Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis

Abstract

Background: Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry.

Results: DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles.

Conclusion: We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Figure 1

Modification of the detergent extraction procedure eliminates 'nuclear' proteins from detergent-resistant membranes. (A) Cholesterol- and sphingolipid-enriched membranes in LNCaP/MyrAkt1 cells were stained with 0.5 μg/mL Alexa 594-CTxB for 10 min prior to staining with anti-S473-P Akt (1:100) and FITC-conjugated secondary Ab (1:100). Nuclei were counterstained with DAPI prior to imaging. Original magnification, 63×. LNCaP cells stably expressing LacZ or MyrAkt1 were extracted using the conventional (1) or modified (2) detergent extraction procedure. Equal amounts (30 μg for detection of SAFB; 10 μg for other target proteins) of Triton-soluble (TS), Triton-insoluble, octylglucoside-soluble (TI) or nuclear (N) fractions were resolved by SDS-PAGE, transferred to nitrocellulose membranes and (B) stained with Ponceau S or (C) blotted with antibodies to the HA epitope tag, phospho-S473 Akt, PCNA, SAFB, hnRNP K and G_iα2.

Figure 2

Quantitative analysis of proteins in detergent-resistant membrane fractions analyzed by mass spectrometry. (A) Proteins in DRM (TI) fractions generated using the conventional or modified methods were analyzed by tandem mass spectrometry and classified as either nuclear, non-nuclear or unannotated based on Gene Ontology cellular component terms. The pie-charts demonstrate the marked reduction in proteins identified as 'nuclear' with the modified extraction procedure. (B) Venn diagrams illustrating the distinct and overlapping protein compositions of TI fractions isolated by each method.