Abstract:The
tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR
signaling and is commonly found downregulated in breast cancer (BC).
Conflicting data from conventional immunoassays such as immunohistochemistry
(IHC) has sparked controversy about PTEN’s role as a prognostic
and predictive biomarker in BC, which can be largely attributed to
the lack of specificity, sensitivity, and interlaboratory standardization.
Here, we present a fully standardized, highly sensitive, robust microflow
immuno-MRM (iMR… Show more
“…CAMP was disproportionately below the LLOQ in the mortality group (95%) compared to the survival group (∼50%) and showed a general trend of down-regulation in the non-survivors, which was significant after imputation of missing values. More sensitive assays may confirm this potential use of CAMP as predictor of survival, for instance by using anti-peptide immuno-enrichment prior to MS quantitation by either LC-MRM (immuno-MRM) ( 54 ) or matrix assisted laser desorption ionization (MALDI) (immuno-MALDI) ( 55 ). Figure 3 Significant differences between COVID-19 survivors and non-survivors plasma proteins and metabolites.…”
“…CAMP was disproportionately below the LLOQ in the mortality group (95%) compared to the survival group (∼50%) and showed a general trend of down-regulation in the non-survivors, which was significant after imputation of missing values. More sensitive assays may confirm this potential use of CAMP as predictor of survival, for instance by using anti-peptide immuno-enrichment prior to MS quantitation by either LC-MRM (immuno-MRM) ( 54 ) or matrix assisted laser desorption ionization (MALDI) (immuno-MALDI) ( 55 ). Figure 3 Significant differences between COVID-19 survivors and non-survivors plasma proteins and metabolites.…”
“…Targeted proteomics has been successful in developing assays for routine clinical use, which provide advantages or additional data when compared to traditional antibody-based techniques. Examples include thyroglobulin for detection of thyroid cancer recurrence, − HER2 quantification for selection of breast cancer therapy, , PTEN as a biomarker for breast cancer, and cytokeratins for evaluating tumor histology. , In much the same way that targeted therapy has moved beyond the one drug-one target thought process, targeted proteomics in cancer must also examine tumor phenotypes in more detail to provide context to the biomarker measurements. As an example, receptor tyrosine kinase quantification needs to be performed as a panel to evaluate the potential for compensatory signaling as well as evaluating whether the tumor has undergone epithelial-to-mesenchymal transition, which provides another mechanism for therapeutic escape.…”
Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use. LC-MRM panels provide the capability to quantify clinical biomarkers and emerging protein markers to establish the context of tumor phenotypes that provide highly relevant supporting information. An application to visualize and communicate targeted proteomics data will empower translational researchers to move protein biomarker panels from discovery to clinical use. Therefore, we have developed a web-based tool for targeted proteomics that provides pathway-level evaluations of key biological drivers (e.g., EGFR signaling), signature scores (representing phenotypes) (e.g., EMT), and the ability to quantify specific drug targets across a sample cohort. This tool represents a framework for integrating summary information, decision algorithms, and risk scores to support Physician-Interpretable Phenotypic Evaluation in R (PIPER) that can be reused or repurposed by other labs to communicate and interpret their own biomarker panels.
“…This, in addition to negligible carryovers, is critical for accurate quantification of compounds by the LC-MS/MS analyses. The method has been adapted for protein biomarker studies using data independent analysis (DIA), parallel reaction monitoring (PRM), and multiple reaction monitoring (MRM) [ 3 , 8 , 9 , 10 ]. However, we are not aware of any reports of the use of the mLC-MS/MS for the analysis of O-glycopeptides.…”
Development of high throughput robust methods is a prerequisite for a successful clinical use of LC-MS/MS assays. In earlier studies, we reported that nLC-MS/MS measurement of the O-glycoforms of HPX is an indicator of liver fibrosis. In this study, we show that a microflow LC-MS/MS method using a single column setup for capture of the analytes, desalting, fast gradient elution, and on-line mass spectrometry measurements, is robust, substantially faster, and even more sensitive than our nLC setup. We demonstrate applicability of the workflow on the quantification of the O-HPX glycoforms in unfractionated serum samples of control and liver disease patients. The assay requires microliter volumes of serum samples, and the platform is amenable to one hundred sample injections per day, providing a valuable tool for biomarker validation and screening studies.
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