RESUMO
In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.
Assuntos
Eletroforese em Gel de Poliacrilamida , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Espectrometria de Massas em Tandem , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas em Tandem/métodos , Membrana Celular/metabolismo , Proteômica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas ras/metabolismo , Proteínas ras/genéticaRESUMO
Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Manejo de Espécimes/métodos , Anticorpos/análise , Anticorpos/imunologia , Western Blotting , Linhagem Celular Tumoral , Células HeLa , Humanos , Células Jurkat , Células MCF-7 , Peptídeos/sangue , Peptídeos/imunologia , Proteoma/genética , Proteoma/imunologia , RNA-Seq/métodos , Reprodutibilidade dos TestesRESUMO
Lung cancer is the leading cause of cancer-related deaths in the USA and worldwide. Yet, about 95% of new drug candidates validated in preclinical phase eventually fail in clinical trials. Such a high attrition rate is attributed mostly to the inability of conventional two-dimensionally (2D) cultured cancer cells to mimic native three-dimensional (3D) growth of malignant cells in human tumors. To ascertain phenotypical differences between these two distinct culture conditions, we carried out a comparative proteomic analysis of a membrane fraction obtained from 3D- and 2D-cultured NSCLC model cell line NCI-H23. This analysis revealed a map of 1,166 (24%) protein species regulated in culture dependent manner, including differential regulation of a subset of cell surface-based CD molecules. We confirmed exclusive expression of CD99, CD146 and CD239 in 3D culture. Furthermore, label-free quantitation, targeting KRas proteoform-specific peptides, revealed upregulation of both wild type and monoallelic KRas4BG12C mutant at the surface of 3D cultured cells. In order to reduce the high attrition rate of new drug candidates, the results of this study strongly suggests exploiting base-line molecular profiling of a large number of patient-derived NSCLC cell lines grown in 2D and 3D culture, prior to actual drug candidate testing.
RESUMO
The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.
RESUMO
RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute's RAS Initiative has a focus on understanding pathways and discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network.
Assuntos
Anticorpos Monoclonais/química , Genes ras , Transdução de Sinais , Linhagem Celular , Impressões Digitais de DNA , Humanos , Indicadores e Reagentes/química , Repetições de Microssatélites , Neoplasias/genéticaRESUMO
The complement system plays an essential role in both innate and adaptive immunity. The traditional understanding of this system comes from studies investigating complement proteins produced by the liver and present in plasma to "complement" the immune cell-mediated response to invading pathogens. Recently, it has been reported that immune cells including, but not limited to, T-cells and monocytes, express complement proteins. This complement is referred to as intracellular (IC) and implicated in the regulation of T-cell activation. The mechanisms and the structure-activity relationship between nanomaterials and IC, however, are currently unknown. Herein, we describe a structure-activity relationship study demonstrating that under in vitro conditions, only polymeric materials with cationic surfaces activate IC in T-cells. The effect also depends on particle size and occurs through a mechanism involving membrane damage, thereby IC on the cell surface serves as a self-opsonization marker in response to the nanoparticle-triggered danger affecting the cell integrity.
Assuntos
Ativação do Complemento , Ativação Linfocitária , Nanopartículas/efeitos adversos , Polímeros/efeitos adversos , Linfócitos T/imunologia , Cátions/efeitos adversos , Cátions/química , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Humanos , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Nanopartículas/química , Polímeros/química , Linfócitos T/efeitos dos fármacosRESUMO
The most widely used cancer animal model is the human-murine tumor xenograft. Unbiased molecular dissection of tumor parenchyma versus stroma in human-murine xenografts is critical for elucidating dysregulated protein networks/pathways and developing therapeutics that may target these two functionally codependent compartments. Although antibody-reliant technologies (e.g., immunohistochemistry, imaging mass cytometry) are capable of distinguishing tumor-proper versus stromal proteins, the breadth or extent of targets is limited. Here, we report an antibody-free targeted cross-species glycoproteomic (TCSG) approach that enables direct dissection of human tumor parenchyma from murine tumor stroma at the molecular/protein level in tumor xenografts at a selectivity rate presently unattainable by other means. This approach was used to segment/dissect and obtain the protein complement phenotype of the tumor stroma and parenchyma of the metastatic human lung adenocarcinoma A549 xenograft, with no need for tissue microdissection prior to mass-spectrometry analysis. An extensive molecular map of the tumor proper and the associated microenvironment was generated along with the top functional N-glycosylated protein networks enriched in each compartment. Importantly, immunohistochemistry-based cross-validation of selected parenchymal and stromal targets applied on human tissue samples of lung adenocarcinoma and normal adjacent tissue is indicative of a noteworthy translational capacity for this unique approach that may facilitate identifications of novel targets for next generation antibody therapies and development of real time preclinical tumor models.
RESUMO
A lack of analytically robust and multiplexed assays has hampered studies of the large, branched phosphosignaling network responsive to DNA damage. To address this need, we developed and fully analytically characterized a 62-plex assay quantifying protein expression and post-translational modification (phosphorylation and ubiquitination) after induction of DNA damage. The linear range was over 3 orders of magnitude, the median inter-assay variability was 10% CV and the vast majority (â¼85%) of assays were stable after extended storage. The multiplexed assay was applied in proof-of-principle studies to quantify signaling after exposure to genotoxic stress (ionizing radiation and 4-nitroquinoline 1-oxide) in immortalized cell lines and primary human cells. The effects of genomic variants and pharmacologic kinase inhibition (ATM/ATR) were profiled using the assay. This study demonstrates the utility of a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to studies on inter-individual variation in the radiation response.