Project description:Human tissue kallikreins (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished only by enzyme-linked immunosorbent assays (ELISA). In this work, we developed a multiplex selected reaction monitoring (SRM) assay for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and then multiplex in a single assay. Assay performance was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in seminal plasma, sweat and cervico-vaginal fluid, with limits of detection ranging from 5 to 500 ng/mL. Analytical performance of the SRM assay was evaluated by measuring endogenous KLKs in relevant clinical samples and results were compared to selected ELISAs. The multiplex SRM assay was proven to be an accurate, reproducible, sensitive and highly specific alternative to the existing antibody-based assays. Finally, we used immunoenrichment-SRM and ELISA to unambiguously detect and quantify seminal plasma levels of kallikrein-4, a highly prostate-specific protein and a potential biomarker of prostate-related diseases. The presented multiplex SRM assay is an alternative analytical tool to study the biological and pathological roles of human KLKs.
Project description:We developed a multiplex pseudo-isobaric dimethyl labeling (m-pIDL) method for proteome quantification to extend the capacity of the fragment ion-based method to 6-plex by one-step dimethyl labeling with several millidalton and dalton mass differences between precursor ions and enlarging the isolation window of precursor ions to 10 m/z during data acquisition.
Project description:Mutations of the fibroblast growth factor receptor (FGFR) family members are frequently observed in metastatic bladder cancer. The development of erdafitinib, a pan-FGFR inhibitor, provides a significant therapeutic advance in bladder cancer, but resistance still limits its efficacy. In this study, we perform an unbiased whole-genome CRISPR-Cas9 synthetic lethal screen on FGFR-mutant bladder cancer cell lines treated with erdafitinib-targeted therapy and identify SRM as a critical contributor to erdafitinib resistance. In polyamine metabolism, SRM catalyzes the production of spermidine, which subsequently promotes the hypusination of eukaryotic translation factor 5A (eIF5A). Moreover, we demonstrate that hypusinated eIF5A (eIF5AHyp) facilitates the efficient translation of HMGA2, which in turn promotes EGFR expression. Notably, pharmacologic inhibition of SRM using MCHA enhances the efficacy of erdafitinib both in vitro and in vivo. Together, these results offer evidence of the synthetic lethality between SRM inhibition and erdafitinib, suggesting that combination treatment is a promising therapeutic strategy to overcome erdafitinib resistance for FGFR-mutant bladder cancer.
Project description:Mutations of the fibroblast growth factor receptor (FGFR) family members are frequently observed in metastatic bladder cancer. The development of erdafitinib, a pan-FGFR inhibitor, provides a significant therapeutic advance in bladder cancer, but resistance still limits its efficacy. In this study, we perform an unbiased whole-genome CRISPR-Cas9 synthetic lethal screen on FGFR-mutant bladder cancer cell lines treated with erdafitinib-targeted therapy and identify SRM as a critical contributor to erdafitinib resistance. In polyamine metabolism, SRM catalyzes the production of spermidine, which subsequently promotes the hypusination of eukaryotic translation factor 5A (eIF5A). Moreover, we demonstrate that hypusinated eIF5A (eIF5AHyp) facilitates the efficient translation of HMGA2, which in turn promotes EGFR expression. Notably, pharmacologic inhibition of SRM using MCHA enhances the efficacy of erdafitinib both in vitro and in vivo. Together, these results offer evidence of the synthetic lethality between SRM inhibition and erdafitinib, suggesting that combination treatment is a promising therapeutic strategy to overcome erdafitinib resistance for FGFR-mutant bladder cancer.
Project description:RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring This SuperSeries is composed of the SubSeries listed below.