Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry.

Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two-dimensional liquid chromatography, TiO2-mediated phosphopeptide enrichment, high-resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.

PAX7 is a required target for microRNA-206-induced differentiation of fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS can be parsed based on clinical outcome into two subtypes, fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS) based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. In both RMS subtypes, tumor cells show histology and a gene expression pattern resembling that of developmentally arrested skeletal muscle. Differentiation therapy is an attractive approach to embryonal tumors of childhood including RMS; however, agents to drive RMS differentiation have not entered the clinic and their mechanisms remain unclear. MicroRNA-206 (miR-206) expression increases through normal muscle development and has decreased levels in RMS compared with normal skeletal muscle. Increasing miR-206 expression drives differentiation of RMS, but the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with gene and proteomic profiling coupled with genetic rescue, we identified key miR-206 targets responsible for the FN-RMS differentiation blockade, PAX7, PAX3, NOTCH3, and CCND2. Specifically, we determined that PAX7 downregulation is necessary for miR-206-induced cell cycle exit and myogenic differentiation in FN-RMS but not in FP-RMS. Gene knockdown of targets necessary for miR-206-induced differentiation alone or in combination was not sufficient to phenocopy the differentiation phenotype from miR-206, thus illustrating that miR-206 replacement offers the ability to modulate a complex network of genes responsible for the developmental arrest in FN-RMS. Genetic deletion of miR-206 in a mouse model of FN-RMS accelerated and exacerbated tumor development, indicating that both in vitro and in vivo miR-206 acts as a tumor suppressor in FN-RMS at least partially through downregulation of PAX7. Collectively, our results illustrate that miR-206 relieves the differentiation arrest in FN-RMS and suggests that miR-206 replacement could be a potential therapeutic differentiation strategy.