Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to ...measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer-related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R(2) > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.
Chimeric antigen receptor (CAR)-modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, ...achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we directly compared TCR and CAR signaling in primary human T cells. Global phosphoproteomic analysis revealed that key T cell signaling proteins-such as CD3δ, CD3ε, and CD3γ, which comprise a portion of the T cell co-receptor, as well as the TCR adaptor protein LAT-were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying a commonplace 4-1BB/CD3ζ CAR sequence to better engage CD3ε and LAT using embedded CD3ε or GRB2 domains resulted in enhanced T cell activation in vitro in settings of a low density of antigen, and improved efficacy in in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling.
Abstract
Background
Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by ...immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies.
Methods
We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity.
Results
HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors.
Conclusions
Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.
Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate ...protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R 2 = 0.94) and immuno-MRM (R 2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.
Clinical Proteomics for Solid Organ Tissues Phipps, William S.; Kilgore, Mark R.; Kennedy, Jacob J. ...
Molecular & cellular proteomics,
11/2023, Letnik:
22, Številka:
11
Journal Article
Recenzirano
Odprti dostop
The evaluation of biopsied solid organ tissue has long relied on visual examination using a microscope. Immunohistochemistry is critical in this process, labeling and detecting cell lineage markers ...and therapeutic targets. However, while the practice of immunohistochemistry has reshaped diagnostic pathology and facilitated improvements in cancer treatment, it has also been subject to pervasive challenges with respect to standardization and reproducibility. Efforts are ongoing to improve immunohistochemistry, but for some applications, the benefit of such initiatives could be impeded by its reliance on monospecific antibody-protein reagents and limited multiplexing capacity. This perspective surveys the relevant challenges facing traditional immunohistochemistry and describes how mass spectrometry, particularly liquid chromatography-tandem mass spectrometry, could help alleviate problems. In particular, targeted mass spectrometry assays could facilitate measurements of individual proteins or analyte panels, using internal standards for more robust quantification and improved interlaboratory reproducibility. Meanwhile, untargeted mass spectrometry, showcased to date clinically in the form of amyloid typing, is inherently multiplexed, facilitating the detection and crude quantification of 100s to 1000s of proteins in a single analysis. Further, data-independent acquisition has yet to be applied in clinical practice, but offers particular strengths that could appeal to clinical users. Finally, we discuss the guidance that is needed to facilitate broader utilization in clinical environments and achieve standardization.
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•Immunohistochemistry workflows are poorly standardized.•Mass spectrometry may complement existing tests for proteins in solid tissues.•Targeted workflows could help clarify borderline immunohistochemistry cases.•Untargeted assays provide relative quantification of 100s to 1000s of proteins.•Guidance is needed to help clinical labs develop robust tools for solid tissues.
For many years, immunohistochemistry has been the primary tool in pathology for protein localization and quantification in solid tissues, but it is poorly standardized and suffers from practical limitations. The field is ripe for novel complementary technologies to help with diagnosis. This perspective focuses on the issues hampering immunohistochemistry and highlights how liquid chromatography-tandem mass spectrometry in particular is well-positioned to complement existing testing. We also discuss where guidance is needed to ensure rigor in clinical environments.
A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one ...analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.
To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one ...discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.
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•A comprehensive proteogenomic analysis of 242 HGSOC tumors was performed•A lack of Chr17-LOH was observed to be associated with refractoriness•A 64-protein signature predicts refractoriness in multiple tumor cohorts•Pathway-based clustering reveals 5 subtypes validated in independent cohorts
Patients with high-grade serous ovarian cancers (HGSOCs) have a poor outcome, with the standard of care not having changed over the decades. A detailed characterization of the proteogenomic landscape of HGSOCs across multiple cohorts and validation studies identifies a distinct signature that predicts with high specificity a subset of patients with chemotherapy-refractory cancers and implicates potential therapeutic vulnerabilities.
A wealth of proteogenomic data has been generated using cancer samples to deepen our understanding of the mechanisms of cancer and how biological networks are altered in association with somatic ...mutation of tumor suppressor genes, such as TP53 and PTEN. To generate functional signatures of TP53 or PTEN loss, we profiled the RNA and phosphoproteomes of the MCF10A epithelial cell line, along with its congenic TP53- or PTEN-knockout derivatives, upon perturbation with the monofunctional DNA alkylating agent methyl methanesulfonate (MMS) vs. mock treatment. To enable quantitative and reproducible mass spectrometry data generation, the cell lines were SILAC-labeled (stable isotope labeling with amino acids in cell culture), and the experimental design included label swapping and biological replicates. All data are publicly available and may be used to advance our understanding of the TP53 and PTEN tumor suppressor genes and to provide functional signatures for bioinformatic analyses of proteogenomic datasets.
Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as ...stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.
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•Extended SIS peptides can control for trypsin digestion in targeted MS-based assays.•Monitoring extended SIS along with tryptic SIS peptides is the basis of a QC metric.•The ratio of extended to tryptic SIS is a useful in-sample QC metric for digestion.•The QC metric also correlates with assay sensitivity and quantification fidelity.•Additional assays can apply this ready-to-use QC panel or identify custom panels.
Extended stable isotope-labeled standard (SIS) peptides (hE), proteolytically digested to release the targeted tryptic SIS peptide (hT) form, have been shown to control for trypsin. We found that by monitoring hE along with hT, the resulting hE/hT ratio is a useful in-sample QC metric for digestion efficiency that correlates with endogenous measurement sensitivity and quantification fidelity. This study describes a ready-to-use QC panel as well as a path for identifying custom QC panels that can be applied to additional mass spectrometry-based assays.
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