A synthetic mixture of real waste packaging plastics representative of the residue from a material recovery facility (plasmix) was submitted to thermal and catalytic pyrolysis. Preliminary ...thermogravimetry experiments coupled with Fourier transform infrared spectroscopy were performed to evaluate the effects of the catalysts on the polymers’ degradation temperatures and to determine the main compounds produced during pyrolysis. The thermal and catalytic experiments were conducted at 370°C, 450°C and 650°C using a bench scale reactor. The oil, gas, and char yields were analyzed and the compositions of the reaction products were compared. The primary aim of this study was to understand the effects of zeolitic hydrogen ultra stable zeolite Y (HUSY) and hydrogen zeolite socony mobil-5 (HZSM5) catalysts with high silica content on the pyrolysis process and the products’ quality. Thermogravimetry showed that HUSY significantly reduces the degradation temperature of all the polymers—particularly the polyolefines. HZSM5 had a significant effect on the degradation of polyethylene due to its smaller pore size. Mass balance showed that oil is always the main product of pyrolysis, regardless of the process conditions. However, all pyrolysis runs performed at 370°C were incomplete. The use of either zeolites resulted in a decrease in the heavy oil fraction and the prevention of wax formation. HUSY has the best performance in terms of the total monoaromatic yield (29 wt-% at 450°C), while HZSM5 promoted the production of gases (41 wt-% at 650°C). Plasmix is a potential input material for pyrolysis that is positively affected by the presence of the two tested zeolites. A more effective separation of polyethylene terephthalate during the selection process could lead to higher quality pyrolysis products.
The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor ...prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.
Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and ...immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens.
Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins.
The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.
Abstract Immunotherapies are revolutionizing cancer care, but many patients do not achieve durable responses and immune-related adverse events are difficult to predict. Quantifying the hundreds of ...proteins involved in cancer immunity has the potential to provide biomarkers to monitor and predict tumor response. We previously developed robust, multiplexed quantitative assays for immunomodulatory proteins using targeted mass spectrometry, providing measurements that can be performed reproducibly and harmonized across laboratories. Here, we expand upon those efforts in presenting data from a multiplexed immuno-oncology (IO)-3 assay panel targeting 43 peptides representing 39 immune- and inflammation-related proteins. A suite of novel monoclonal antibodies was generated as assay reagents, and the fully characterized antibodies are made available as a resource to the community. The publicly available dataset contains complete characterization of the assay performance, as well as the mass spectrometer parameters and reagent information necessary for implementation of the assay. Quantification of the proteins will provide benefit to correlative studies in clinical trials, identification of new biomarkers, and improve understanding of the immune response in cancer.
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.
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.
To date, most of the proteomic analyses on lung cancer tissue samples have been performed using surgical specimens, which are obtained after a diagnosis is made. To determine if a proteomic signature ...obtained from bronchoscopic biopsy samples could be found to assist with diagnosis, 50 lung cancer bronchoscopic biopsy samples and 13 adjacent normal lung tissue samples were analyzed using histology‐directed, matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Lung tissue samples were cryosectioned, and sinapinic acid was robotically deposited on areas of each tissue section enriched in epithelial cells, either tumor or normal. Mass spectra were acquired using a MALDI‐time of flight instrument. Small cell lung cancers (SCLCs) demonstrated clearly different protein profiles from normal lung tissue and from non‐small cell lung cancers (NSCLCs). Calcyclin (m/z= 10 094.7) was identified to be underexpressed in small cell lung cancers, as compared with non‐small cell lung cancers and normal lung tissue. An immunohistochemistry study using 152 NSCLCs and 21 SCLCs confirmed significantly reduced calcyclin stain in SCLCs. Thus, protein profiles obtained from bronchoscopic biopsy samples via MALDI MS distinguish cancerous epithelium from normal lung tissue and between NSCLCs and SCLCs.
Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) ...with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.
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.
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.
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