß-1. Our results revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, is relatively the efficient and reliable protein extraction method for a wide range of MW subcellular markers, cytoplasmic GAPDH and transmembrane integrin ß-1 proteins. We anticipate that this simple and cost-effective protein extraction protocol might be extremely useful across a broad range of subcellular proteins in different biologic samples.
Asunto(s)
Neoplasias de la Mama/patología , Citosol/química , Biología Molecular/métodos , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias Ováricas/patología , Tampones (Química) , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/aislamiento & purificación , Humanos , Immunoblotting , Integrina beta1/inmunología , Integrina beta1/aislamiento & purificación , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/aislamiento & purificación , Proteínas de Neoplasias/inmunologíaRESUMEN
The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding. While the binding specificities of individual bromodomains have been studied by our lab and others, the results have failed to reach a consensus. The acetyl-histone binding features of the full-length protein is unknown and is the motivation for this work. The hypothetical HMG and BAH domains have not been studied and the actual function of these regions is currently unknown. Thus, the precise interactions of this large and complex protein are not well-studied. Advances in understanding this large protein have been hindered by the inability to express and purify recombinant full-length BAF180 protein. Currently, only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. Here, we report the successful expression, purification of full-length biologically active BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full-length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.
Asunto(s)
Histonas/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Pichia/genética , Factores de Transcripción/genética , Sitios de Unión , Clonación Molecular , Proteínas de Unión al ADN , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histonas/química , Histonas/genética , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Pichia/metabolismo , Regiones Promotoras Genéticas , Análisis por Matrices de Proteínas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND & OBJECTIVES: Ocular and adnexal tumours are important causes of morbidity in India and globally. Immunohistochemistry (IHC) is a vital molecular pathology tool, which helps to diagnose a tumour with more accuracy. The present study was undertaken to document the profile of ocular and adnexal tumour with IHC at a tertiary eye care center in Northeast India. METHODS: This was a prospective and laboratory-based study. Histopathological and IHC study of the ocular and adnexal tumour was carried out from 2012 to 2014. Selection of pathological cases was made on the result of the histological diagnosis. All samples were subjected to IHC using kits for different antibodies as per indications. RESULTS: In total, 645 tumours were included in our study, with 449 benign conditions and 196 were malignant tumours. Total IHCs were done in 87 tumours and 238 of antibodies were used. Non-Hodgkin's lymphomas (B-cell, low-to-intermediate type and mucosal-associated lymphoid tumours) were the most common tumor. INTERPRETATION & CONCLUSIONS: Clinical utility of the IHCs in different ophthalmic tumours can enable pathologists to make an accurate diagnosis and thus help in the overall management of the patient care. IHC may be carried out using various methods and some of the methods practiced are time consuming and tedious. In this study, kit methods were used which were found to be simpler and less time-consuming.
Asunto(s)
Neoplasias del Ojo/diagnóstico , Neoplasias del Ojo/genética , Ojo/metabolismo , Proteínas de Neoplasias/aislamiento & purificación , Linfocitos B/metabolismo , Linfocitos B/patología , Ojo/patología , Neoplasias del Ojo/epidemiología , Neoplasias del Ojo/patología , Femenino , Humanos , Inmunohistoquímica , India/epidemiología , Masculino , Proteínas de Neoplasias/genética , Centros de Atención TerciariaRESUMEN
Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.