Quantitative MS Workflow for a High-Quality Secretome Analysis by a Quantitative Secretome-Proteome Comparison.

Cells secrete proteins to communicate with their environment. Therefore, it is interesting to characterize the proteins which are released from cells under certain experimental conditions the so-called secretome. Here, often proteins from conditioned medium of cultured cells are analyzed, but these additionally might include also contaminating proteins of serum that have not been sufficiently removed or proteins from dying cells. To provide high-quality secretome data and minimize potential contaminants, we describe a quantitative comparison of conditioned medium and the cellular proteome. The described workflow comprises cell cultivation, sample preparation, and final data analysis which is based on the comparison of data from label-free mass spectrometric quantification of proteins from the conditioned medium with corresponding cellular proteomes enabling the detection of bona fide secreted proteins.

The migration behavior of human glioblastoma cells is influenced by the redox-sensitive human macrophage capping protein CAPG.

The macrophage capping protein CAPG belongs to the gelsolin superfamily which modulates actin dynamics by capping the growing end of actin filaments in a Ca2+- and PIP2-dependent manner resulting in polymerization inhibition of actin filaments. In the last years, additional functions for CAPG in transcription regulation were described and higher CAPG amounts have been linked to increased invasiveness and migration behavior in different human tumor entities like e.g. glioblastoma. Nevertheless, there is a lack of knowledge how additional functions of CAPG are regulated. As CAPG contains several cysteine residues which may be accessible to oxidation we were especially interested to investigate how alterations in the cysteine oxidation state may influence the function, localization, and regulation of CAPG. In the present study, we provide strong evidence that CAPG is a redox-sensitive protein and identified two cysteines: C282 and C290 as reversibly oxidized in glioblastoma cell lines. Whereas no evidence could be found that the canonical actin capping function of CAPG is redox-regulated, our results point to a novel role of the identified cysteines in the regulation of cell migration. Along with this, we found a localization shift out of the nucleus of CAPG and RAVER1, a potential interaction partner identified in our study which might explain the observed altered cell migration properties. The newly identified redox sensitive cysteines of CAPG could perspectively be considered as new targets for controlling tumor invasive properties.