Chip-based sensing for release of unprocessed cell surface proteins in vitro and in serum and its (patho)physiological relevance Data Supplements for Manuscript in the American Journal of Physiology - Endocrinology and Metabolism

To study the possibility that certain components of eukaryotic plasma membranes are released under certain (patho)physiological conditions, a chip-based sensor was developed for the detection of cell surface proteins, which are anchored at the outer leaflet of eukaryotic plasma membranes by a covalently attached glycolipid, exclusively, and might be prone to spontaneous or regulated release on basis of their amphiphilic character. For this, unprocessed full-length glycosylphosphatidylinositol-anchored proteins (GPI-AP) together with associated phospholipids were specifically captured and detected by a chip- and microfluidic channel-based sensor leading to changes in phase and amplitude of surface acoustic waves (SAW) propagating over the chip surface. Unprocessed GPI-AP in complex with lipids were found to be released from rat adipocyte plasma membranes immobilized on the chip which was dependent on the flow rate and composition of the buffer stream. The complexes were identified in the incubation medium of primary rat adipocytes, in correlation to the cell size, and in rat as well as human serum. With rats, the measured changes in SAW phase shift, reflecting specific mass/size or amount of the unprocessed GPI-AP in complex with lipids, and SAW amplitude, reflecting their viscoelasticity, enabled the differentiation between the lean and obese (high fat diet) state, and the normal (Wistar) and hyperinsulinemic (ZF) as well as hyperinsulinemic hyperglycemic (ZDF) state. Thus chip-based sensing for complexes of unprocessed GPI-AP and lipids reveals the inherently labile anchorage of GPI-AP at plasma membranes and their susceptibility for release in response to (intrinsic/extrinsic) cues of metabolic relevance and may therefore be useful for monitoring of (pre-)diabetic disease states.Data Supplements for Manuscript in American Journal of Physiology - Endocrinology and Metabolism