RESUMEN
The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Péptidos/análisis , Proteoma/análisis , Amidinotransferasas/análisis , Amidinotransferasas/genética , Amidinotransferasas/metabolismo , Amoníaco-Liasas/análisis , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Anexina A2/análisis , Anexina A2/genética , Anexina A2/metabolismo , Expresión Génica , Glutamato Formimidoiltransferasa/análisis , Glutamato Formimidoiltransferasa/genética , Glutamato Formimidoiltransferasa/metabolismo , Hepatocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Enzimas Multifuncionales , Proteínas Oncogénicas/análisis , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Peroxiredoxina VI/análisis , Peroxiredoxina VI/genética , Peroxiredoxina VI/metabolismo , Proteína Desglicasa DJ-1 , Proteolisis , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Técnicas de Cultivo de Tejidos , Tripsina/química , Canal Aniónico 2 Dependiente del Voltaje/análisis , Canal Aniónico 2 Dependiente del Voltaje/genética , Canal Aniónico 2 Dependiente del Voltaje/metabolismoRESUMEN
The mammalian polycystic kidney disease (PKD) gene family comprises eight members whose role in cell physiology is still poorly understood. Two of the founding members of the PKD family, PKD1 and PKD2, are responsible for the majority of cases of autosomal dominant polycystic kidney disease. The present study focuses on a PKD1 homologue, mouse polycystic kidney disease and receptor for egg jelly (PKDREJ) and its putative role in mammalian fertilization. To examine PKDREJ tissue distribution multiple-tissue Northern blot analysis was performed. We observed that PKDREJ expression is confined to mouse testis. A PKDREJ transcript was detected in spermatogenic cells by in situ hybridization with mouse testicular tissue. Upon heterologous expression PKDREJ was retained in intracellular membrane compartments and unlike PKD1 did not undergo cleavage in the G-protein-coupled receptor proteolytic site domain (GPS). Immunocytochemical experiments on isolated epididymal mouse spermatozoa using PKDREJ-specific polyclonal antibodies revealed that the protein is localized in the acrosomal region and on the inner aspect of the falciform-shaped head. To precisely characterize PKDREJ expression in the acrosomal region, transmission electron microscopy was performed. Immunogold labeling was only visible at the plasma membrane of the mouse sperm head. Collectively, these data suggest PKDREJ to be a sperm plasma membrane protein presumably contributing to transmembrane signaling in mammalian spermatozoa.