Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction.

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability

Membrane-initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expression in retinal glial cells.

Neurosteroids, such as progesterone, influence central nervous system development and function by regulating a broad spectrum of physiological processes. Here, we investigated membrane-initiated actions of progesterone in the retina and identified the membrane-associated progesterone receptor component 1 (PGRMC1). We found PGRMC1 expressed mainly in retinal Muller glia (RMG) and retinal pigment epithelium, and localized uniquely to microsomal and plasma membrane fractions. In RMG, membrane-impermeable progesterone conjugate induced calcium influx and subsequent phosphatidylinositol 3-kinase-mediated phosphorylation of PKC and ERK-1/2. Induction by progesterone also led to PKC-dependent activation of VEGF gene expression and protein synthesis, suggesting a contribution of membrane-initiated hormone effects to VEGF induced neovascularization within retina