RESUMEN
Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a two-stage methanol-assisted solubilization to digest lysates prepared from 8-µm-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts. Coupling this approach with a nanoflow reversed-phase liquid chromatography (LC) and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance MS resulted in the identification of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. The disease driving H-Ras protein along with vimentin were unambiguously identified by LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry and western blotting. The pathway analysis involving proteins identified by MS showed strong association of proteomic data with cardiovascular disease. More importantly, the MS identification and subsequent cross-validation of Wnt3a and ß-catenin, in conjunction with IHC identification of phosphorylated GSK-3ß and nuclear localization of ß-catenin, provided evidence of Wnt/ß-catenin canonical pathway activation secondary to Ras activation in the course of pathogenic myocardial hypertrophic transformation. Our method yields results indicating that the described proteomic approach permits molecular discovery and assessment of differentially expressed proteins regulating H-Ras induced hypertrophic cardiomyopathy. Selected proteins and pathways can be further investigated using immunohistochemical techniques applied to serial tissue sections of similar or different origin.
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Cardiomiopatía Hipertrófica/metabolismo , Miocardio/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Secuencia de Aminoácidos , Animales , Cardiomiopatía Hipertrófica/genética , Cromatografía Liquida , Análisis por Conglomerados , Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación Missense , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteoma/genética , Proteómica , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Vimentina/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMEN
Differential (18)O/(16)O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H(2)(18)O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs have made trypsin-catalyzed (18)O postdigestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed (18)O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the (18)O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual (18)O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then (18)O/(16)O ratios derived via evolutionary programming. The algorithm is tested using trypsin-catalyzed (18)O postdigestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both accuracy and precision are improved utilizing this rigorous mathematical approach. We further demonstrate the effectiveness of this method to accurately calculate (18)O/(16)O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its nonmyristylated mutant.
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Algoritmos , Marcaje Isotópico/métodos , Péptidos/química , Secuencia de Aminoácidos , Animales , Bovinos , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Datos de Secuencia Molecular , Isótopos de Oxígeno/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Tripsina/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
A method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer is described. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with nonmetastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and preoperative plasma were analyzed using 2D-liquid chromatography-mass spectrometry (LC-MS). The lists of identified proteins were filtered to discover proteins that (i) were found in the tumor but not normal tissue, (ii) were identified in matching plasma, and (iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase in the blood of the patient in the study as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/sangre , Neoplasias Renales/sangre , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/diagnóstico , Cromatografía Liquida , Humanos , Neoplasias Renales/diagnóstico , Espectrometría de MasasRESUMEN
Although there are a number of causes of traumatic brain injury (TBI), the armed conflict in Iraq and Afghanistan has brought this disorder to the attention of the global community. A biomarker that would enable army medics to rapidly diagnose the severity of TBI on the battle-field would be a huge asset. Unfortunately, the study of TBI has not historically attracted the proteomic research community's interest as other disorders have, such as cancer. On the positive side, however, many of the analytical and technological challenges that were overcome in the development of biofluid proteomic methods are now being applied to the study of TBI. In this review, we discuss and highlight select examples of discovery-driven proteomic studies focused on finding effective biomarkers for TBI.
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Lesiones Encefálicas/diagnóstico , Proteómica/métodos , Biomarcadores , Humanos , Proteínas/análisis , Índices de Gravedad del TraumaRESUMEN
The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements. Collectively, the stroma forms an essential microenvironment, which is indispensable for the survival and growth of the malignant neoplasm. This cellular heterogeneity makes molecular profiling of solid tumors via mass spectrometry (MS)-based proteomics a daunting task. Laser capture microdissection (LCM) is commonly used to obtain distinct histological cell types (e.g., tumor parenchymal cells, stromal cells) from tumor tissue and attempt to address the tumor heterogeneity interference with downstream liquid chromatography (LC) MS analysis. To provide optimal LC-MS analysis of micro-scale and/or nano-scale tissue sections, we modified and optimized a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) protocol for the LC-MS analysis of LCM-procured fresh-frozen tissue specimens. Presented is a detailed in-gel digestion protocol adjusted specifically to maximize the proteome coverage of amount-limited LCM samples, and facilitate in-depth molecular profiling. Following LCM, targeted tissue sections are further fractionated using silver-stained 1D-SDS-PAGE to resolve and visualize tissue proteins prior to in-gel digestion and subsequent LC-MS analysis.
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Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Secciones por Congelación/métodos , Captura por Microdisección con Láser/métodos , Neoplasias/metabolismo , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Separación Celular/métodos , Humanos , Proteínas/aislamiento & purificación , Proteómica/métodos , Plata/químicaRESUMEN
BACKGROUND: Transcriptome analysis of breast cancer discovered distinct disease subtypes of clinical significance. However, it remains a challenge to define disease biology solely based on gene expression because tumor biology is often the result of protein function. Here, we measured global proteome and transcriptome expression in human breast tumors and adjacent non-cancerous tissue and performed an integrated proteotranscriptomic analysis. METHODS: We applied a quantitative liquid chromatography/mass spectrometry-based proteome analysis using an untargeted approach and analyzed protein extracts from 65 breast tumors and 53 adjacent non-cancerous tissues. Additional gene expression data from Affymetrix Gene Chip Human Gene ST Arrays were available for 59 tumors and 38 non-cancerous tissues in our study. We then applied an integrated analysis of the proteomic and transcriptomic data to examine relationships between them, disease characteristics, and patient survival. Findings were validated in a second dataset using proteome and transcriptome data from "The Cancer Genome Atlas" and the Clinical Proteomic Tumor Analysis Consortium. RESULTS: We found that the proteome describes differences between cancerous and non-cancerous tissues that are not revealed by the transcriptome. The proteome, but not the transcriptome, revealed an activation of infection-related signal pathways in basal-like and triple-negative tumors. We also observed that proteins rather than mRNAs are increased in tumors and show that this observation could be related to shortening of the 3' untranslated region of mRNAs in tumors. The integrated analysis of the two technologies further revealed a global increase in protein-mRNA concordance in tumors. Highly correlated protein-gene pairs were enriched in protein processing and disease metabolic pathways. The increased concordance between transcript and protein levels was additionally associated with aggressive disease, including basal-like/triple-negative tumors, and decreased patient survival. We also uncovered a strong positive association between protein-mRNA concordance and proliferation of tumors. Finally, we observed that protein expression profiles co-segregate with a Myc activation signature and separate breast tumors into two subgroups with different survival outcomes. CONCLUSIONS: Our study provides new insights into the relationship between protein and mRNA expression in breast cancer and shows that an integrated analysis of the proteome and transcriptome has the potential of uncovering novel disease characteristics.
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Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteómica , Neoplasias de la Mama/metabolismo , Cromatografía Liquida , Femenino , Humanos , Espectrometría de Masas , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.
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Métodos Analíticos de la Preparación de la Muestra , Biomarcadores Farmacológicos/análisis , Proteínas Sanguíneas/aislamiento & purificación , Terapia Molecular Dirigida , Proteómica , Manejo de Especímenes/métodos , Espectrometría de Masas en Tándem/métodos , Carcinoma de Células Renales/metabolismo , Cromatografía Liquida/métodos , Humanos , Neoplasias Renales/metabolismoRESUMEN
Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.
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Neoplasias del Colon/química , Formaldehído/química , Proteínas de Neoplasias/análisis , Proteómica , Fijación del Tejido , Cromatografía Liquida , Neoplasias del Colon/patología , Humanos , Espectrometría de Masas , Adhesión en Parafina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.
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Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Glicosilación , Células HEK293 , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
The advent of proteomics has brought with it the hope of discovering novel biomarkers that can be used to diagnose diseases, predict susceptibility, and monitor progression. Much of this effort has focused on the mass spectral identification of the thousands of proteins that populate complex biosystems such as serum and tissues. A revolutionary approach in proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, and prostate cancer. This technology is capable of analyzing hundreds of clinical samples per day and has the potential to be a novel, highly sensitive diagnostic tool for the early detection of diseases, or as a predictor of response to therapy.
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Biomarcadores de Tumor/análisis , Neoplasias/diagnóstico , Proteómica/métodos , Humanos , Espectrometría de Masas/métodos , Neoplasias/genética , Análisis por Matrices de Proteínas , Reproducibilidad de los ResultadosRESUMEN
The advent of systems biology approaches that have stemmed from the sequencing of the human genome has led to the search for new methods to diagnose diseases. While much effort has been focused on the identification of disease-specific biomarkers, recent efforts are underway toward the use of proteomic and metabonomic patterns to indicate disease. We have developed and contrasted the use of both proteomic and metabonomic patterns in urine for the detection of interstitial cystitis (IC). The methodology relies on advanced bioinformatics to scrutinize information contained within mass spectrometry (MS) and high-resolution proton nuclear magnetic resonance (1H-NMR) spectral patterns to distinguish IC-affected from non-affected individuals as well as those suffering from bacterial cystitis (BC). We have applied a novel pattern recognition tool that employs an unsupervised system (self-organizing-type cluster mapping) as a fitness test for a supervised system (a genetic algorithm). With this approach, a training set comprised of mass spectra and 1H-NMR spectra from urine derived from either unaffected individuals or patients with IC is employed so that the most fit combination of relative, normalized intensity features defined at precise m/z or chemical shift values plotted in n-space can reliably distinguish the cohorts used in training. Using this bioinformatic approach, we were able to discriminate spectral patterns associated with IC-affected, BC-affected, and unaffected patients with a success rate of approximately 84%.
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Infecciones Bacterianas/diagnóstico , Cistitis Intersticial/diagnóstico , Cistitis/diagnóstico , Proteómica , Infecciones Bacterianas/orina , Biología Computacional , Cistitis/orina , Cistitis Intersticial/orina , Femenino , Humanos , Masculino , Espectrometría de Masas , Resonancia Magnética Nuclear BiomolecularRESUMEN
The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and/or cancer biomarker research. Finally, this perspective presents the background, rationale and strategy for using tissue-directed high-resolution/accuracy MS-based shotgun proteomics to detect genuine tumor proteins in the peripheral blood of a patient diagnosed with nonmetastatic cancer, employing concurrent liquid chromatography-MS analysis of immunodepleted clinical tissue and blood specimens.
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Biomarcadores de Tumor/análisis , Espectrometría de Masas , Anticuerpos/inmunología , Biomarcadores de Tumor/sangre , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/aislamiento & purificación , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Cromatografía Líquida de Alta Presión , Perfilación de la Expresión Génica , Humanos , Neoplasias Renales/sangre , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , ProteómicaRESUMEN
The discovery of effective cancer biomarkers is essential for the development of both advanced molecular diagnostics and new therapies/medications. Finding and exploiting useful clinical biomarkers for cancer patients is fundamentally linked to improving outcomes. Towards these aims, the heterogeneous nature of tumors represents a significant problem. Thus, methods establishing an effective functional linkage between laser capture microdissection (LCM) and mass spectrometry (MS) provides for an enhanced molecular profiling of homogenous, specifically targeted cell populations from solid tumors. Utilizing frozen tissue avoids molecular degradation and bias that can be induced by other preservation techniques. Since clinical samples are often of a small quantity, tissue losses must be minimized. Therefore, all steps are carried out in the same single tube. Proteins are identified through peptide sequencing and subsequent matching against a specific proteomic database. Using such an approach enhances clinical biomarker discovery in the following ways. First, LCM allows for the complexity of a solid tumor to be reduced. Second, MS provides for the profiling of proteins, which are the ultimate bio-effectors. Third, by selecting for tumor proper or microenvironment-specific cells from clinical samples, the heterogeneity of individual solid tumors is directly addressed. Finally, since proteins are the targets of most pharmaceuticals, the enriched protein data streams can then be further analyzed for potential biomarkers, drug targets, pathway elucidation, as well as an enhanced understanding of the various pathologic processes under study. Within this context, the following method illustrates in detail a synergy between LCM and MS for an enhanced molecular profiling of solid tumors and clinical biomarker discovery.
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Secciones por Congelación , Captura por Microdisección con Láser/métodos , Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Biomarcadores de Tumor/análisis , Separación Celular , Humanos , Espectrometría de Masas , Neoplasias/química , Proteínas/químicaRESUMEN
Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.
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Biomarcadores de Tumor/análisis , Marcaje Isotópico , Melanoma/metabolismo , Isótopos de Oxígeno/química , Proteínas/análisis , Tripsina/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Membrana Celular/metabolismo , Cromatografía Liquida , Humanos , Espectrometría de Masas , Proteínas/química , Proteómica/métodosRESUMEN
The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make â¼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs. Post-digestion (18)O exchange is a simple and efficient method for differential (18)O/(16)O stable isotope labeling of two biologically distinct specimens, allowing relative quantitation of proteins in complex mixtures when coupled with shotgun MS-based proteomics. Due to minimal sample consumption and unrestricted peptide tagging, (18)O/(16)O stable isotope labeling is particularly suitable for amount-limited protein specimens typically encountered in membrane and clinical proteomics. This chapter describes a protocol that relies on shotgun proteomics for quantitative profiling of the detergent-insoluble membrane proteins isolated from HeLa cells, differentially transfected with plasmids expressing HIV Gag protein and its myristylation-defective N-terminal mutant. Whilst this protocol depicts solubilization of detergent-insoluble membrane proteins coupled with post-digestion (18)O labeling, it is amenable to any complex membrane protein mixture. Described approach relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (v/v) methanol followed by differential (18)O/(16)O labeling of protein digests in 20% (v/v) methanol buffer. After mixing, the differentially labeled peptides are fractionated using off-line strong cation exchange (SCX) followed by on-line reversed phase nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS identification/quantiation of peptides/proteins. The use of methanol-based buffers in the context of the post-digestion (18)O exchange/labeling eliminates the need for detergents or chaotropes that interfere with LC separations and peptide ionization. Sample losses are minimized because solubilization, digestion, and stable isotope labeling are carried out in a single tube, avoiding any sample transfer or buffer exchange between these steps.
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Proteínas de la Membrana/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Cromatografía por Intercambio Iónico , Cromatografía de Fase Inversa , Interpretación Estadística de Datos , Células HeLa , Humanos , Marcaje Isotópico , Datos de Secuencia Molecular , Peso Molecular , Isótopos de Oxígeno , Mapeo Peptídico , Proteolisis , Proteómica , Programas Informáticos , Espectrometría de Masas en Tándem , Tripsina/químicaRESUMEN
Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics.
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Comunicación Celular , Membrana Eritrocítica/metabolismo , Proteínas de la Membrana/biosíntesis , Proteoma/biosíntesis , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Transducción de Señal , Adulto , Anciano , Citoesqueleto/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Fumar/metabolismoRESUMEN
The heterogeneous nature of solid tumors represents a common problem in mass spectrometry (MS)-based analysis of fresh-frozen tissue specimens. Here, we describe a method that relies on synergy between laser capture microdissection (LCM) and MS for enhanced molecular profiling of solid tumors. This method involves dissection of homogeneous histologic cell types from thin fresh-frozen tissue sections via LCM, coupled with liquid chromatography (LC)-MS analysis. Such an approach enables an in-depth molecular profiling of captured cells. This is a bottom-up proteomic approach, where proteins are identified through peptide sequencing and matching against a specific proteomic database. Sample losses are minimized, since lysis, solubilization, and digestion are carried out directly on LCM caps in buffered methanol using a single tube, thus reducing sample loss between these steps. The rationale for the LCM-MS coupling is that once the optimal method parameters are established for a solid tumor of interest, homogeneous histologic tumor/tissue cells (i.e., tumor proper, stroma, etc.) can be effectively studied for potential biomarkers, drug targets, pathway analysis, as well as enhanced understanding of the pathological process under study.
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Rayos Láser , Microdisección/métodos , Neoplasias/patología , Proteoma/metabolismo , Separación Celular/métodos , Cromatografía de Fase Inversa/métodos , Criopreservación , Interpretación Estadística de Datos , Humanos , Espectrometría de Masas/métodos , Neoplasias/química , Neoplasias/metabolismo , Fragmentos de Péptidos/química , Proteoma/química , Análisis de la Célula Individual/métodos , Coloración y Etiquetado/métodos , Tripsina/químicaRESUMEN
The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens approximately 50,000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins. Prepared samples were analyzed by LC/MS/MS resulting in more than 500 unique protein identifications. Decoy database searching revealed a false-positive rate between 5 and 10%. Subcellular localization analysis for stromal cells revealed plasma membrane 14%, cytoplasm 39%, nucleus 11%, extracellular space 27%, and unknown 9%; and tumor cell results were 5%, 58%, 26%, 4%, and 7%, respectively. Western blot analysis confirmed specific linkage of validated proteins to underlying pathology and their potential role in solid tumor heterogeneity. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery.
RESUMEN
The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability gamma1 CH2 was cloned and a panel of cysteine mutants was constructed. Human gamma1 CH2 unfolded at a higher temperature (T(m) = 54.1 degrees C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 degrees C). One mutant (m01) was remarkably stable (T(m) = 73.8 degrees C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.
Asunto(s)
Regiones Constantes de Inmunoglobulina/química , Inmunoglobulina G/química , Ingeniería de Proteínas , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Disulfuros , Glicosilación , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Mutantes/química , Docilidad , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus-1) as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1 deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/- mice displayed significantly higher neutrophil accumulation in lipopolysaccharide-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1 double-deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.