Slow Off-Rate Modified Aptamer (SOMAmer) Proteomic Analysis of Patient-Derived Malignant Glioma Identifies Distinct Cellular Proteomes.
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ABSTRACT:
SUBMITTER: Thanasupawat T
PROVIDER: S-EPMC8431317 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An important precondition for the successful development of diagnostic assays of cerebrospinal fluid (CSF) biomarkers of age-related neurodegenerative diseases is an understanding of the dynamic nature of the CSF proteome during the normal aging process. In this study, a novel proteomic technology was used to quantify hundreds of proteins simultaneously in the CSF from 90 cognitively normal adults 21 to 85 years of age. SomaLogic's highly multiplexed proteomic platform can measure more than 800 proteins simultaneously from small volumes of biological fluids using novel slow off-rate modified aptamer (SOMAmer) protein affinity reagents with sensitivity, specificity, and dynamic ranges that meet or exceed those of enzyme-linked immunosorbent assays. In the first application of this technology to CSF, we detected 248 proteins that possessed signals greater than twofold over background. Several novel correlations between detected protein concentrations and age were discovered that indicate that both inflammation and response to injury in the central nervous system may increase with age. Applying this powerful proteomic approach to CSF provides potential new insight into the aging of the human central nervous system that may have utility in discovering new disease-related changes in the CSF proteome.
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Project description:Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.
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Project description:Despite extensive preclinical research on immunotherapeutic approaches, malignant glioma remains a devastating disease of the central nervous system for which standard of care treatment is still confined to resection and radiochemotherapy. For peripheral solid tumors, immune checkpoint inhibition has shown substantial clinical benefit, while promising preclinical results have yet failed to translate into clinical efficacy for brain tumor patients. With the advent of high-throughput sequencing technologies, tumor antigens and corresponding T cell receptors (TCR) and antibodies have been identified, leading to the development of chimeric antigen receptors (CAR), which are comprised of an extracellular antibody part and an intracellular T cell receptor signaling part, to genetically engineer T cells for antigen recognition. Due to efficacy in other tumor entities, a plethora of CARs has been designed and tested for glioma, with promising signs of biological activity. In this review, we describe glioma antigens that have been targeted using CAR T cells preclinically and clinically, review their drawbacks and benefits, and illustrate how the emerging field of transgenic TCR therapy can be used as a potent alternative for cell therapy of glioma overcoming antigenic limitations.
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Project description:BackgroundWe used a large-scale, high-throughput DNA aptamer-based discovery proteomic platform to identify circulating biomarkers of cardiac remodeling and incident heart failure (HF) in community-dwelling individuals.MethodsWe evaluated 1895 FHS (Framingham Heart Study) participants (age 55±10 years, 54% women) who underwent proteomic profiling and echocardiography. Plasma levels of 1305 proteins were related to echocardiographic traits and to incident HF using multivariable regression. Statistically significant protein-HF associations were replicated in the HUNT (Nord-Trøndelag Health) study (n=2497, age 63±10 years, 43% women), and results were meta-analyzed. Genetic variants associated with circulating protein levels (pQTLs) were related to echocardiographic traits in the EchoGen (n=30 201) and to incident HF in the CHARGE (n=20 926) consortia.ResultsSeventeen proteins associated with echocardiographic traits in cross-sectional analyses (false discovery rate <0.10), and 8 of these proteins had pQTLs associated with echocardiographic traits in EchoGen (P<0.0007). In Cox models adjusted for clinical risk factors, 29 proteins demonstrated associations with incident HF in FHS (174 HF events, mean follow-up 19 [limits, 0.2-23.7] years). In meta-analyses of FHS and HUNT, 6 of these proteins were associated with incident HF (P<3.8×10-5; 3 with higher risk: NT-proBNP [N-terminal proB-type natriuretic peptide], TSP2 [thrombospondin-2], MBL [mannose-binding lectin]; and 3 with lower risk: ErbB1 [epidermal growth factor receptor], GDF-11/8 [growth differentiation factor-11/8], and RGMC [hemojuvelin]). For 5 of the 6 proteins, pQTLs were associated with echocardiographic traits (P<0.0006) in EchoGen, and for RGMC, a protein quantitative trait loci was associated with incident HF (P=0.001).ConclusionsA large-scale proteomics approach identified new predictors of cardiac remodeling and incident HF. Future studies are warranted to elucidate how biological pathways represented by these proteins may mediate cardiac remodeling and HF risk and to assess if these proteins can improve HF risk prediction.
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Project description:End stage renal disease (ESRD) is characterized by complex metabolic abnormalities, yet the clinical relevance of specific biomarkers remains unclear. The development of multiplex diagnostic platforms is creating opportunities to develop novel diagnostic and therapeutic approaches. SOMAscan is an innovative multiplex proteomic platform which can measure >1300 proteins. In the present study, we performed SOMAscan analysis of plasma samples and validated the measurements by comparison with selected biomarkers. We compared concentrations of SOMAscan-measured prostate specific antigen (PSA) between males and females, and validated SOMAscan concentrations of fibroblast growth factor 23 (FGF23), FGF receptor 1 (FGFR1), and FGFR4 using Enzyme-Linked immunosorbent assay (ELISA). The median (25th and 75th percentile) SOMAscan PSA level in males and females was 4304.7 (1815.4 to 7259.5) and 547.8 (521.8 to 993.4) relative fluorescence units (p = 0.002), respectively, suggesting biological plausibility. Pearson correlation between SOMAscan and ELISA was high for FGF23 (R = 0.95, p < 0.001) and FGFR4 (R = 0.69, p < 0.001), indicating significant positive correlation, while a weak correlation was found for FGFR1 (R = 0.13, p = 0.16). In conclusion, there is a good to near-perfect correlation between SOMAscan and standard immunoassays for FGF23 and FGFR4, but not for FGFR1. This technology may be useful to simultaneously measure a large number of plasma proteins in ESRD, and identify clinically important prognostic markers to predict outcomes.
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Project description:Target engagement is a key concept in drug discovery and its direct measurement can provide a quantitative understanding of drug efficacy and/or toxicity. Failure to demonstrate target occupancy in relevant cells and tissues has been recognised as a contributing factor to the low success rate of clinical drug development. Several techniques are emerging to quantify target engagement in cells; however, in situ measurements remain challenging, mainly due to technical limitations. Here, we report the development of a non-covalent clickable probe, based on SCH772984, a slow off-rate ERK1/2 inhibitor, which enabled efficient pull down of ERK1/2 protein via click reaction with tetrazine tagged agarose beads. This was used in a competition setting to measure relative target occupancy by selected ERK1/2 inhibitors. As a reference we used the cellular thermal shift assay, a label-free biophysical assay relying solely on ligand-induced thermodynamic stabilization of proteins. To validate the EC50 values measured by both methods, the results were compared with IC50 data for the phosphorylation of RSK, a downstream substrate of ERK1/2 used as a functional biomarker of ERK1/2 inhibition. We showed that a slow off-rate reversible probe can be used to efficiently pull down cellular proteins, significantly extending the potential of the approach beyond the need for covalent or photoaffinity warheads.
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