Comparative Secretomics Gives Access to High Confident Secretome Data: Evaluation of Different Methods for the Determination of Bona Fide Secreted Proteins.

Secretome analysis is broadly applied to understand the interplay between cells and their microenvironment. In particular, the unbiased analysis by mass spectrometry-based proteomics of conditioned medium has been successfully applied. In this context, several approaches have been developed allowing to distinguish proteins actively secreted by cells from proteins derived from culture medium or proteins released from dying cells. Here, three different methods comparing conditioned medium and lysate by quantitative mass spectrometry-based proteomics to identify bona fide secreted proteins are evaluated. Evaluation in three different human cell lines reveals that all three methods give access to a similar set of bona fide secreted proteins covering a broad abundance range. In the analyzed primary cells, that is, mesenchymal stromal cells and normal human dermal fibroblasts, more than 70% of the identified proteins are linked to classical secretion pathways. Furthermore, 4-12% are predicted to be released by unconventional secretion pathways. Interestingly, evidence of release by ectodomain shedding in a large number of the remaining candidate proteins is found. In summary, it is convinced that comparative secretomics is currently the method of choice to obtain high-confident secretome data and to identify novel candidates for unconventional protein secretion which have been neglected so far.

The Proteomic Landscape of Cysteine Oxidation That Underpins Retinoic Acid-Induced Neuronal Differentiation.

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.