RESUMEN
Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics.
RESUMEN
Extracellular vesicles (EVs) are a new therapeutic modality with the promise to treat many diseases through their ability to deliver diverse molecular cargo. As with other emerging modalities transitioning into the industrialization phase, all aspects of the manufacturing process are rich with opportunities to enhance the ability to deliver these medicines to patients. With the goal of improving cell culture EV productivity, we have utilized high throughput siRNA screens to identify the underlying genetic pathways that regulate EV productivity to inform rational host cell line engineering and media development approaches. The screens identified multiple metabolic pathways of potential interest; one of which was validated and shown to be a ready implementable, cost-effective strategy to increase EV titers. We show that both EV volumetric and specific productivity from HEK293 and CHO-S were increased in a dose and cell line-dependent manner up to ninefold when cholesterol synthesis was inhibited by the inclusion of statins in the cell culture media. In addition, we show in response to statin treatment, elevation of EV markers in mesenchymal stem cell (MSC) cell culture media suggesting this approach can also be applicable to MSC EVs. Furthermore, we show that the EVs produced from statin-treated HEK293 cultures are effectively loaded by both endogenous and exogenous loading methods and have equivalent in vitro or in vivo potency relative to EVs from untreated cultures.
