Lens Proteomic Analysis of Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells

Zhen Wang and Kevin L. Schey

Department of and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, United States

Correspondence: Kevin L. Schey, PURPOSE. Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty Department of Biochemistry, Mass acids, , and sphingolipids—key components of lipid rafts. Thus, lipid rafts are Spectrometry Research Center, PMB expected to constitute a significant portion of fiber cell membranes and play important roles 407916, Nashville, TN 37240-7916, in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. USA; [email protected]. METHODS. Quantitative proteomics, both label-free and iTRAQ methods, were used to Submitted: September 25, 2015 characterize lens fiber cell lipid raft . Detergent-resistant, lipid raft membrane (DRM) Accepted: November 24, 2015 fractions were isolated by sucrose gradient centrifugation. To confirm localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-b-cyclodextrin was quantified Citation: Wang Z, Schey KL. Proteo- by iTRAQ analysis. mic analysis of lipid raft-like detergent- resistant membranes of lens fiber RESULTS. A total of 506 proteins were identified in raft-like detergent-resistant membranes. cells. Invest Ophthalmol Vis Sci. Proteins identified support important functions of raft domains in fiber cells, including 2015;56:8349–8360. DOI:10.1167/ trafficking, , and cytoskeletal organization. In cholesterol-sensitivity iovs.15-18273 studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains.

CONCLUSIONS. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on organization in these cells. Keywords: proteomics, mass spectrometry, lipid raft

ell membranes are composed of a complex array of and it is generally regarded as a key lipid component of lipid C and these lipids do not distribute randomly throughout the rafts.8 membrane but, instead, form distinct membrane domains via Plasma membranes of lens fiber cells are distinguished from lipid-lipid and lipid-protein interactions.1 The lipid raft hypoth- other eukaryotic cell membranes by their unique lipid esis was initially proposed in 1997 by Simons and Ikonen2 as a composition. Lens fiber cell membranes contain high concen- principle of membrane subcompartmentalization. Later in 2006 trations of long-chain saturated fatty acids and high abundances at the Keystone Symposium on Lipid Rafts and Cell Function, of sphingomyelin and cholesterol.9–11 This unique lipid lipid rafts were defined as small, heterogeneous, highly composition gives rise to a unique lens fiber dynamic membrane microdomains (10–200 nm) that are that is important for lens transparency.11–13 The abundance of enriched in cholesterol and sphingolipids and that compart- sphingomyelin and cholesterol contributes to lens membrane mentalize cellular processes.3 As is now increasingly appreci- rigidity and structural order. Considering this unusually high ated, lipid rafts may play important roles in various cellular concentration of cholesterol and sphingolipid, it is expected processes, such as signal transduction, membrane trafficking, that lipid raft domains are highly abundant and play important cell adhesion, cytoskeletal rearrangement, and many other roles in lens fiber cell membrane. Evidence for immiscible membrane functions.4 cholesterol-rich and -poor domains in normal lens fiber cell The concept of lipid sorting into membrane microdomains membrane was reported,14 and cholesterol-rich domains were was initially introduced to explain the generation of the more pronounced and better defined in cataractous lens.15 glycosphingolipid-rich apical membrane in epithelial cells.5 Previously, lipid raft domains were isolated and lipids13 and Later, this idea was expanded to include cholesterol-rich raft -116 content was examined. Sorting of AQP0 and microdomains.2,6,7 Based on the lipid raft hypothesis,2 sphin- connexins to raft and nonraft domains was also reported.17 golipids associate laterally with one another in the exoplasmic However, the structure and function of lipid rafts in lens fiber leaflet and any void space is filled with cholesterol. Cholesterol cells are largely unknown. is also present in the cytoplasmic leaflet and fills the void space Lipid rafts have been isolated from a variety of tissues relying created by interdigitating fatty acid chains. Cholesterol is on their insolubility in nonionic detergents at low temperature. known to increase the thickness and stiffness of lipid bilayers Although detergent insolubility in itself does not accurately

Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. iovs.arvojournals.org j ISSN: 1552-5783 8349

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reflect preexisting raft domains, studying detergent-resistant hours. After digestion, the sample was dried by a speedvac membranes (DRMs) has produced results consistent with other (Thermo Fisher, Milford, MA, USA) and reconstituted in 0.1% methods, such as direct imaging.18,19 Thus, detergent resis- formic acid. The supernatant was collected by centrifuging at tance remains an interesting and useful tool for assigning 20,000g for 5 minutes and the remaining pellets were potential membrane raft association. In this report, we isolated extracted by 50 lL 99.9% acetonitrile (ACN), 0.1% formic DRM from bovine lens fiber cells and performed quantitative acid. The ACN extract was dried by a speedvac and proteomic studies of DRM and detergent-soluble membrane reconstituted in 5% ACN (0.1% formic acid). Equal volumes (DSM) fractions. Our results demonstrated that a significant of ACN extract and 0.1% formic acid extract were mixed before percentage of AQP0, MP20, AQP5, and a small fraction of liquid chromatography–tandem mass spectrometry (LC-MS/ connexin 50 and connexin 46 reside in lipid raft domains. MS) analysis. For isotope-coded tags for relative and absolute quantitation (iTRAQ) analysis, fractions 3 and 4 were diluted 15 times by MATERIALS AND METHODS water and centrifuged at 200,000g for 30 minutes. The pellets were washed twice with water and reconstituted in 500 mM Bovine lenses (1 year or older) were obtained from PelFreez triethylammonium bicarbonate (TEAB; pH 8.0) containing 10% Biologicals (Rogers, AK, USA). Sequence-grade modified ACN. Proteins were reduced with 50 mM TCEP (Tris-[2- trypsin was obtained from Promega (Madison, WI, USA). Brij carboxyethyl]phosphine) at 608C for 1 hour, alkylated with 200 98 was purchased from Acros Organics (Morris Plains, NJ, mM MMTS (methyl methanethiosulfonate) at room tempera- USA). Methy-b-cyclodextrin and other chemicals were pur- ture for 10 minutes, and digested with sequencing-grade chased from Sigma-Aldrich Corp. (St. Louis, MO, USA). All trypsin overnight. Peptides were then labeled with iTRAQ HPLC grade solvents were purchased from Fisher (Fair Lawn, reagents according to the manufacturer’s instructions (AB NJ, USA). Sciex, Foster City, CA, USA). Labeling reagents were reconsti- tuted in ethanol such that each protein sample was labeled at a Preparation of Detergent-Resistant Membrane final concentration of 90% ethanol and labeling was performed for 2 hours. The 35 mM MbCD-treated sample was labeled with Frozen bovine lenses were decapsulated and dissected into the 114 iTRAQ reagent and the untreated sample was labeled cortex (the superficial soft tissue) and nucleus (the remaining with 116 iTRAQ reagent. The iTRAQ-labeled MbCD-treated hard gummy tissue) before homogenization. Tissue was sample and one-fifth of iTRAQ-labeled untreated sample were homogenized in homogenizing buffer (200 mM sucrose, 150 mixed, acidified with trifluoroacetic acid, and were subse- mM NaCl, 50 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol [DTT], quently desalted by a modified Stage-tip method20 before LC- 1 mM phenylmethylsulfonyl fluoride [PMSF] in 10 mM Hepes) MS/MS analysis. and centrifuged at 100,000g for 20 minutes and the superna- tant was discarded. The pellets were resuspended in homog- Liquid Chromatography–Electrospray Ionization/ enizing buffer, divided into two samples, and centrifuged at 100,000g for 20 minutes; 1 mL homogenizing buffer, with or MS/MS Analysis without 15 mM or 35 mM methyl-b-cyclodextrin (MbCD), was Tryptic peptides were either directly separated on a one- added to the pellets, and the samples were vortexed and dimensional fused silica capillary column (20 cm 3 100 lm) incubated at 378C for 30 minutes. The samples were then packed with Jupiter resin (3-lm mean particle size, 300 A˚ pore further centrifuged and the pellets were collected; 500 lL1% size; Phenomenex, Torrance, CA, USA) or analyzed by Brij 98 and 15 mM octylglucoside (OG) in homogenizing buffer MudPIT.21 One-dimensional liquid chromatography was used was added to each pellet and incubated on ice for 30 minutes. with the following gradient at a flow rate of 0.5 lL/min: 0 to 2 Each sample was then mixed with 500 lL 83.2% sucrose in minutes: 2% ACN (0.1% formic acid), 2 to 70 minutes: 2% to homogenizing buffer containing 1% Brij 98 to make final 35% ACN (0.1% formic acid), 70 to 90 minutes: 35% to 90% sucrose concentration of 45% and loaded at the bottom of the ACN (0.1% formic acid) balanced with 0.1% formic acid. The centrifuge tube. Thirty-five percent and 5% sucrose prepared in eluate was directly infused into a Velos Pro linear ion trap mass homogenizing buffer were layered on the top of the sample spectrometer (ThermoFisher, San Jose, CA, USA) equipped sequentially and centrifuged at 160,000g using a SW55Ti rotor with a nanoelectrospray source. For MudPIT analysis, peptides in a Beckman L90K ultracentrifuge (Fullerton, CA, USA) for 18 were loaded onto a custom packed biphasic C18/SCX trap hours at 48C. Ten fractions (0.5 mL/fraction) were collected column (4 cm 3 150 lm, Jupiter C18, 5 lm, 300 A˚ media across the sucrose gradient and fractions 3 to 10 were used for followed by 4 cm 3 150 lm, Luna SCX, 5 lm, 100 A˚ media; further analysis. Phenomenex). The trap column was coupled to a capillary analytical column (20 cm 3 100 lm, Jupiter C18, 3 lm, 300 A˚ Preparation of Samples for MS Analysis media). MudPIT analysis was done with a 13-step salt pulse gradient (0 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 Proteins in 100 lL each fraction were precipitated by mM, 250 mM, 300 mM, 500 mM, 750 mM, 1 M, and 2 M chloroform/methanol and the resulting protein pellets were ammonium acetate). Following each salt pulse, peptides were solubilized in 1% SDS for protein assay. The total protein eluted from the analytical column with a 90-minute reverse- concentration was measured by a bicinchoninic acid assay phase solvent gradient (2%–45% ACN, 0.1% formic acid; 2%– (BCA assay; Thermo Scientific, Rockford, IL, USA). For 95% ACN, 0.1% formic acid for last two salt pulses). The eluate proteomic analysis, 200 lL from each fraction was reduced was directly electrosprayed into a Velos Pro mass spectrometer with DTT (final concentration of 10 mM) at 568C for 1 hour (ThermoFisher). The instrument was operated in a data- and alkylated with iodoacetamide (final concentration 50 mM) dependent mode with one precursor scan event to identify at room temperature for 45 minutes. The samples were the top 15 most abundant ions in each MS scan, which were concentrated in a Speedvac to 100 lL and proteins in each then selected for fragmentation. Dynamic exclusion (repeat sample were then precipitated by chloroform/methanol. The count 1, exclusion list size 300, and exclusion duration 15 resulting proteins were suspended in 10% acetonitrile in 50 seconds) was enabled to allow detection of less abundant ions. mM Tris buffer, pH 8.0. Trypsin was added (enzyme/substrate For quantification of fatty acylated AQP0, the samples were run ¼ 1/100; Promega), and the sample was digested at 378C for 18 on a one-dimensional column as above with modified gradient

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picking and quantitation. At least seven product ions were used for each peptide and the total peak area from all product ions selected was normalized to the total peak area from all product ions of the AQP0 peptide 188 to 196. For iTRAQ data analysis, mass spectra were processed using the Spectrum Mill software package (version B.04.00; Agilent Technologies, Santa Clara, CA, USA). The MS/MS spectra acquired on the same precursor m/z (60.01 m/z) within 61 second in retention time were merged. The MS/MS spectra of poor quality that failed the quality filter by not having a sequence tag length greater than 1 were excluded from searching. A minimum matched peak intensity require- ment was set to 50%. For peptide identification, MS/MS spectra were searched against a Uniprot bovine database (Oct 18, 2013). Additional search parameters included trypsin enzyme specificity with a maximum of three missed cleavages, 620 ppm precursor mass tolerance, 620 ppm (HCD) product mass tolerance, and fixed modifications including MMTS alkylation of cysteines and iTRAQ labeling of lysines and peptide N-termini. Oxidation of methionine was allowed as a variable modification. FIGURE 1. The relative protein abundance in DRM fractions prepared Autovalidation was performed such that peptide assignments to using 1% Triton X-100 or 1% Brij 98. The DRMs were prepared using mass spectra were designated as valid following an automated either 1% Triton X-100 plus 15 mM OG or 1% Brij 98 plus 15 mM OG as procedure during which score thresholds were optimized described in the Materials and Methods section. Proteins at the 5% to separately for each precursor charge state and the maximum 35% sucrose interface were digested by trypsin and analyzed by LC-MS/ target-decoy–based FDR was set to 1.0%. To obtain iTRAQ MS. The abundance of each protein was represented as the peak area of protein ratios, which represent control/MbCD-treated ratios, the one peptide from this protein. The relative abundance of a certain protein in two different detergent-treated samples was plotted as a median was calculated for all peptides assigned to each protein. ratio of the peak areas. Because only one-fifth of the control sample was used, the final iTRAQ ratio was obtained by multiplying by a factor of 5. (0–50 minutes: 2%–30% ACN, 50–65 minutes: 30%–95% ACN, 65–80 minutes: 95% ACN balanced with 0.1% formic acid) and RESULTS the mass spectrometer was set to acquire one MS1 scan followed by targeted MS/MS scans for fatty acylated AQP0 Isolation of Lipid Raft-Like DRMs peptides and unmodified AQP0 peptides. The iTRAQ-labeled samples were analyzed using MudPIT analysis as described The most commonly used method for isolation of raft-like above with eight salt pulse steps (0, 50 mM, 100 mM, 200 mM, DRMs uses the nonionic detergent Triton X-100; however, 300 mM, 500 mM, 1 M, and 2 M ammonium acetate). Peptides sucrose gradient centrifugation of the lens Triton X-100– were introduced via nano-electrospray into a Q Exactive mass resistant fraction yields only trace amounts of DRM from lens 16 spectrometer (Thermo Scientific, San Jose, CA, USA). The Q fiber cell membrane. Consistently, we found that there was Exactive was operated in data-dependent mode acquiring no visible band at the 5% to 35% sucrose interface if the fiber higher-energy collisional dissociation (HCD) MS/MS scans (R ¼ cell membrane was solubilized with 1% Triton X-100. 17,500) after each MS1 scan (R ¼ 70,000) on the 18 most Therefore, octyl-b-glucopyranoside (OG) was included in the 16 2 abundant ions using an MS1 ion target of 1 3 106 ions and an preparation as previously reported. In addition, Mg þ and Kþ MS2 target of 1 3 105 ions. The maximum ion time for MS/MS ions were included in the detergent solubilization buffer as scans was set to 100 ms, the HCD-normalized collision energy described previously by Chen et al.25 to mimic the intracellular was set to 26, dynamic exclusion was set to 30 seconds, and environment and stabilize the inner leaflet of the membranes peptide match and isotope exclusion were enabled. during detergent solubilization. Even though Triton X-100 was the preferred detergent for Data Analysis membrane raft isolation, Triton X-100 was also proposed to potentially induce the artificial formation of detergent-resistant Tandem mass spectra were analyzed using a suite of custom- structures.26 A milder detergent, the polyoxyethylene ether developed bioinformatics tools. All MS/MS spectra were Brij 98, has been used for solubilizing nonraft domains because converted to mzML files by Scansifter, a tool under development its mono-unsaturated ether moiety was predicted to preferen- at Vanderbilt University Medical Center, and searched on a 2500- tially solubilize loosely packed fluid phase lipids.27,28 Advan- node Linux cluster supercomputer using a custom version of tages of Brij 98 include isolation of lipid rafts at 378C and the TagRecon algorithm.22 Trypsin specificity was used with a reduced aggregation of detergent-insoluble membranes follow- maximum two missed cleavage sites. The data were searched ing detergent extraction. Thus, we tested both Triton X-100 against a Uniprot bovine database (October 18, 2013) with a and Brij 98 in combination with OG for isolation of DRM. The static modification of carbamidomethylation of cysteine residues fraction at the 5% to 35% sucrose interface was collected from and variable modifications of oxidation of methionine and both preparations, and proteins in this fraction were analyzed deamination of N-terminal glutamine residues. The search by MS. Figure 1 shows the relative abundance of several results were filtered by IDPicker23 by controlling protein false- abundant lens proteins and raft marker proteins in the Triton X- discovery rate (FDR) to less than 1%. For quantitative peak area 100–treated sample compared with the Brij 98–treated sample. measurements, selected ion chromatograms were generated and The Brij 98–treated sample yielded higher amounts of raft peaks areas were calculated using the Genesis peak algorithm marker proteins (FLOT1, FLOT2, ANK2, CXADR, CAV1) in the within the Xcalibur software 2.2 SP 1.48 (ThermoFisher). For high-buoyancy fraction compared with the Triton X-100– quantification of the relative abundance of fatty acylated AQP0, treated sample, but did not significantly increase the presence the resulting raw files were imported into Skyline24 for peak- of soluble putative nonraft proteins, such as crystallins

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FIGURE 2. The total protein concentration in different fractions collected from raft-like DRM preparations using 1% Brij 98 plus 15 mM OG. Proteins in 100 lL each fraction were precipitated by chloroform/methanol. The resulting pellets were solubilized in 1% SDS and protein concentration was measured by BCA assay. The average protein concentration of each fraction from two experiments was plotted. Open circles: protein concentration; filled circles: protein concentration after MbCD treatment; filled squares: density.

(CRYAA, CRYAB), and cytoskeletal proteins, such as and . In addition, the Brij 98 treatment dramatically increased the amount of AQP0 and AQP5 in the low-density fraction. Increasing evidence supports the lipid raft localization of AQP5,29–31 whereas the localization of AQP0 in lipid rafts has been reported previously.16,17 These results suggest that Brij 98 provides better yields of lipid raft marker proteins in the low-density fractions; therefore, further studies were per- FIGURE 3. Distribution of lipid raft markers across sucrose density formed only on DRM isolated after Brij 98 solubilization. gradient. Proteins in each fraction were digested by trypsin and In the course of method optimization, we noticed that the analyzed by LC-MS/MS. The data were searched against a Uniprot detergent-protein ratio affects the amount of proteins in the bovine database. The spectral count from each protein was normalized high buoyancy fractions (data not shown). During detergent to the total spectral count and the relative abundance of each protein was represented using the normalized spectral count. The y-axis shows solubilization, the total protein concentration was controlled the normalized spectral counts for some raft marker proteins. to be 3 mg/mL. Under this condition, the total protein concentration of different fractions collected across the sucrose density gradient is shown in Figure 2. Fractions 4 Identification of Lens Lipid Raft-Like DRM Proteins and 5 correspond to the 5% to 35% sucrose interface. Only a by LC-MS/MS small fraction of the total proteins was present in the high- buoyancy fractions (fractions 3–5); however, the detergent- To analyze proteins that are present in the high-buoyancy DRM soluble fractions were dominated by highly abundant soluble fractions, fractions 3 and 4 were pooled, digested by trypsin, crystallins. As expected, removing cholesterol by MbCD and analyzed by MudPIT. Fraction 5 typically had higher significantly reduced the yield of raft-like DRM and shifted protein concentrations than fraction 4 and contained signifi- proteins from the DRM to the DSM. cant amounts of raft marker proteins; however, the normalized To evaluate whether our preparation yielded bona fide raft- spectral counts for most raft markers were lower in fraction 5 like DRM, the distribution of several lipid raft markers across than fraction 4, indicating contamination with nonraft pro- different sucrose density gradient was studied. The relative teins. Therefore, only fractions 3 and 4 were used for abundance of a protein in each fraction was represented by its identifying proteins in rafts to reduce contamination by nonraft normalized spectral count in the LC-MS/MS data; a rough proteins. This experiment was repeated twice. In total, there estimate of protein abundance.32 This experiment was were 598 proteins detected in the pooled high-buoyancy lipid repeated three times. Consistently, many lipid raft markers raft fraction (Supplementary Table S1) and 506 proteins (84%) were repeatedly detected in the DRM faction, such as flotillin 1 detected in both analyses. Functional and localization annota- and 2, erlin 1 and 2, G proteins, and 2, HRAS, and tions were assigned for 506 proteins that were identified in KRAS. Using this preparation, most of these proteins are highly both analyses using the information in the Uniprot database enriched in lipid raft-like low-density DRM, and are much less (http://www.uniprot.org, in the public domain) as well as after abundant or not detected in the DSM fractions. The normalized analysis by the DAVID Bioinformatic Resources.33 A search for spectral counts for some raft markers in different fractions membrane raft proteins in the Uniprot database yielded 251 showed a similar trend as shown in Figure 3. Some of these unique proteins and 40 of these proteins were detected in the proteins, such as KRAS, and GNAI2, have bimodal distribu- DRM fraction. These proteins are listed in the Table 1. Among tions, suggesting their presence in both DRM and DSM the detected 506 proteins, 79 proteins are listed as lipidated membranes. proteins and 140 proteins (27.7%) were listed as glycosylated

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TABLE 1. Lipid Raft Proteins Detected in DRM Fractions of Lens Fiber 14.8% (75 proteins) can be found in the raft proteome full list Cells including some well-studied raft proteins, such as AQP5, ANK2, KRAS, and STX2. Seventy-two proteins from our lens Name Protein Description study are not present in the lipid raft database, including 15 ANXA2 Annexin A2 proteins that are uniquely expressed in the lens. BSG Basigin Pathway analysis was also carried out using DAVID 33 CAV1 Caveolin-1 Bioinformatic Resources. The top enriched GO terms and CDH2 Cadherin-2 pathways are listed in Figure 4. Combing both cell membrane CTSD Cathepsin D and organelle membrane lists, most proteins (75%) detected in CXADR Coxsackievirus and adenovirus receptor our study localize to membranes. Similar to previous reports, homolog mitochondrial and (ER) proteins were DAG1 also detected.35–37 Results from enriched biological process EFNB1 Ephrin-B1 and pathway analysis provides evidence that supports impor- EGFR Epidermal growth factor receptor tant raft-related functions, such as membrane transport and ERLIN1 Erlin-1 trafficking,38,39 signal transduction,40,41 cell junctions,42,43 and ERLIN2 Erlin-2 organization.41,44 EZR Ezrin FAIM2 Protein lifeguard 2 Cholesterol Dependence FLOT1 Flotillin-1 FLOT2 Flotillin-2 Lens fiber cell plasma membranes are unique due to their GJA1 Gap junction alpha-1 protein extremely high relative content of cholesterol.10,45 Cholesterol GJA3 Gap junction alpha-3 protein represents up to 40% of the total lipid10 and the cholesterol-to- GNAI1 Guanine nucleotide-binding protein G(i) subunit phospholipid molar ratio ranges from 1 to 4 in the lens plasma alpha-1 membrane.16,45 Both 5 mM and 10 mM MbCD treatment, GNAI2 Guanine nucleotide-binding protein G(i) subunit commonly used to remove cholesterol, removed only a small alpha-2 fraction of cholesterol from lens fiber cell membranes as GNAI3 Guanine nucleotide binding protein, alpha evidenced by the presence of the high-buoyancy band at the inhibiting activity polypeptide 3 5% to 35% sucrose interface (data not shown). Therefore, a GPC1 Glypican-1 series of concentrations of MbCD was tested for cholesterol GSN removal. The results indicate that cholesterol can be depleted ITGB1 Integrin beta-1 with 35 mM MbCD treatment under our experiment condi- KRAS GTPase KRas tions. Considering that MbCD treatment could remove LAMTOR1 Ragulator complex protein LAMTOR1 cholesterol in nonraft domains46 and 15 mM MbCD treatment LAT Blood–brain barrier large neutral amino acid can remove the high-buoyancy band at the 5% to 35% sucrose transporter interface, 15 mM MbCD was used for most of our studies; 35 PAG1 Phosphoprotein associated with mM MbCD was used for iTRAQ experiments to ensure removal glycosphingolipid-enriched microdomains 1 of any residual lipid raft cholesterol. PGK1 Phosphoglycerate 1 To quantify the effect of cholesterol removal on the protein PRNP Major prion protein content of the DRM fraction and, thereby, validate the lens PSEN1 Presenilin-1 lipid raft proteome, a quantitative proteomics approach was RAB5A Ras-related protein -5A taken. Similar to a previous report from the M. Mann group,47 RALA Ras related v-ral simian leukemia viral oncogene the iTRAQ method was used. Samples were extracted with 35 homolog A mM MbCD or homogenizing buffer (control) before detergent Ras-related protein Rap-2b RAP2B solubilization. Pooled sucrose gradient fractions 3 and 4 were Erythrocyte band 7 integral membrane protein STOM digested and analyzed. Liquid chromatography MS/MS analysis STOML2 Stomatin-like protein 2, mitochondrial was used to produce sequence-specific ions for protein STX12 -12 identification and iTRAQ reporter ion signals for quantification; STX2 Syntaxin-2 200 proteins were quantified and the detailed list can be found TMED10 Transmembrane emp24 domain-containing in Supplementary Table S2. Among the 200 proteins quanti- protein 10 fied, 170 were detected in the label-free MudPIT analysis. TMED2 Transmembrane emp24 domain-containing protein 2 Plotting the control/MbCD-treated ratio did not show distinct 47 VDAC2 Voltage-dependent anion-selective channel discontinuities in iTRAQ ratios as previously reported protein 2 (Supplementary Fig. S1); however, the top 71 proteins had control/MbCD-treated ratios greater than 25 and included important lipid raft markers such as flotillins, KRAS, STX2, ezrin, integrin, and major prion protein. Table 2 lists the top 71 proteins in the Uniprot protein database (shown in the proteins as well as their predicted cellular localization and Supplementary Table S1). Recently, a mammalian raft pro- modifications. Most of these proteins localize to plasma teome database was established based on multiple proteomic membrane and some of them are cytoskeletal proteins that 34 studies in a variety of tissue or cells. In this database, a high- are modified by glycosylation or lipidation. Many proteins in confidence lipid raft protein list was generated to include only this list are involved in cell junctions. The sedimentation of proteins that are sensitive to cholesterol depletion or proteins several major lens proteins present in the high-buoyancy, low- that are detected in raft domains by more than one biochemical density DRM fractions, including AQP0 and connexins, was method.34 A comparison of the proteins detected in this study very sensitive to cholesterol removal. Furthermore, although with the proteins in the raft proteome database revealed that peptides from lens membrane intrinsic protein MP20 (LIM2) 70.9% (359 proteins) of the proteins detected in the DRM of were not detected by MudPIT analysis, a complementary one- lens fiber cells are present in the high-confidence list. Another dimensional LC-MS/MS experiment showed MP20 had a higher-

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FIGURE 4. Enriched GO categories and pathways. Enriched GO categories and pathways in raft-like DRM fractions were analyzed using David Bioinformatics Resources; P < 0.0001 was considered enriched GO categories. Selected enriched categories were plotted with categories that have more proteins on the top.

control/MbCD-treated ratio than AQP0, indicating a higher the higher-density DSM fractions without MbCD treatment. A sensitivity to cholesterol removal (data not shown). lower percentage of connexin 50 (GJA8) is present in the DRM Table 3 lists the proteins that have a control/MbCD-treated fractions compared with AQP0; however, this portion of ratio less than 15. The proteins in this list mainly localize to connexin 50 shifts to the DSM fraction on MbCD treatment. cytoplasm and organelle membranes, such as nucleus, Connexin 46 followed the same trend as connexin 50 (data not , ER, and Golgi membranes. Two widely used shown). nonraft marker proteins, calnexin and transferrin receptor protein 2, appear in Table 3. Proteins in Table 3 are weakly Fatty Acylation of AQP0 affected by cholesterol removal and, therefore, are most likely nonraft proteins that cosedimented with raft proteins. Proteins Previously we reported that AQP0 undergoes palmitoylation that have control/MbCD-treated ratios between 15 and 25 and oleoylation on Met1 and K238 residues through an amide include some raft marker proteins such as -2, several G bond and that modified AQP0 cannot be extracted by Triton X- proteins, and Rab family proteins (RAB9A, RALB, RAP2B). 100, suggesting its possible localization with lipid raft Several proteins involved in SNARE interaction in vesicular domains.48 In this study, we quantified the level of oleic transport are also in this group, such as SNAP23, STX7, VAMP2, acid–modified AQP0 relative to total AQP0 signal across the and VAMP5. This group also includes some major lens-soluble sucrose gradient (Fig. 6). The result shown in Figure 6 is an proteins, such as alpha B crystallin and several beta crystallins. average of three analyses of three sets of samples. Oleic acid– We interpret the presence of these soluble crystallins in lipid modified (acylated) AQP0 on Met1 and K238 was highly raft fractions and with control/MbCD-treated ratios that are enriched in fraction 4 and fraction 5, respectively. After MbCD somewhat sensitive to cholesterol removal as most likely due treatment, fatty acylated AQP0 also moved to the DSM to association with lipid raft proteins. fractions. This result further confirmed that fatty acylation of The iTRAQ experiment provided information about how AQP0 acts as one of the signals to target AQP0 to lipid raft cholesterol removal affects the amount of proteins in raft-like domains. DRM fractions. We also used a label-free, peak area approach to detect the shifts in protein sedimentation throughout the sucrose gradient on cholesterol removal. The abundance of DISCUSSION each protein was represented as the peak area of one unique peptide from this protein in each sucrose gradient fraction. Methodologic advances continue to provide compelling Figure 5 shows the abundance of some important lens evidence to support the important functions of lipid rafts; proteins. This experiment was repeated a second time and however, lipid raft characterization remains challenging the same trend was detected (data not shown). The lipid raft because individual lipid domains cannot be isolated in a pure markers flotillin 1 and 2 are primarily detected in the DRM form. The DRM fraction has been widely used for studying fractions without MbCD treatment; however, they shift to the proteins targeting to lipid raft-like domains. Given the high higher-density DSM fractions on MbCD treatment. Even though concentration of cholesterol in lens tissue, the detected the signal for aA crystallin in DRM fraction is strong, the cholesterol-rich domains in lens fiber cell membranes, and amount of aA crystallin in the DRM fraction was only a very the effects of cholesterol on membrane protein function, it is small fraction of total aA crystallin in the sample. A significant important to understand the details of membrane protein percentage of total AQP0, MP20, and AQP5 is present in the environments in the lens. In this work, we optimized methods low-density DRM fractions, and, after MbCD treatment, protein for studying raft-like DRMs from bovine lens fiber cells and our abundances in the DRM fractions decreased dramatically. Note isolation method revealed proteins whose solubility is highly that some fraction of AQP0, AQP5, and MP20 are detected in sensitive to MbCD treatment. In this study, 71 proteins were

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TABLE 2. Proteins With iTRAQ Ratios Greater Than 25, Their Cellular Localization, and Modifications

Gene Name Protein iTRAQ Ratios Cellular Localization and Modifications

MIP Lens fiber major intrinsic protein 261 Transmembrane, fatty acylation by multiple fatty acids GJA8 Gap junction protein 229 Transmembrane, cell junction CADM1 Cell adhesion molecule 1 189 Transmembrane, glycosylation, cell junction DAG1 Dystroglycan 95 Membrane, membrane raft SHF SH2 domain-containing adapter protein F 94 No information for cellular localization CTNNB1 beta-1 93 Cytoskeleton, cell junction, glycosylation CLU Clusterin 74 ER, mitochondrion, glycosylation JAM3 Junctional adhesion molecule C 68 Transmembrane, cell junction S100A14 Protein S100-A14 68 Cytoplasm, cell junction ZNFX1 NFX1-type zinc finger-containing protein 1 64 No inforamtion for cellular localization LCTL Lactase-like protein 60 Transmembrane, glycosylation BCAP31 B-cell receptor-associated protein 31 60 Transmembrane, ER NRCAM Neuronal cell junction molecule 58 Transmembrane, cell junction UBB Polyubiquitin-B 56 Cytoskeleton, cell junction, glycosylation GPRC5B G protein–coupled receptor, family C, group 5, 51 Transmembrane, glycosylation, membrane raft member B NAALAD2 N-acetylated-alpha-linked acidic dipeptidase 2 50 Transmembrane, glycosylation GJA3 Gap junction alpha-3 protein 48 Transmembrane, cell junction PALM Paralemmin-1 48 Cytoskeleton, lipid-anchor, palmitoylation, prenylation KRAS GTPase KRas 47 Membrane, lipid-anchor, palmitoylation, prenylation, Membrane raft RAC1 Ras-related C3 substrate 1 47 Membrane, lipid-anchor, ADP-ribosylation, prenylation BASP1 Brain acid soluble protein 1 46 Membrane, lipid-anchor, myristoylation STX2 Syntaxin-2 46 Transmembrane, membrane raft SLC2A1 Solute carrier family 2, facilitated glucose 45 Transmembrane transporter member 1 NCAM1 Neural cell adhesion molecule 1 44 Transmembrane, GPI-anchor, glycosylation PHB Prohibitin 43 Membrane, mitochondrion, cytoplasm VAPA Vesicle-associated membrane protein-associated 42 Transmembrane, ER protein A PALM2 Paralemmin-2 40 Cytoskeleton, lipid-anchor, palmitoylation, prenylation GNG12 Guanine nucleotide-binding protein G(I)/G(S)/G(O) 40 Membrane, lipid-anchor, prenylation subunit gamma-12 CDH13 Cadherin-13 39 Membrane, GPI-anchor, glycosylation, cell junction ANK3 Ankyrin-3 39 Membrane, cytoskeleton VDAC2 Voltage-dependent anion-selective channel protein 2 39 Mitochondrion RAB5B Ras-related protein Rab-5B 39 Membrane, lipid-anchor, prenylation ATP6V0D1 V-type proton ATPase subunit d 1 37 Membrane PTPRU Receptor-type tyrosine-protein phosphatase U 37 Transmembrane, glycosylation, cell junction YES1 Tyrosine-protein kinase Yes 36 Membrane, palmitoylation, myrisotylation STX4 Syntaxin-4 35 Transmembrane CTNNA1 Catenin alpha-1 34 Membrane, cytoskeleton TMED10 Transmembrane emp24 domain-containing protein 34 Transmembrane, glycosylation, membrane raft 10 EZR Ezrin 34 Membrane, cytoskeleton, membrane raft LAMC2 Laminin subunit gamma-2 33 Extracellular, cell adhesion, glycosylation VAMP3 Vesicle-associated membrane protein 3 33 Transmembrane NEXN Nexilin 33 Cytoskeleton, cell junction PHB2 Prohibitin-2 33 Membrane, mitochondrion PRX Periaxin 33 Membrane DNASE1L1 Deoxyribonuclease-1-like 1 33 ER, glycosylation RDX Radixin 33 Membrane, cytoskeleton SDK2 Protein sidekick-2 32 Transmembrane, glycosylation, cell junction CXADR Coxsackievirus and adenovirus receptor homolog 32 Transmembrane, glycosylation, palmitoylation, cell junction, membrane raft FLOT2 Flotillin-2 31 Membrane, palmitoylation, membrane raft, cell junction RHOA Transforming protein RhoA 31 Membrane, lipid-anchor, prenylation, cell junction FLOT1 Flotillin-1 31 Membrane, palmitoylation, membrane raft, cell junction

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TABLE 2. Continued

Gene Name Protein iTRAQ Ratios Cellular Localization and Modifications

CEACAM18 CEAantigen-related cell adhesion molecule 18 30 Glycosylation Histone H4 30 Nucleus CTNND1 Catenin delta-1 30 Membrane, cell junction EPHA2 Ephrin type-A receptor 2 29 Transmembrane, glycosylation, cell junction KIAA1217 Sickle tail protein homolog 29 Cytoplasm, glycosylation CHMP6 Charged multivesicular body protein 6 29 Membrane, lipid-anchor, myristoylation ERLIN2 Erlin-2 28 Transmembrane, ER, glycosylation, membrane raft ITGB1 Integrin beta-1 28 Transmembrane, membrane raft, glycosylation CD47 Leukocyte surface antigen CD47 28 Transmembrane, glycosylation, cell junction TM7SF2 Delta(14)-sterol reductase 28 Transmembrane CRYGN Gamma-crystallin N 27 No information for cellular localization BSG Basigin 27 Transmembrane, membrane raft, glycosylation AQP5 AQP5 26 Transmembrane, glycosylation CTNNA2 Catenin alpha-2 26 Cytoskeleton, cell junction PFKL 6-phosphofructokinase, liver type 26 Cytoplasm, glycosylation PRNP Major prion protein 26 Membrane, GPI-anchor, glycosylation, membrane raft CRYAA Alpha-crystallin A chain 25 Cytoplasm, glycosylation BFSP1 Filensin 25 Cytoskelaton, myristoylation ACTB , cytoplasmic 1 25 Cytoskelation RAP2B Ras-related protein Rap-2b 25 Membrane, lipid raft, palmitoylation GPI, glycosylphosphatidylinositol.

quantified by iTRAQ to be highly sensitive to cholesterol. MP20. In addition, MP20 also has a CRAC/CARC sequence that These proteins included AQP0, AQP5, MP20, and connexins. could directly interact with cholesterol.50 The MP20 is found in Previously, AQP0 was reported to localize to both raft and the cytoplasm of young fiber cells and traffics to the plasma nonraft domains,17 and our results are consistent with this membrane concomitantly with the loss of intracellular finding. A significant portion, approximately 40%, of the total organelles in maturing lens fiber cells.59 Because lipid raft AQP0 sedimented to DRM fractions. A previous study indicated domains play important roles in protein trafficking and cell AQP0 homo-oligomerization is a key factor that targets AQP0 to adhesion, understanding how MP20 targets to lipid raft domain lipid raft domains.17 Later, AQP0 was found to be modified by will help to elucidate the function of this protein in the lens. fatty acids, such as oleic acid and palmitic acid, and that fatty Our results suggest that significantly lower amounts of acylation acted as a potential raft-targeting modification.48 In connexins reside in DRM domains compared with AQP0, a this study, we confirmed that fatty acylated AQP0 was enriched result consistent with a previous report that connexins are in the raft-like DRM domains; however, a significant amount of located primarily in nonraft domains.17 The small fraction of AQP0 in the DRM was not modified by fatty acids, suggesting total connexins present in the high-buoyancy DRM was that other mechanisms exist to target AQP0 to membrane rafts, strongly cholesterol sensitive. The observation of two forms possibly AQP0 oligomerization. In addition, the presence of a of connexins in lens fiber cells, DRM and DSM residing, is cholesterol-binding or sphingolipid-binding sequence of trans- supported by a report of cholesterol-rich gap junction and membrane proteins can increase their concentration in lipid cholesterol-free connexons.60 Connexin family members have raft.49 The AQP0 has four CRAC/CARC sequences that have the been shown to interact with caveolin-1 and to target to lipid potential to bind with cholesterol.50 Further study is needed to raft domains to regulate gap junctional intercellular communi- confirm whether AQP0 directly binds with cholesterol. A cation43,61,62; however, previous results showed inconsistency previous report showed that AQP0 water permeability was related to the targeting of Cx46 and Cx50 to lipid raft affected by membrane lipid composition and that increased domains.17,61,62 Based on our experiments, the overall cholesterol content decreased AQP0 water permeability.51 This distribution of Cx46 and Cx50 across the sucrose gradient result also suggested a direct AQP0-cholesterol interaction. was similar, with most of the protein present in the nonraft Among the major lens proteins, MP20 was found to be DSM fraction; however, the small fraction present in the DRM highly sensitive to cholesterol removal; MP20 is a member of fraction was sensitive to MbCD treatment. the PMP22/EMP/MP20/Claudin family, a diverse group of The aA-crystallin was among the proteins that are sensitive proteins with roles in intercellular adhesion and protein to MbCD treatment, whereas most b- and c-crystallins have trafficking.52,53 The PMP22 is a known constituent of lipid much lower MbCD sensitivity. Previously, a-crystallin was the rafts in peripheral nerve myelin and may play a role in the only crystallin reported to noncovalently bind with bovine lens linkage of the actin cytoskeleton with the plasma membrane, membranes,11,63 and this membrane association property of a- likely through regulating the cholesterol content of lipid crystallin was attributed to either direct lipid binding64 or rafts.54,55 Localization of lens MP20 in lipid rafts has not been association with AQP0.63,65 reported. Previously, MP20 was found to form large oligomers Compelling evidence supports the central role of mem- in the presence of native lens lipids.56 Oligomerization, as brane rafts in subcellular membrane transport, trafficking, and reported for AQP0, could target MP20 to lipid raft domains. In signaling.38,39 Rafts are believed to have ideal features for addition, almost all tetraspanins are modified by the posttrans- acting as a sorting mechanism in membrane trafficking due to lational addition of palmitate to membrane-proximal cysteine their proposed size and their capacity to sequester both lipids residues.57 Palmitoylation of MP20 has not been reported, but and proteins.38 In our candidate lipid raft protein list, CSS-Palm software58 predicts palmitoylation of cysteine 11 in membrane trafficking–related processes were significantly

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TABLE 3. Proteins With iTRAQ Ratios Less Than 15, Their Cellular Localization, and Modifications

Gene Name Protein Name iTRAQ Ratios Cellular Localization and Modifications

CRYBA1 Beta-crystallin A3 14 Cytoplasm, nucleus CYFIP2 Cytoplasmic FMR1-interacting protein 2 14 Cytoplasm MICAL3 Protein-methionine sulfoxide oxidase MICAL3 14 Cytoplasm, cytoskeleton CPD Uncharacterized protein 14 Transmembrane, glycosylation, palmitoylation EPB41L3 Band 4.1-like protein 3 14 Cytoskeleton LMCD1 LIM and cysteine-rich domains protein 1 14 Cytoplasm, nucleus TMEM143 143 14 Transmembrane, mitochondrion LMAN1 Uncharacterized protein 14 ER, Golgi membrane ANXA2 Annexin A2 14 Extracellular EEF2 Elongation factor 2 14 Cytoplasm, nucleus LUC7L2 Uncharacterized protein 14 Nucleus HSPA1A Heat shock 70 kDa protein 1A 13 Cytoplasm HSPB1 Heat shock protein beta-1 13 Cytoplasm ANXA1 Annexin A1 13 Cytoplasm, nucleus, basolateral cell membrane PLEC Uncharacterized protein 13 Cytoplasm, cytoskeleton FLNC -C 13 Cytoplasm, cytoskeleton TUBB4B Tubulin beta-4B chain 13 Cytoplasm, cytoskeleton YKT6 homolog YKT6 13 Cytoplasm, lipid-anchor, palmitoylation, prenylation RPS2 40S ribosomal protein S2 13 Cytoplasm, nucleus BFSP2 Phakinin 13 Cytoskeleton LNP Protein lunapark 13 Transmembrane, ER, myristoylation ATP5A1 ATP synthase subunit alpha, mitochondrial 13 Mitochondrion FLNA Filamin-A 13 Cytoplasm, cytoskeleton TMED2 Transmembrane emp24 domain-containing protein 2 12 Transmembrane, ER, Golgi ATP2A2 Sarcoplasmic/endoplasmic reticulum ATPase 2 12 Transmembrane, ER CA14 Carbonic anhydrase 14 12 Transmembrane, glycosylation ARVCF Armadillo repeat protein deleted in velo-card 12 Cytoplasm, membrane SEC22B Vesicle-trafficking protein SEC22b 12 Transmembrane, ER EPB41 Protein 4.1 12 Cytoplasm, cytoskeleton ZNF19 Zinc finger protein 19 12 Nucleus PDCD6IP Programmed cell death 6-interacting protein 11 Cytoplasm, cytoskeleton JUP Junction 11 Cytoplasm, cytoskeleton, membrane, glycosylation ZMPSTE24 CAAX prenyl 1 homolog 11 Transmembrane, ER VIM Vimentin 10 Cytoskeleton GSTM1 Glutathione S-transferase Mu 1 10 Cytoplasm LOC101905591 Uncharacterized protein 10 Cytoskeleton MLEC MLEC protein 9 Transmembrane, ER ALDH3A2 Aldehyde dehydrogenase 9 Transmembrane, ER BCL2L13 Bcl-2-like protein 13 9 Transmembrane, mitochondrion EPB41L1 Band 4.1-like protein 1 9 Cytoplasm, cytoskeleton DSP 9 Cytoplasm, cytoskeleton SERBP1 SERPINE1 mRNA binding protein 1 8 Cytoplasm, nucleus SNCG Gamma-synuclein 8 Cytoplasm, cytoskeleton LGSN Lengsin 8 Membrane SLC9C2 Uncharacterized protein 8 Transmembrane PBRM1 Uncharacterized protein 8 Nucleus TFR2 Transferrin receptor protein 2 8 Transmembrane canx Canx protein 8 Transmembrane, ER DSG1 Desmoglein-1 8 Transmembrane

enriched based on DAVID bioinformatic analysis. For example, also detected, including caveolin-1, Cell division control significantly enriched biological processes included protein protein 42 homolog, Transforming protein RhoA, ADP-ribosy- transport, vesicle-mediated transport, and Golgi vesicle trans- lation factor 1, and flotillins. These results strongly support the port. The pathway of SNARE interactions in vesicular transport concept that lipid raft domains play an important role in was also significantly enriched. Proteins detected in the DRM subcellular trafficking within lens fiber cells. The most fraction of lens fiber cells that are involved in SNARE important role of rafts at the cell surface may be their function interactions included Vesicle-trafficking protein SEC22b in signal transduction and the small GTPases are central to (SEC22b), Synaptobrevin homolog YKT6 (YKT6), Synaptosom- many signaling processes.40 In our list of proteins detected in al-associated protein (SNAP23), Vesicle transport through the DRM fractions, enriched biological processes include small interaction with t-SNAREs homolog 1B (VTI1B), Vesicle GTPase-mediated signal transduction. In addition, organization transport protein USE1 (USE1), several vesicle-associated and clustering of lipid rafts into more active signaling platforms membrane proteins, and multiple types of . Proteins depends on interaction with and dynamic rearrangement of in raft domains that are involved in endocytic trafficking37 are the cytoskeleton41 and many of the structural and functional

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FIGURE 6. Quantification of oleic acid–modified AQP0 in different fractions. Chloroform/methanol-precipitated proteins from each frac- tion were washed with 4 M urea followed by trypsin digestion and analysis by LC-MS/MS as described in the Materials and Methods section. The raw data were imported into Skyline24 for peak-picking and quantification. The total peak area from at least seven product ions was used for each peptide. Signal from the oleic acid–modified AQP0 peptides (peptide 1–5 or 234–259) were normalized by the signal for AQP0 peptide 188 to 196.

organization, it will be interesting to study the role of lens fiber

FIGURE 5. Distribution of some major lens proteins across the sucrose cell lipid rafts in differentiation as well as in cell migration and density gradient and effects of MbCD treatment. Proteins in each elongation. Considering different lipid composition between fraction were digested by trypsin and analyzed by LC-MS/MS as human and bovine lens,66 as well as lipid changes with aging,67 described in the Materials and Methods section. Protein abundance further characterization of the lipid raft proteome in the was represented as the peak area of one peptide. Total peak area human lens is warranted. It also will be interesting to study corresponding to proteins from 100 lL each fraction was calculated whether lipid rafts play a role in lens protein aggregation and plotted. The y-axis shows the peak areas of corresponding during lens aging, given their increased affinity for oligomeric peptides. proteins, particularly in neurodegenerative diseases.68–70

properties of rafts require an intact actin cytoskeleton.44 Acknowledgments Cytoskeleton organization was one of the enriched biological processes and regulation of actin cytoskeleton was one of the Supported by National Institutes of Health Grants EY-13462 (KLS) enriched pathways. and P30 EY-008126. The authors acknowledge support from the In conclusion, we have identified 506 proteins in high- Vanderbilt University Proteomics Facility in the Mass Spectrometry buoyancy DRM fractions of lens fiber cell membranes and Research Center. more than 85% of identified proteins have been reported to Disclosure: Z. Wang, None; K.L. Schey, None have lipid raft localization in other cells. The iTRAQ analysis of the high-buoyancy DRM proteins showed variable cholesterol References removal dependencies suggesting the presence of true lipid raft proteins and some lipid raft–associated proteins. Enriched 1. Bretscher MS. Membrane structure: some general principles. GO categories and pathways strongly support that lipid raft Science. 1973;181:622–629. domains play important roles in the lens; roles that have not 2. Simons K, Ikonen E. Functional rafts in cell membranes. been widely explored. Considering the important functions of Nature. 1997;387:569–572. lipid raft domains in other cell types, including roles in 3. Pike L. Rafts defined: a report on the Keystone Symposium on trafficking, signal transduction, apoptosis, and cytoskeletal Lipid Rafts and Cell Function. J Lipid Res. 2006;47:1597–1598.

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