Clinical proteomics · 2026
Targeted proteomics for the detection of androgen receptor variants in preclinical models of castration-resistant prostate cancer.
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Sychev Zoi E, Larson Gabrianne, Ludwig Megan L, Shiba Seiei, Tietz Kiel, Russo Joshua W, Balk Steven, Corey Eva, Plymate Stephen, Antonarakis Emmanuel S, Dehm Scott M, Seegmiller Jesse C, Drake Justin M
Аннотация
BACKGROUND: The emergence of Androgen Receptor variants (AR-Vs) during hormonal treatment is believed to be one of the key mechanisms of resistance. AR-Vs lack the ligand-binding domain and function as constitutively active transcription factors, rendering AR-targeted endocrine-based therapeutics ineffective in tumors that express AR-Vs. The goal of this study was to develop a targeted multiplex assay to identify known and unexplored AR-V proteins and help guide the stratification of PCa patients based on their AR-V protein profiles.
METHODS: A targeted multiple reaction monitoring (MRM) assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify total AR proteins using the AR N-terminal domain (AR-Nterm), a well-characterized AR splice variant AR-V7 (AR-V7), a putative, previously identified but uncharacterized protein isoform (AR-V12) and isoforms that code for an exon 3 duplicated junction seen in AR-V2 and AR- full length with two copies of exon 3. Specimens included three AR-positive PCa cell lines (22Rv1, VCaP16, LNCaP), two AR-negative PCa cell lines (PC3, DU145), and a patient-derived xenograft (PDX) model of CRPC. Endogenous target peptides were quantified according to tier-2 targeted mass spectrometry "fit-for-purpose" guidelines to ensure analytical rigor.
RESULTS: Analytical performance was characterized across a linearity range of 4.15 fmol to 4000 fmol followed by evaluations of precision, accuracy, selectivity, stability, ion suppression and repeatability studies. The overall coefficient of variation (CV) was < 20% across seven independent validation experiments. Endogenous AR-V peptides were accurately quantified using calibration curves, achieving acceptable linear regression coefficients (R2 > 0.95). Four targets, including AR-Nterm and 3 AR-variants, were identified and quantified in PCa cell lines and in a PDX pre-clinical model.
CONCLUSION: This study establishes a robust, validated MRM targeted proteomics assay capable of reproducibly detecting and quantifying both known and previously uncharacterized AR-Vs at the protein level . The assay demonstrated < 20% analytical variation at the lower limit of quantification of 4.25 femtomole (fmol) for AR-Nterm and 8.25 fmol for AR-V7, AR Exon3-Dup/AR-V2 and AR-V12 and, showed robust analytical performance in preclinical models and is ready for further evaluation in clinical specimens. This provides a clinically applicable platform for the comprehensive profiling of AR-V proteins in human subjects, enabling improved stratification of patients with CRPC and supporting precision therapeutic strategies.
METHODS: A targeted multiple reaction monitoring (MRM) assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify total AR proteins using the AR N-terminal domain (AR-Nterm), a well-characterized AR splice variant AR-V7 (AR-V7), a putative, previously identified but uncharacterized protein isoform (AR-V12) and isoforms that code for an exon 3 duplicated junction seen in AR-V2 and AR- full length with two copies of exon 3. Specimens included three AR-positive PCa cell lines (22Rv1, VCaP16, LNCaP), two AR-negative PCa cell lines (PC3, DU145), and a patient-derived xenograft (PDX) model of CRPC. Endogenous target peptides were quantified according to tier-2 targeted mass spectrometry "fit-for-purpose" guidelines to ensure analytical rigor.
RESULTS: Analytical performance was characterized across a linearity range of 4.15 fmol to 4000 fmol followed by evaluations of precision, accuracy, selectivity, stability, ion suppression and repeatability studies. The overall coefficient of variation (CV) was < 20% across seven independent validation experiments. Endogenous AR-V peptides were accurately quantified using calibration curves, achieving acceptable linear regression coefficients (R2 > 0.95). Four targets, including AR-Nterm and 3 AR-variants, were identified and quantified in PCa cell lines and in a PDX pre-clinical model.
CONCLUSION: This study establishes a robust, validated MRM targeted proteomics assay capable of reproducibly detecting and quantifying both known and previously uncharacterized AR-Vs at the protein level . The assay demonstrated < 20% analytical variation at the lower limit of quantification of 4.25 femtomole (fmol) for AR-Nterm and 8.25 fmol for AR-V7, AR Exon3-Dup/AR-V2 and AR-V12 and, showed robust analytical performance in preclinical models and is ready for further evaluation in clinical specimens. This provides a clinically applicable platform for the comprehensive profiling of AR-V proteins in human subjects, enabling improved stratification of patients with CRPC and supporting precision therapeutic strategies.