Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis
© Adam et al; licensee BioMed Central Ltd. 2008
Received: 24 January 2008
Accepted: 05 June 2008
Published: 05 June 2008
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.
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.
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.
Lipid rafts and caveolae, membrane microdomains that are enriched in cholesterol and sphingolipids, have been implicated in diverse physiologic mechanisms, such as signal transduction, trafficking and lipid transport [1, 2]. The invaginated and vesicular structures known as caveolae are a type of microdomain that harbor caveolin family proteins; however, non-caveolar 'flat' lipid rafts have also been shown to exist in cells that do not express caveolins . Lipid rafts were originally defined as detergent-resistant membranes (DRMs) because of their relative insolubility in cold non-ionic detergents . However, equating DRMs with lipid rafts is now believed to be inaccurate due to controversy over the biophysical and biochemical nature of rafts, and whether they even exist in vivo [3, 5–7]. In addressing these concerns, several recent papers have proposed a revision of the raft hypothesis. These studies suggest that, although DRMs do not correspond to lipid rafts as they exist in vivo, the presence of a given protein within a DRM, and its loss from that fraction when DRMs are perturbed by cholesterol depletion, for example, strongly suggests the potential for raft association of that protein in vivo [8–10]. In addition, it has been noted in a recent publication that, despite its limitations, isolation of DRMs is the only biochemical approach for assessing protein interactions with rafts .
DRMs are thought to be diverse in protein content and in functional roles. However, these membrane domains are still poorly understood and are currently being characterized using proteomics tools . The cell signaling function of DRMs is thought to arise from the ability of these microdomains to selectively retain or exclude specific proteins, resulting in the formation of multiprotein complexes that process biochemical information across specific signaling axes. Because cholesterol-rich microdomains may be functionally altered by pathophysiologic alterations in lipid metabolism , systematic analysis of proteins present in DRMs is likely to provide novel insights into multiple signaling mechanisms that operate in the normal state and in disease.
The biophysical and biochemical properties of DRMs present unique challenges to studies of their protein composition. Membrane proteins possess hydrophobic regions that render them poorly soluble during extraction [13, 14]. In addition, DRMs represent a minor and transient component of the total membrane surface. The classical method of isolating DRM-enriched fractions is by flotation of cell extracts, prepared in cold detergents such as Triton X-100 or under detergent-free conditions, in a sucrose density gradient . This approach exploits the detergent insolubility of DRMs at low temperatures as well as their light buoyant density. However, gradient centrifugation procedures are time-consuming and labor-intensive, requiring processing times on the order of 24 h. Moreover, the resultant dilute, sucrose-containing fractions have limited utility for proteomic analysis without additional processing steps .
We have employed a DRM isolation technique originally described by Solomon et al. [17, 18] that exploits the differential solubility of detergent-resistant microdomains in cold, non-ionic detergents [19–24]. This approach is rapid and yields fractions that are comparable to those isolated by flotation on density gradients. We have validated our observations from the detergent extraction procedure using alternative approaches, including sucrose density ultracentrifugation and immunofluorescence imaging [21, 23, 24]. Both unpublished and published data from us and others revealed the presence of nuclear proteins in DRM fractions isolated either by flotation in density gradients or by differential detergent solubility [16, 25–30](N.K.M. and R.M.A, unpublished observations). However, whether this unanticipated finding arose from intracellular trafficking of proteins between nuclei and DRMs, or, alternatively, was a reflection of nuclear contamination during extraction has not been determined. In this study, we describe a modification of the existing differential detergent extraction procedure that minimizes nuclear contamination of DRMs. Proteomic analysis of the resulting fractions suggests that the presence of 'nuclear' proteins in DRMs is likely to result from shuttling between the nucleus and other subcellular sites.
Results and discussion
To determine the effect of pelleting nuclei and/or intact cells on the overall protein profiles of isolated fractions, we initially compared the conventional extraction method with the modified procedure. As starting material for the extraction, we generated membrane preparations from LNCaP/MyrHA-Akt1 or LNCaP/LacZ cells in log-phase growth by mechanical disruption of cells under detergent-free conditions, as described in Methods. In the conventional method, homogenized samples were centrifuged at 16,000 × g in a Beckman microcentrifuge for 10 min at 4°C. In the modified approach, samples were centrifuged at 500 × g for 5 min at 4°C to pellet intact cells and nuclei; importantly, no brake was applied during the deceleration of the centrifuge. The supernatant was decanted carefully and centrifuged at 16,000 × g in a Beckman microcentrifuge for 10 min at 4°C to pellet membranes. The pellet remaining from the low-speed spin, comprising predominantly nuclei, was lysed in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate and 0.1% SDS plus protease and phosphatase inhibitors. In each case (conventional or modified), pellets from the high-speed centrifugation steps were then subjected to successive detergent extraction in Triton X-100 and octylglucoside to yield Triton-soluble (TS) and Triton-insoluble/octylglucoside-soluble (TI) fractions, as described in Methods.
Following determination of the protein concentration of fractions by MicroBCA assay, equal amounts of protein from each fraction were resolved by SDS-PAGE and transferred to nitrocellulose membranes. To verify the presence of protein in each fraction, membranes were stained with 0.1% (w/v) Ponceau S in 5% acetic acid (v/v) prior to blotting with the indicated antibodies. The staining pattern revealed both quantitative and qualitative differences in the overall protein profiles of TS and TI fractions generated by the two methods (Figure 1B). We confirmed the distribution of MyrAkt1 in TS, TI and nuclear (N) fractions by HA immunoblot, and observed robust enrichment of MyrAkt1 in TI fractions in both cases (Figure 1C). A small amount of MyrAkt1 was detected in the nuclear pellet from the low speed centrifugation, consistent with previous reports . To determine the impact of the modified extraction procedure on protein distribution, we probed the membranes for selected proteins with defined subcellular localizations, including proliferating cell nuclear antigen (PCNA), a nuclear protein  and the small G-protein, Giα2, that resides in lipid rafts [34, 35]. As shown in Figure 1C, PCNA, although present in the TI fraction following the conventional extraction method, was undetectable in TI fractions prepared with the modified approach. As anticipated, the nuclear fraction showed robust signal corresponding to PCNA. In contrast, Giα2 was present in TI fractions generated with either extraction method, consistent with its localization to DRMs. These data were reproducible across three independent biological replicates and similar findings were obtained with fractions isolated from LNCaP cells stably expressing the irrelevant gene (LacZ). We also probed for two additional proteins that were identified as putative raft-resident binding partners of MyrAkt1 transiently expressed in HEK293 cells (N.K.M. and R.M.A, unpublished observations): scaffold attachment factor B (SAFB), a protein implicated in transcriptional regulation and chromatin organization ; and heterogeneous ribonucleoprotein K (hnRNP K), a pleiotropic nucleic acid binding protein that regulates gene and protein expression [37, 38]. Although SAFB was present in TI fractions generated by conventional means, it was absent from this material following elimination of nuclei, strongly suggesting that its presence in the TI fraction arose from nuclear contamination. In contrast, hnRNP K was detected in TS and TI fractions with both extraction methods, as well as in nuclei. These observations are consistent with localization of hnRNP K to multiple distinct subcellular sites .
Proteins identified by tandem mass spectrometry.
(A) Proteins associated with DRMs
VAMP (Vesicle-associated membrane protein)-associated protein B and Ca
YES tyrosine kinase
Ras-related protein Rap-1b
Tubulin beta-4q chain
Splice Isoform 1 of Fibronectin precursor
[16, 25, 39]
[16, 29, 39, 40]
GPI-anchored protein p137a
Mitogen-activated protein kinase kinase 1 interacting protein 1
Alpha subunit of GsGTP binding protein
Voltage-dependent anion-selective channel protein 1
Ubiquinol-cytochrome c reductase complex 14 kDa protein
G-protein gamma-12 subunit variant
Stomatin-like protein 2
Guanine nucleotide-binding protein G(i) alpha-1 subunit
Heat shock 70 kDa protein 6
ATP synthase alpha chain, mitochondrial precursor
Ubiquinol-cytochrome c reductase complex 7.2 kDa protein
(B) Nuclear proteins shown to exist in other subcellular locations
Evidence for shuttling
Splice Isoform 1 of Programmed cell death protein 8, mitochondrial precursor
Nucleoside diphosphate kinase A
Ras-GTPase-activating protein binding protein 1
Fragile X mental retardation syndrome-related protein 1
Fragile X mental retardation syndrome-related protein 2
40S ribosomal protein S6
Transcriptional activator protein PUR-alpha
FK506-binding protein 3
39S ribosomal protein L19, mitochondrial precursor
Yes: UniProt 49406
Mitochondrial import inner membrane translocase subunit TIM8 A
Similar to heterogeneous nuclear ribonucleoprotein A1 isoform a
Polymerase delta interacting protein 2
DEAD-box protein 3, X-chromosomal
40S ribosomal protein S19
Splice Isoform 5 of Interleukin enhancer-binding factor 3
Splice Isoform 1 of Plasminogen activator inhibitor 1 RNA-binding protein
Similar to Laminin receptor 1
Yes: UniProt P08865
Interestingly, of the 21 proteins annotated as nuclear in localization, that were found exclusively in the modified TI fraction, evidence from the literature and protein databases suggests that 16 of these are also present in additional subcellular locations, and may shuttle between the nucleus and other sites in the cell [46–58]. Thus, only 5 proteins (shaded in Table 1B), out of a total of 156 specifically identified in the modified TI fraction in this experiment, were present exclusively in nuclei. In addition, at least 60% of the 33 nuclear proteins identified with both approaches are also present in other subcellular locations. In contrast, only about 30% of the 108 nuclear proteins specifically identified in the conventional TI fraction are annotated to be present in additional locations.
These findings strongly suggest that the modified DRM extraction method generates fractions that are largely free of nuclear contamination. They also imply that, given the high frequency with which so-called 'nuclear proteins' are found in other organelles, due to shuttling between compartments, nuclear proteins are likely to be detected by most extraction procedures, even when performed under the most stringent conditions. Canonical nuclear proteins, such as nuclear hormone receptors, can shuttle between the cytoplasm and nuclear compartments as part of their hormone-dependent functions. Recently, the androgen receptor was found to process androgenic signals non-genomically by forming a complex with Akt1 in lipid raft microdomains . The identification of such proteins in DRMs may thus reflect a legitimate physiologic process and not an experimental artifact.
In summary, we describe a rapid, robust method for isolation of detergent-resistant membrane fractions that are compatible with downstream proteomic analyses with minimal additional processing. The present approach allows for a significant reduction of nuclear protein contamination of DRM samples. We believe the results presented here indicate that the DRM isolation procedure we describe can be applied with confidence to settings where a robust characterization of DRM constituents by mass spectrometry is desired, and also to more traditional biochemical methods involving antibody-based analyses.
Membrane preparations and isolation of detergent resistant membrane fractions
Populations of LNCaP/MyrHA-Akt1 or LNCaP/LacZ cells in log-phase growth were rinsed twice with phosphate-buffered saline (PBS), resuspended in Buffer M [50 mM HEPES pH 7.4, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA plus protease inhibitor cocktail (Complete Mini, Roche Applied Science), 1 mM Na3VO4, 1 mM NaF and 1 mM Na4P2O7.10 dH2O] and homogenized at 4°C using a Potter-Elvehjem tissue grinder (12 strokes, 1800 rpm). 107-108 cells were used for each extraction method. High-speed (16,000 × g) pellets from either the conventional or modified approach were subjected to successive detergent extraction essentially as described [19, 21, 24]. Briefly, pellets were resuspended in Buffer A [25 mM 2-(N-morpholino)-ethanesulfonic acid, 150 mM NaCl, pH 6.5] and samples combined with an equal volume of Buffer A containing 2% Triton X-100, and phosphatase and protease inhibitors. Samples were incubated on ice for 60 min, centrifuged at 16,000 × g for 20 min at 4°C and supernatants collected as Triton-soluble (TS) material. Pellets were rinsed briefly with Buffer A and resuspended in Buffer B [10 mM Tris-Cl, pH 7.6, 150 mM NaCl, 60 mM β-octylglucoside and phosphatase and protease inhibitors]. Samples were incubated on ice for 30 min, centrifuged at 16,000 × g for 20 min at 4°C and supernatants collected as Triton-insoluble (TI) material.
Tryptic Digestion and Mass Spectrometry Analysis
Four micrograms of each TI fraction was subjected to in-solution tryptic digestion. Three MS experiments were performed: TI fraction from the conventional extraction procedure; TI fraction from the modified extraction procedure; and extraction buffer control with a single replicate for each. Approximately 2 μL protein extracts or extraction buffer as control, were mixed with 8 μL 0.2% RapiGest™ SF (Waters Corporation, Milford, MA) in 50 mM ammonium bicarbonate, boiled for 5 min and cooled to room temperature. Dithiothreitol was added to 5 mM final concentration, samples heated at 60°C for 30 min and cooled to room temperature. Iodoacetamide was added to 15 mM final concentration and samples were incubated protected from light for 30 min prior to addition of 100 ng MS-grade trypsin and further incubation at 37°C for 2 h. To hydrolyze the RapiGest™ surfactant, trifluoroacetic acid was added to a final concentration of 0.5% and samples were incubated at 37°C for 45 min. Following centrifugation at 14,000 × g for 10 min, the supernatants were transferred to screw-top vials. Acetonitrile and acetic acid were added to a final concentration of 7.5% and 1.5%, respectively. Samples were analyzed by online C18 nanoflow reversed-phase HPLC (Eksigent nanoLC·2D™) hyphenated to a Thermo Scientific LTQ Orbitrap mass spectrometer. The digest was loaded onto a 100 μm i.d. × 15 cm C18 column and the peptides were separated at 200 nL/min with 80 min gradients from 5 to 31.5% acetonitrile in 0.4% formic acid. Survey spectra were acquired in the orbitrap with the resolution set to a value of 30,000. Up to five of the most intense ions per cycle were fragmented and analyzed in the linear trap.
Database searching was performed using the Mascot search engine (Matrix Science, v.2.1). All MS datasets were searched against the International Protein Index (IPI) human protein database of the International Protein Index (v3.10; 57479 sequences). Protein modifications were selected as carbamidomethyl (C) (fixed) and oxidation (M) (variable). Up to one missed cleavage was allowed. The mass tolerance was set as ± 20 ppm for MS spectra and ± 0.5 Da for MS/MS spectra. The following criteria were used to generate a high-confidence data set: at least two peptides must be identified for each protein, with a score not less than 33, corresponding to a significance level of p < 0.01 as determined by reverse database searching. Known contaminants (e.g. keratins and trypsin) were removed from the protein lists. Applying these parameters, no false positives were identified through searching the reversed IPI_Human database.
The authors thank Dr. Michael Freeman, Dr. Heidi Rayala and Dr. Keith Solomon for helpful discussions. This work was funded in part by NIH grant R21 DK66412 (to R.M.A.), the Children's Urological Foundation (to R.M.A.) and the American Italian Cancer Foundation (to D.D.V.).
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