Proteomic patterns as a potential diagnostic technology has been well established for several cancer conditions and other diseases. The use of machine learning techniques such as decision trees, ...neural networks, genetic algorithms, and other methods has been the basis for pattern determination. Cancer is known to involve signaling pathways that are regulated through PTM of proteins. These modifications are also detectable with high confidence using high-resolution MS. We generated data using a prOTOFtrade mark sign mass spectrometer on two sets of patient samples: ovarian cancer and cutaneous t-cell lymphoma (CTCL) with matched normal samples for each disease. Using the knowledge of mass shifts caused by common modifications, we built models using peak pairs and compared this to a conventional technique using individual peaks. The results for each disease showed that a small number of peak pairs gave classification equal to or better than the conventional technique that used multiple individual peaks. This simple peak picking technique could be used to guide identification of important peak pairs involved in the disease process.
Reference materials are vital to benchmarking the reproducibility of clinical tests and essential for monitoring laboratory performance for clinical proteomics. The reference material utilized for ...mass spectrometric analysis of the human proteome would ideally contain enough proteins to be suitably representative of the human proteome, as well as exhibit a stable protein composition in different batches of sample regeneration. Previously, The Clinical Proteomic Tumor Analysis Consortium (CPTAC) utilized a PDX-derived comparative reference (CompRef) materials for the longitudinal assessment of proteomic performance; however, inherent drawbacks of PDX-derived material, including extended time needed to grow tumors and high level of expertise needed, have resulted in efforts to identify a new source of CompRef material. In this study, we examined the utility of using a panel of seven cancer cell lines, NCI-7 Cell Line Panel, as a reference material for mass spectrometric analysis of human proteome. Our results showed that not only is the NCI-7 material suitable for benchmarking laboratory sample preparation methods, but also NCI-7 sample generation is highly reproducible at both the global and phosphoprotein levels. In addition, the predicted genomic and experimental coverage of the NCI-7 proteome suggests the NCI-7 material may also have applications as a universal standard proteomic reference.
A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National ...Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.
Proteomics in Clinical Trials and Practice Azad, Nilofer S.; Rasool, Nabila; Annunziata, Christina M. ...
Molecular & cellular proteomics,
October 2006, Letnik:
5, Številka:
10
Journal Article
Recenzirano
Odprti dostop
The study of clinical proteomics is a promising new field that has the potential to have many applications, including the identification of biomarkers and monitoring of disease, especially in the ...field of oncology. Expression proteomics evaluates the cellular production of proteins encoded by a particular gene and exploits the differential expression and post-translational modifications of proteins between healthy and diseased states. These biomarkers may be applied towards early diagnosis, prognosis, and prediction of response to therapy. Functional proteomics seeks to decipher protein-protein interactions and biochemical pathways involved in disease biology and targeted by newer molecular therapeutics. Advanced spectrometry technologies and new protein array formats have improved these analyses and are now being applied prospectively in clinical trials. Further advancement of proteomics technology could usher in an era of personalized molecular medicine, where diseases are diagnosed at earlier stages and where therapies are more effective because they are tailored to the protein expression of a patient’s malignancy.
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 (∼85%) 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.
With the numerous reports of new technologies and biomarkers reported in the literature, it may be surprising that there are not an equal number of new products available to the clinical diagnostic ...laboratory. Powerful potential tools such as protein microarrays and MS patterns have been extensively published yet commercialization and acceptance of these technologies has yet to happen. The reasons for this are a combination of industry risk avoidance, academic focus on discovery, and a lack of appreciation for the high standards and regulation that surrounds the clinical diagnostic laboratory. The development and validation of a new technology or biomarker ensures that a test is reproducible, controllable, and has a defined accuracy and clinical predictive result but this information is only obtained through somewhat mundane but necessary experimental work. The use of design of experiment principles helps to define material parameters to ensure performance. The organization and documentation of this work through a quality system is both mandated and practical. All of this must be done before a test can reach the market with the safety and effectiveness review of regulatory agencies.
Abstract only
KRAS is a frequently mutated oncogene and activating missense mutations have been shown to have a causative role in tumor formation. More than a third of human colon cancers are driven ...by KRAS mutations. Here, we integrated genomic, transcriptomic and proteomic information to study isogenic cell lines and primary tumor samples with defined KRAS genotypes. We combined RNA‐seq data with top‐down and bottom‐up proteomic analyses to measure differential expression within the proteome, and KRAS proteoforms specifically. Integration of these three complementary omics datasets provided us with a list of molecular entities showing differential expression in response to the mutational status of KRAS. Overall, the results show a strong enrichment for kinases, consistent with the established effect of KRAS mutations in constitutive activation of the MAPK signaling pathway. In addition, proteins associated with cytoskeleton reorganization seemed to be upregulated in the presence of the KRAS G13D mutant allele. This could be involved in increasing cell motility and lead to a more invasive phenotype. Furthermore, we developed a targeted top‐down assay for the detection and quantitation of endogenous KRAS proteoforms to measure allele‐specific modification patterns. Such quantitative measurements (both targeted and untargeted) provide new insights into colorectal cancer phenotypes through the measurement of cancer‐specific protein variants.
Support or Funding Information
This work was supported by Federal Funds from the National Cancer Institute (Office of Cancer Clinical Proteomics Research), National Institutes of Health, under Contract No. HHSN261200800001E, and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health.
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a ...catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
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•Systematic identification of colon cancer-associated proteins and phosphosites•Proteomics-supported neoantigens and cancer/testis antigens in 78% of the tumors•Rb phosphorylation is an oncogenic driver and a putative target in colon cancer•Glycolysis inhibition may render MSI tumors more sensitive to checkpoint blockade
A systematic proteogenomic analysis of colon cancer reveals vulnerabilities of potential clinical value inaccessible from genomic assessment alone.
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.
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•Comprehensive proteogenomics resource from prospectively collected breast tumors•Proteogenomics defines ERBB2 and Rb status with clinical implications•Acetylproteome profiling yields insights into subtype-specific cancer metabolism•Immune profiling nominates subsets of luminal tumors for immune therapy
Breast cancer is a highly heterogeneous disease with variable outcomes and subtype-driven treatment approaches, making precision medicine a considerable challenge. Proteogenomic analyses of 122 primary breast cancers provide insights into clinically relevant biology, including cell cycle dysregulation, tumor immunogenicity, aberrant metabolism, and heterogeneity in therapeutic target expression.
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