A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development ...of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX4720. These assays replace over 60 western blots with quantitative mass-spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism-of-action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov.
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•Quantitative protein assays are required to understand cancer signaling networks•We develop a suite of multiplexed mass-spectrometry-based assays•The assays offer specific and precise quantification of key networks and PTMs•The assays provide a resource for mechanism-of-action and pharmacodynamic measurements
A lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays by using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor.
Whiteaker et al. describe a suite of mass-spectrometry-based assays for quantification of protein expression and phosphorylation in receptor tyrosine kinase, AKT, and MAP-kinase networks. The assays provide a resource for replacing over 60 commonly used cancer signaling and tumor biology western blots with high molecular specificity and quantitative rigor.
Mass spectrometry (MS)-based profiling was used to determine whether ion fingerprints could distinguish women with stage 1 breast cancer from women without breast cancer.
The initial study population ...consisted of 310 subjects: 155 women with yearly negative breast examination and negative mammography findings for at least 4 years, and 155 women undergoing surgery for pathology-proven stage 1 invasive ductal carcinoma. High-resolution SELDI-TOF (surface-enhanced laser desorption ionization-time of flight) analysis was performed on serum obtained from blood samples collected before mammography in controls, and before surgery in patients with breast cancer. Samples were divided into a training (109 controls and 109 cancers) and blinded (46 controls and 46 cancers) testing set; each group had similar age demographics. In addition, an independent study set of 46 serum samples was analyzed 14 months after the initial study to validate the robustness of the classifier.
A discriminatory profile consisting of seven ion peaks found in the training set, when applied to the blinded test set, achieved a sensitivity and specificity of 95.6% and 86.5%, respectively. This same seven-peak profile achieved a 96.5% sensitivity and 85.7% specificity, with correct identification of all of 17 T1a tumors when applied to the validation study set.
Mass spectrometry profiling of human serum generated a robust classifier composed of seven low-molecular-weight ions that yielded a highly sensitive and specific diagnostic procedure for the discrimination of women with stage 1 breast cancer compared with women without breast cancer in this research study set.
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.
Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic ...Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.
Clinical Proteomics and Biomarker Discovery JOHANN JR, DONALD J.; MCGUIGAN, MICHAEL D.; PATEL, AMIT R. ...
Annals of the New York Academy of Sciences,
June 2004, Letnik:
1022, Številka:
1
Journal Article
Recenzirano
: Early detection of disease generally provides much‐improved outcomes by a definitive medical procedure or through lifestyle modification along with specific medical management strategies. For serum ...biomarkers, which are central to the diagnosis of many diseases, to become truly useful sentinels of pathogenesis, their sensitivity and specificity in both early detection and recurrence monitoring must be improved. Currently, the detection and monitoring of disease markers is based on solitary proteins, and this approach is not always reliable. New classes of biomarkers derived from mass spectroscopy analysis of the low molecular weight proteome have shown improved abilities in the early detection of disease and hence in patient risk stratification and outcome. The development of a modular platform technology with sufficient flexibility and design ions allowing for concurrent experimentation, test, and refinement will help speed the progress of mass spectroscopy‐derived proteomic pattern‐based diagnostics from the scientific laboratory to the medical clinic. For acceptance by scientists, physicians, and regulatory personnel, new bioinformatic tools are essential system components for data management, analysis, and intuitive display of these new and complex data. Clinically engineered mass spectroscopy systems are essential for the further development and validation of multiplexed biomarkers that have shown tremendous promise for the early detection of disease.
Proteomic patterns have been discovered for a variety of cancers and cancer related diseases. The platforms used have been both mass spectrometry and microarrays and the incorporation of computer ...informatics has resulted in innovative possibilities for novel diagnostics.
Extensive genomic characterization of human cancers presents the problem of inference from genomic abnormalities to cancer phenotypes. To address this problem, we analysed proteomes of colon and ...rectal tumours characterized previously by The Cancer Genome Atlas (TCGA) and perform integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. Messenger RNA transcript abundance did not reliably predict protein abundance differences between tumours. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA 'microsatellite instability/CpG island methylation phenotype' transcriptomic subtype, but had distinct mutation, methylation and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates, including HNF4A (hepatocyte nuclear factor 4, alpha), TOMM34 (translocase of outer mitochondrial membrane 34) and SRC (SRC proto-oncogene, non-receptor tyrosine kinase). Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology.
Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful ...application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.
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.
The potential of using mass spectrometry profiling as a diagnostic tool has been demonstrated for a wide variety of diseases. Various cancers and cancer-related diseases have been the focus of much ...of this work because of both the paucity of good diagnostic markers and the knowledge that early diagnosis is the most powerful weapon in treating cancer. The implementation of mass spectrometry as a routine diagnostic tool has proved to be difficult, however, primarily because of the stringent controls that are required for the method to be reproducible. The method is evolving as a powerful guide to the discovery of biomarkers that could, in turn, be used either individually or in an array or panel of tests for early disease detection. Using proteomic patterns to guide biomarker discovery and the possibility of deployment in the clinical laboratory environment on current instrumentation or in a hybrid technology has the possibility of being the early diagnosis tool that is needed.
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