Interval-Based Secretomics Unravels Acute-Phase Response in Hepatocyte Model Systems

EXCRETE enables deep proteomics of the microbial extracellular environment

“…Therefore, we sought to develop a fast, robust, and unbiased workflow that allows easy exoproteome concentration and subsequent removal of contaminants. To this end, we took inspiration from solid-phase enhanced protein aggregation protocols (e.g., SP3, PAC) that have already been applied to exoproteomic samples, albeit of human model systems 34,49 . These protocols rely on a phenomenon where precipitated and aggregated proteins bind to a solid phase irrespective of its surface chemistry.…”

“…Miniaturization of EXCRETE will allow for the analysis of dozens of secretomes per day and opens the door to automation of exoproteome analysis on robotic liquid handling platforms 55 . EXCRETE allows for deep exoproteomic profiling of cyanobacteria from a range of habitats Solid-phase enhanced protein aggregation protocols have already been successfully applied to analyze the exoproteome of human tissue cultures 34,49 . However, in contrast to these, bacteria grow in a wide range of environments that can be rich in salt, lipids, and polysaccharides.…”

“…Solid-phase enhanced protein aggregation protocols have already been successfully applied to analyze the exoproteome of human tissue cultures 34,49 . However, in contrast to these, bacteria grow in a wide range of environments that can be rich in salt, lipids, and polysaccharides.…”

Secretome Analysis: Reading Cellular Sign Language to Understand Intercellular Communication

“…This has been employed, for example, to investigate substrates of beta-secretase 2 (BACE2) in a model for cancer metastasis in melanoma ( 49 ) or to show that the secretome pattern of (SILAC-labeled) astrocytes was altered within a few hours, depending on the type of (unlabeled) neurons that were added in coculture, including proteins that were secreted by classical, unconventional, and shedding mechanisms ( 50 ). To partially circumvent potential negative effects due to extended periods of serum starvation, one approach is to decouple cell treatment (exposure to cytokines) from serum depletion, by only removing serum from growth media during the last 2 h before harvest of conditioned media ( 51 ). In this way, the authors could identify profiles of cytokine-induced protein secretion over a 72 h period.…”

An Introduction to Analytical Challenges, Approaches, and Applications in Mass Spectrometry–Based Secretomics

“…The experimental design has also a critical influence on the depth and sensitivity of the analysis. For example, hepatocyte model cell lines such as HepaRG cells are highly secretory active upon cytokine treatments ( 18 ), allowing the execution of serum-free secretomics experiments in a 12-well plate format and still enable the identification of a multitude of different acute-phase response proteins (ng/ml range). However, the identification and quantification of cytokines, for example, which are usually only present in very low abundances (pg/ml range), may require millions of immune cells to adjust to the detection limit for the LC-MS instrumentation and therefore might require the use of big culture dishes.…”