RESUMEN
Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the spatial proteome of an antibody-producing plasma cell-derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in plasma cell-derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein-expressing cell lines. Data are available via ProteomeXchange with identifier PXD029115.
Asunto(s)
Mieloma Múltiple , Proteómica , Cricetinae , Animales , Humanos , Proteómica/métodos , Células CHO , Proteoma/metabolismo , Cricetulus , Células Plasmáticas/química , Células Plasmáticas/metabolismo , Línea Celular Tumoral , Proteínas Recombinantes/metabolismo , Retículo Endoplásmico/metabolismo , Inmunoglobulina G , Preparaciones FarmacéuticasRESUMEN
Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carboplatino/farmacología , Cistadenocarcinoma Seroso/tratamiento farmacológico , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Anciano , Proteínas de la Ataxia Telangiectasia Mutada/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Daño del ADN , ADN de Neoplasias/genética , Resistencia a Antineoplásicos , Proteína Potenciadora del Homólogo Zeste 2/deficiencia , Proteína Potenciadora del Homólogo Zeste 2/genética , Femenino , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Propofol is a commonly used intravenous general anesthetic. Multi-capillary column (MCC) coupled Ion-mobility spectrometry (IMS) can be used to quantify exhaled propofol, and thus estimate plasma drug concentration. Here, we present results of the calibration and analytical validation of a MCC/IMS pre-market prototype for propofol quantification in exhaled air. Calibration with a reference gas generator yielded an R2≥0.99 with a linear array for the calibration curve from 0 to 20 ppbv. The limit of quantification was 0.3 ppbv and the limit of detection was 0.1 ppbv. The device is able to distinguish concentration differences >0.5 ppbv for the concentration range between 2 and 4 ppbv and >0.9 ppbv for the range between 28 and 30 ppbv. The imprecision at 20 ppbv is 11.3% whereas it is 3.5% at a concentration of 40 ppbv. The carry-over duration is 3min. The MCC/IMS we tested provided online quantification of gaseous propofol over the clinically relevant range at measurement frequencies of one measurement each minute.