Selective proteome‐wide detection of hydrophobic integral membrane proteins using a novel fluorescence‐based staining technology

Abstract

Integral proteins containing two or more alpha-helical membrane-spanning domains are underrepresented in two-dimensional gels. While sodium dodecyl sulfate (SDS)-polyacrylamide gels separate these proteins, staining profiles are usually dominated by high-abundance hydrophilic proteins in the specimen. A fluorescence-based stain is presented that selectively highlights integral proteins containing two or more alpha-helical transmembrane domains but does not detect lipoproteins or proteins with hydrophobic pockets, such as albumin. The stain detects as little as 5-10 ng of bacteriorhodopsin, a seven-helix transmembrane protein. Stained proteins are detected using a laser scanner or charge-coupled device (CCD) camera imaging system. Fluorescence intensity of stained bands is linear with protein quantity over at least two orders of magnitude. After visualizing transmembraneous proteins, the total protein profile may be revealed using a general protein stain. Analysis of the multisubunit protein F1F0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.

Proteomic analyses of native brain KV4.2 channel complexes

“…This complication could reflect the fact that these are low abundance proteins or, alternatively, that they are proteins with lower affinity interactions (with the targeted K V 4.2 protein). Finally, it is also important to note, as described in previous studies, that some proteins, and particularly transmembrane proteins,35 do not stain well in gel, which will ultimately result in excluding these proteins from mass spectrometric analyses.…”

Rice proteomics: A cornerstone for cereal food crop proteomes

“…Integral membrane proteins, having important roles in signal transduction, typically contain one or more hydrophobic, transmembrane domains that intermingle with the hydrophobic portion of lipid bilayer membranes. Generating good proteomic profiles of these highly hydrophobic proteins on 2-DGE is almost impossible, mainly due to their very poor resolution in IEF, which arises from poor solubilization by nonionic detergents, precipitation near their pI points, and significant isoelectric point heterogenetity due to glycosylation that results in streaking in IEF (Ito & Akiyama, 1985;Hartinger et al, 1996;Ito, Matsuo, & Akiyama, 1999;Lescuyer et al, 2003;Hart, Schulenberg, & Patton, 2004). Like in mammalian systems, FIGURE 11.…”

“…One deals with the detection of membrane proteins in-gel (Hart, Schulenberg, & Patton, 2004), and the other deals with a doubled SDS-PAGE (dSDS-PAGE)-based 2-D electrophoresis system (Rais, Karas, & Schagger, 2004). Although, SDS-PAGE is more suitable for fractionating integral membrane proteins than 2-DGE, due to better solubilization properties of anionic detergents, there was a lack of specific visualization techniques for integral membrane proteins against a background of high-abundance hydrophilic cytoplasmic proteins.…”

“…Although, SDS-PAGE is more suitable for fractionating integral membrane proteins than 2-DGE, due to better solubilization properties of anionic detergents, there was a lack of specific visualization techniques for integral membrane proteins against a background of high-abundance hydrophilic cytoplasmic proteins. To help overcome this problem, researchers at the proteomic section of Molecular Probes, Inc. (Eugene, OR), have recently reported a novel fluorescence-based staining technology for the selective proteome-wide detection of hydrophobic integral membrane proteins on denaturing SDS-PAGE (Hart, Schulenberg, & Patton, 2004). The stain, Pro-Q Amber gel stain (Pro-Q AGS) selectively stains integral membrane proteins based on their innate amphiphilicity, and detects proteins containing two or more a-helical transmembrane domains; it does not detect lipoproteins or proteins with hydrophobic pockets.…”

Application of proteomic technology in eye research: a mini review

“…Using size‐ and charge‐matched fluorescent CyDyes, two‐dimensional fluorescence difference gel electrophoresis (2‐D DIGE) technique has been developed recently, which provides a quantitative dimension to the traditional 2D PAGE technique 14,15 . In addition, the identification of transmembrane proteins and post‐translational modified proteins, such as phosphorylated or glycosylated proteins on 2D gels are also possible using fluorescent gel staining method 16–18 …”