RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteogenômica , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Carcinoma Ductal Pancreático/diagnóstico , Estudos de Coortes , Células Endoteliais/metabolismo , Epigênese Genética , Feminino , Dosagem de Genes , Genoma Humano , Glicólise , Glicoproteínas/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias Pancreáticas/diagnóstico , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Prognóstico , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Especificidade por Substrato , Transcriptoma/genéticaRESUMO
The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this "proteogenomics" approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases. Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinogênese/patologia , Terapia de Alvo Molecular , Proteogenômica , Desaminases APOBEC/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Estudos de Coortes , Dano ao DNA , Reparo do DNA , Feminino , Humanos , Imunoterapia , Metabolômica , Pessoa de Meia-Idade , Mutagênese/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Receptor ErbB-2/metabolismo , Proteína do Retinoblastoma/metabolismo , Microambiente Tumoral/imunologiaRESUMO
To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteogenômica , Adenocarcinoma de Pulmão/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas de Fusão Oncogênica , Fenótipo , Fosfoproteínas/metabolismo , Proteoma/metabolismoRESUMO
To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass-spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSCs). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease, such as how different copy-number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, and the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC. VIDEO ABSTRACT.
Assuntos
Proteínas de Neoplasias/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Proteoma , Acetilação , Instabilidade Cromossômica , Reparo do DNA , DNA de Neoplasias , Feminino , Dosagem de Genes , Humanos , Espectrometria de Massas , Fosfoproteínas/genética , Processamento de Proteína Pós-Traducional , Análise de SobrevidaRESUMO
The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top-down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4BC185∗ proteoform is unable to associate with the plasma membrane, resulting in a decrease in mitogen-activated protein kinase signaling pathway activation. Collectively, our study provides a reference set of functionally distinct KRAS proteoforms and the colorectal cancer contexts in which they are present.
Assuntos
Neoplasias Colorretais , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Humanos , Neoplasias Colorretais/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Proteômica , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
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.
Assuntos
Neoplasias Colorretais/enzimologia , Mutação de Sentido Incorreto , Proteínas de Neoplasias/análise , Mutação Puntual , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/análise , Sequência de Aminoácidos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cromatografia Líquida , Neoplasias Colorretais/genética , Cisteína/química , Humanos , Metilação , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/isolamento & purificação , Nitrosação , Prenilação , Conformação Proteica , Proteômica/métodos , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em TandemRESUMO
Methodologies that facilitate high-throughput proteomic analysis are a key step toward moving proteome investigations into clinical translation. Data independent acquisition (DIA) has potential as a high-throughput analytical method due to the reduced time needed for sample analysis, as well as its highly quantitative accuracy. However, a limiting feature of DIA methods is the sensitivity of detection of low abundant proteins and depth of coverage, which other mass spectrometry approaches address by two-dimensional fractionation (2D) to reduce sample complexity during data acquisition. In this study, we developed a 2D-DIA method intended for rapid- and deeper-proteome analysis compared to conventional 1D-DIA analysis. First, we characterized 96 individual fractions obtained from the protein standard, NCI-7, using a data-dependent approach (DDA), identifying a total of 151,366 unique peptides from 11,273 protein groups. We observed that the majority of the proteins can be identified from just a few selected fractions. By performing an optimization analysis, we identified six fractions with high peptide number and uniqueness that can account for 80% of the proteins identified in the entire experiment. These selected fractions were combined into a single sample which was then subjected to DIA (referred to as 2D-DIA) quantitative analysis. Furthermore, improved DIA quantification was achieved using a hybrid spectral library, obtained by combining peptides identified from DDA data with peptides identified directly from the DIA runs with the help of DIA-Umpire. The optimized 2D-DIA method allowed for improved identification and quantification of low abundant proteins compared to conventional unfractionated DIA analysis (1D-DIA). We then applied the 2D-DIA method to profile the proteomes of two breast cancer patient-derived xenograft (PDX) models, quantifying 6,217 and 6,167 unique proteins in basal- and luminal- tumors, respectively. Overall, this study demonstrates the potential of high-throughput quantitative proteomics using a novel 2D-DIA method.
Assuntos
Peptídeos/análise , Proteínas/análise , Proteômica , Humanos , Espectrometria de MassasRESUMO
Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.
Assuntos
Genoma Humano , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteoma/química , Proteômica/métodos , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas , Fenótipo , Biossíntese de Proteínas , Isoformas de Proteínas/química , Ubiquitina/químicaRESUMO
Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this "guilt-by-association" (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies.
Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteômica/métodos , Algoritmos , Mapeamento Cromossômico , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Espectrometria de Massas , Análise de Sequência com Séries de Oligonucleotídeos , Mapas de Interação de Proteínas , NavegadorRESUMO
BACKGROUND: Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries' residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide. METHODS: To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time. RESULTS: All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset. CONCLUSIONS: These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.
RESUMO
Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the "peptide-to-protein" inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0-30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a â¼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0-30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.
Assuntos
Neoplasias da Mama/metabolismo , Xenoenxertos/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Animais , Neoplasias da Mama/genética , Cromatografia Líquida de Alta Pressão , Feminino , Genótipo , Humanos , Camundongos , Peso Molecular , Peptídeos/genética , Peptídeos/metabolismo , Polimorfismo de Nucleotídeo Único , Proteoma/química , Proteoma/genética , Espectrometria de Massas em Tandem , Transplante HeterólogoRESUMO
The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm ). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.
Assuntos
Xenoenxertos/química , Proteômica/métodos , Proteômica/normas , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Cromatografia Líquida , Interpretação Estatística de Dados , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Redes e Vias Metabólicas , Variações Dependentes do Observador , Proteoma , Proteômica/instrumentação , Controle de Qualidade , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normasRESUMO
Advances in both targeted and unbiased MS-based proteomics are now at a mature stage for comprehensively and reproducibly characterizing a large part of the cancer proteome. These developments combined with the extensive genomic characterization of several cancer types by large-scale initiatives such as the International Cancer Genome Consortium and Cancer Genome Atlas Project have paved the way for proteogenomic analysis of omics datasets and integration methods. The advances serve as the basis for the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium and this article highlights its current work and future steps in the area of proteogenomics.
Assuntos
Genômica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Animais , HumanosRESUMO
The global human proteomics community in 2014 is fully engaged in projects that aim to create a better understanding of human biology and its complexities and to provide products from this new knowledge that will in some way benefit humanity. Human proteomics, like any other scientific enterprise, needs to identify areas of direction and development, both for the near future in completing current research projects and into the long-term for the engagement with even more complex challenges. In this Editorial we highlight and discuss four important areas that we collectively believe require attention and demand a collective response going forward. These four areas are: (1) Provide high-quality standardized, sensitive, specific, quantitative, and readily accessible protein, peptide, or other biomarkers of health, disease, response to therapy into the approval processes of regulatory agencies (e.g., U.S. Food and Drug Administration; FDA), and obtaining approval from the relevant agencies for their use in a clinical or other testing settings. (2) Implement standard processes for collecting, processing, and storing human clinical samples in biorepositories and enforcement of measures to ensure subject integrity including informed consent for the downstream use of samples and in registrations of subject identities within study databases. (3) Test and validate mass spectrometry technology platforms that hold much promise for creating opportunities for obtaining new important knowledge at levels of detection previously not achievable. (4) Organize clinical discovery operations and activities in an intuitive manner to meet the challenges of increased interests in the science we provide and diminishing levels of centrally financed resource and infrastructure support.
Assuntos
Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Proteômica/normas , Biomarcadores/análise , Biomarcadores/metabolismo , Pesquisa Biomédica/métodos , Pesquisa Biomédica/normas , Pesquisa Biomédica/tendências , Humanos , Proteoma/metabolismo , Proteômica/tendências , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Protein biomarker discovery and validation in current omics era are vital for healthcare professionals to improve diagnosis, detect cancers at an early stage, identify the likelihood of cancer recurrence, stratify stages with differential survival outcomes, and monitor therapeutic responses. The success of such biomarkers would have a huge impact on how we improve the diagnosis and treatment of patients and alleviate the financial burden of healthcare systems. In the past, the genomics community (mostly through large-scale, deep genomic sequencing technologies) has been steadily improving our understanding of the molecular basis of disease, with a number of biomarker panels already authorized by the U.S. Food and Drug Administration (FDA) for clinical use (e.g., MammaPrint, two recently cleared devices using next-generation sequencing platforms to detect DNA changes in the cystic fibrosis transmembrane conductance regulator (CFTR) gene). Clinical proteomics, on the other hand, albeit its ability to delineate the functional units of a cell, more likely driving the phenotypic differences of a disease (i.e., proteins and protein-protein interaction networks and signaling pathways underlying the disease), "staggers" to make a significant impact with only an average â¼ 1.5 protein biomarkers per year approved by the FDA over the past 15-20 years. This statistic itself raises the concern that major roadblocks have been impeding an efficient transition of protein marker candidates in biomarker development despite major technological advances in proteomics in recent years.
Assuntos
Biomarcadores Tumorais/análise , Espectrometria de Massas/métodos , Neoplasias/metabolismo , Proteoma/análise , Proteômica/métodos , Biomarcadores Tumorais/metabolismo , Pesquisa Biomédica/métodos , Pesquisa Biomédica/normas , Pesquisa Biomédica/tendências , Biologia Computacional/métodos , Biologia Computacional/normas , Biologia Computacional/tendências , Humanos , Espectrometria de Massas/instrumentação , Neoplasias/diagnóstico , Proteoma/metabolismo , Proteômica/normas , Proteômica/tendências , Reprodutibilidade dos TestesRESUMO
Clinical samples contained in biorepositories represent an important resource for investigating the many factors that drive human biology. The biological and chemical markers contained in clinical samples provide important measures of health and disease that when combined with such medical evaluation data can aid in decision making by physicians. Nearly all disciplines in medicine and every "omic" depend upon the readouts obtained from such samples, whether the measured analyte is a gene, a protein, a lipid, or a metabolite. There are many steps in sample processing, storage, and management that need to understood by the researchers who utilize biorepositories in their own work. These include not only the preservation of the desired analytes in the sample but also good understanding of the moral and legal framework required for subject protection irrespective of where the samples have been collected. Today there is a great deal of effort in the community to align and standardize both the methodology of sample collection and storage performed in different locations and the necessary frameworks of subject protection including informed consent and institutional review of the studies being performed. There is a growing trend in developing biorepositories around the focus of large population-based studies that address both active and silent nonsymptomatic disease. Logistically these studies generate large numbers of clinical samples and practically place increasing demand upon health care systems to provide uniform sample handling, processing, storage, and documentation of both the sample and the subject as well to ensure that safeguards exist to protect the rights of the study subjects for deciding upon the fates of their samples. Currently the authority to regulate the entire scope of biorepository usage exists as national practice in law in only a few countries. Such legal protection is a necessary component within the framework of biorepositories, both now and in the future. In this brief overview, we provide practical information to the potential users of biorepositories about some of the current developments in both the methodology of sample acquisition and in the regulatory environment governing their use.
Assuntos
Bancos de Espécimes Biológicos/normas , Pesquisa Biomédica/normas , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Bancos de Espécimes Biológicos/ética , Bancos de Espécimes Biológicos/legislação & jurisprudência , Pesquisa Biomédica/ética , Pesquisa Biomédica/legislação & jurisprudência , Revisão Ética , Humanos , Consentimento Livre e Esclarecido , Legislação Médica , Padrões de Referência , Manejo de Espécimes/ética , Doadores de TecidosRESUMO
During the past several decades, the understanding of cancer at the molecular level has been primarily focused on mechanisms on how signaling molecules transform homeostatically balanced cells into malignant ones within an individual pathway. However, it is becoming more apparent that pathways are dynamic and crosstalk at different control points of the signaling cascades, making the traditional linear signaling models inadequate to interpret complex biological systems. Recent technological advances in high throughput, deep sequencing for the human genomes and proteomic technologies to comprehensively characterize the human proteomes in conjunction with multiplexed targeted proteomic assays to measure panels of proteins involved in biologically relevant pathways have made significant progress in understanding cancer at the molecular level. It is undeniable that proteomic profiling of differentially expressed proteins under many perturbation conditions, or between normal and "diseased" states is important to capture a first glance at the overall proteomic landscape, which has been a main focus of proteomics research during the past 15-20 years. However, the research community is gradually shifting its heavy focus from that initial discovery step to protein target verification using multiplexed quantitative proteomic assays, capable of measuring changes in proteins and their interacting partners, isoforms, and post-translational modifications (PTMs) in response to stimuli in the context of signaling pathways and protein networks. With a critical link to genotypes (i.e., high throughput genomics and transcriptomics data), new and complementary information can be gleaned from multi-dimensional omics data to (1) assess the effect of genomic and transcriptomic aberrations on such complex molecular machinery in the context of cell signaling architectures associated with pathological diseases such as cancer (i.e., from genotype to proteotype to phenotype); and (2) target pathway- and network-driven changes and map the fluctuations of these functional units (proteins) responsible for cellular activities in response to perturbation in a spatiotemporal fashion to better understand cancer biology as a whole system.
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Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor, and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung, and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC) with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step toward building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Sanguíneas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteômica/estatística & dados numéricos , Manejo de Espécimes/estatística & dados numéricos , Algoritmos , Biomarcadores Tumorais/metabolismo , Proteínas Sanguíneas/metabolismo , Estudos de Coortes , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Projetos de Pesquisa , Tamanho da Amostra , Sensibilidade e EspecificidadeRESUMO
Vasopressin's action in renal cells to regulate water transport depends on protein phosphorylation. Here we used mass spectrometry-based quantitative phosphoproteomics to identify signaling pathways involved in the short-term V2-receptor-mediated response in cultured collecting duct cells (mpkCCD) from mouse. Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) with two treatment groups (0.1 nM dDAVP or vehicle for 30 min), we carried out quantification of 2884 phosphopeptides. The majority (82%) of quantified phosphopeptides did not change in abundance in response to dDAVP. Analysis of the 273 phosphopeptides increased by dDAVP showed a predominance of so-called "basophilic" motifs consistent with activation of kinases of the AGC family. Increases in phosphorylation of several known protein kinase A targets were found. In addition, increased phosphorylation of targets of the calmodulin-dependent kinase family was seen, including autophosphorylation of calmodulin-dependent kinase 2 at T286. Analysis of the 254 phosphopeptides decreased in abundance by dDAVP showed a predominance of so-called "proline-directed" motifs, consistent with down-regulation of mitogen-activated or cyclin-dependent kinases. dDAVP decreased phosphorylation of both JNK1/2 (T183/Y185) and ERK1/2 (T183/Y185; T203/Y205), consistent with a decrease in activation of these proline-directed kinases in response to dDAVP. Both ERK and JNK were able to phosphorylate residue S261of aquaporin-2 in vitro, a site showing a decrease in phosphorylation in response to dDAVP in vivo. The data support roles for multiple vasopressin V2-receptor-dependent signaling pathways in the vasopressin signaling network of collecting duct cells, involving several kinases not generally accepted to regulate collecting duct function.
Assuntos
Túbulos Renais Coletores/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Receptores de Vasopressinas/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Técnicas de Cultura de Células , Células Cultivadas , Desamino Arginina Vasopressina/farmacologia , Marcação por Isótopo , MAP Quinase Quinase 4/metabolismo , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/química , Fosforilação/efeitos dos fármacos , Proteômica , Receptores de Vasopressinas/efeitos dos fármacos , Transdução de SinaisRESUMO
Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents.