Interval-based secretomics unravels the time-dependent acute phase response in hepatocyte model systems

ABSTRACT: Mass spectrometry based secretomics approaches preferentially utilize serum-free culture conditions to circumvent serum induced interference and enable sufficient analytical depth. However, this negatively affects a wide range of cellular functions and thus reduces the biological relevance. Moreover, the experimental time range is limited by the viability of cells under serum free culture conditions which results in potentially missing out a large fraction of transcriptionally regulated secretion events and feedback-loops. We present an “interval-based” secretomics workflow, that addresses the shortcomings of the serum-free secretomics approach, by probing protein secretion rates in short serum-free time windows. We applied this approach to unravel the time-resolved secretion in the hepatocyte model systems HepG2 and HepaRG after stimulation of the acute-phase response (APR), a cellular reaction to inflammatory stimuli that typically manifests over days. The integration of relative quantification using tandem mass tags (TMT) enabled precise monitoring of time dependent changes in the secretome over days. Cell line and cytokine specific changes were observed: IL1b directed the APR towards pathogen defense over three distinct chronological phases. In contrast, IL6 directed the APR towards regeneration. Cell surface shedding was pronounced upon IL1b stimulation and small molecule inhibition of ADAM and matrix metalloproteases identified induced as well as constitutive shedding events. Inhibition of ADAM proteases by TAPI-0 not only led to reduced shedding of the sorting receptor SORT1, but also an attenuated cytokine response suggesting a direct link between cell surface shedding and cytokine secretion rates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

SUBMITTER: Sascha Knecht  

LAB HEAD: Marcus Bantscheff

PROVIDER: PXD027821 | Pride | 2022-05-04

REPOSITORIES: Pride