Proteomic Analysis of Lipid Microdomains from Lipopolysaccharide

Proteomic Analysis of Lipid Microdomains from Lipopolysaccharide-Activated Human Endothelial Cells Aly Karsan,*,†,‡ Josip Blonder,§ Jennifer Law,† Elisa Yaquian,† David A. Lucas,§ Thomas P. Conrads,§ and Timothy Veenstra§ Department of Medical Biophysics and Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, British Columbia, Canada V5Z 1L3, Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, and Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702-1201 Received September 30, 2004

The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells. Keywords: lipopolysaccharide • endothelium • caveolae • lipid rafts • lipid-rich microdomains • inflammation

Introduction

orchestrating the inflammatory response triggered by sepsis or endotoxemia.4,5

Receptors of the innate immune system are expressed on various cell types including the endothelium.1 Because of the broad specificity of the innate immune system, receptors of this system have a wide spectrum of recognition for common structures on multiple pathogens.1 A structure that is common to Gram-negative bacteria is lipopolysaccharide (LPS).2 LPS is a critical glycolipid component of the outer wall of Gramnegative bacteria, and many of the cellular signals activated by Gram-negative bacteria are attributed to LPS.2 LPS is the prototypic activator of innate immune responses, and many of the inflammatory and clinical effects of Gram-negative sepsis are elicited by LPS.3 The endothelium plays a pivotal role in

Various receptors have been suggested to play a role in transducing LPS signals.6 The more recent focus has been on three cell surface proteins, CD14, Toll-like receptor-4 (TLR4), and MD-2, which have been shown to play a major role in recognizing and transmitting the LPS signal.7 Delivery of LPS by LPS-binding protein to a complex of TLR4, CD14 and MD2, results in a cascade of intracellular signaling events.7 Membrane-bound CD14 has been localized to discrete lipid microdomains in macrophages, which are critical for LPS signal transduction.8 However, there are distinctions between polarized epithelial and endothelial cells compared to leukocytes, in that the latter cell type display membrane-bound CD14, whereas endothelial/epithelial cells rely on soluble CD14 in serum to receive the LPS signal.9

* To whom correspondence should be addressed. Tel: (604) 877-6248. Fax: (604) 877-6002. E-mail: [email protected]. † Department of Medical Biophysics and Pathology and Laboratory Medicine, British Columbia Cancer Agency. ‡ Department of Pathology and Laboratory Medicine, University of British Columbia. § Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick. 10.1021/pr049824w CCC: $30.25

 2005 American Chemical Society

Though, once thought to be homogeneous, it is clear from recent evidence that the plasma membrane is spatially organized by asymmetric localization of cholesterol- and sphingolipid-rich islands.10,11 These discrete microdomains, broadly referred to as lipid rafts, are themselves heterogeneous.12 In Journal of Proteome Research 2005, 4, 349-357

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research articles addition to the morphologically flat lipid rafts, endothelial and other cells that express the protein Caveolin-1 display flaskshaped, lipid-rich microdomains, termed caveolae.13,14 To avoid confusion, we will refer to these discrete microdomains collectively as lipid microdomains. One of the most important properties of lipid microdomains is that they can include or exclude proteins to variable extents.11 By accumulating proteins within a spatially discrete area, lipid microdomains can act as concentrating platforms for individual receptors activated by ligand binding.11 The importance of lipid microdomains in LPS signaling has recently been demonstrated in macrophages, and the involvement of specific proteins has been identified.15 However, the entire cohort of proteins involved in the process of LPS signaling remains to be established. In endothelial cells, the composition of lipid microdomains has revolved around the investigation of specific proteins, but a comprehensive global survey of proteins that are concentrated in these microdomains is still lacking. In this study, we initiated a global proteome analysis of lipid microdomain proteins in LPS-stimulated endothelial cells to begin to understand how the components of these microdomains modulate or initiate the LPS signal. Unlike cytoplasmic proteins, global proteome analyses of membrane proteins has been difficult due to their poor solubility in aqueous buffers used in conventional, two-dimensional-PAGE based, proteomic investigations.16 Here, we describe an organic solvent method to solubilize and tryptically digest purified lipid microdomain fractions, that when combined with gas-phase fractionation in the mass-to-charge (m/z) dimension (GPFm/z) during microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (µRPLC-MS/MS), enables the identification of membrane proteins from LPS-activated human umbilical vein endothelial cells (HUVEC).

Materials and Methods Materials. Triton X-100 and Brij 35 were obtained from Fisher Scientific (Nepean, Ontario, Canada). Sequencing grademodified trypsin was purchased from Promega (Madison, WI). Ammonium bicarbonate (NH4CO3) and bacterial lipopolysaccharide (LPS) (E. coli 0111:B4) was purchased from Sigma (St. Louis, MO). Trifluoroacetic acid (F3CCOOH) and formic acid (HCOOH) were from Fluka (Milwaukee, WI). Tricholoroacetic acid (Cl3CCOOH) was purchased from Sigma. High-performance liquid chromatography (HPLC)-grade methanol (CH3OH) and acetonitrile (CH3CN) were obtained from EM Science (Darmstadt, Germany). Antibodies directed against Caveolin1, poly(ADP-ribose) polymerase (PARP) and CD31 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and against Dysferlin from Medicorp Inc. (Montreal, Quebec, Canada). Ultrapure water was obtained using a Barnstead purification system (Dubuque, IA). Human Umbilical Vein Endothelial Cell (HUVEC) Culture. Primary human umbilical vein endothelial cells (HUVEC) were isolated and cultured as previously described.17,18 Four 15-cm tissue culture dishes of confluent HUVEC (∼2 × 108 cells) were treated for 5 min with bacterial lipopolysaccharide (100 ng/ mL) and harvested for density gradient separation. Preparation of Detergent-Resistant Membranes (Lipid Microdomains). Cells were washed four times with cold phosphatebuffered saline and HUVEC on each 15-cm dish lysed in 400 µL ice-cold lysis buffer containing 50 mM HEPES pH 7.4/150 mM NaCl, protease inhibitors, and various concentrations of nonionic detergents. Cell lysates were sheared by five successive 350

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Figure 1. Isolation of detergent-resistant membranes. (A) Cartoon showing the sucrose density layers and the location of specific fractions. (B) Caveolin-1 immunoblots of protein fractions isolated from sucrose density-gradient separation of HUVEC lysed in different nonionic detergents as labeled. (C) Pooled sucrose density-gradient fractions of HUVEC lysed in 1% Brij 35 and centrifuged to pellet the nuclei. Poly (ADP-ribose) polymerase (PARP), a nuclear protein, is not present in the low-density fraction.

passages through 26 gauge needles and then centrifuged at 3000 × g for 5 min at 4 °C to pellet nuclei. The supernatant was mixed with an equal volume of 80% sucrose (w/v) in icecold lysis buffer without detergent, and transferred to 11 × 34 mm ultracentrifuge tubes (Beckman Instruments, Inc., Palo Alto, CA). The samples were then overlaid with 1.3 mL of 30% sucrose and 500 µL mL of 5% sucrose and centrifuged using a TLS-55 swinging bucket rotor on a TL-100 tabletop ultracentrifuge (Beckman) at 100 000 × g for 18 h (Figure 1A). All of the procedures were done at 4 °C. Following centrifugation, 11 fractions of 200 µL each were collected, starting at the top of the gradient. Aliquots of each fraction were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer to prepare for immunoblotting or further prepared for mass spectrometry (MS). For MS and some of the immunoblotting experiments, fractions 1-6 and 7-11 were pooled separately into single fractions. Fractions 1 through 6 corresponding to the 5% to 30% sucrose interface were referred to as lipid microdomain fractions. Extraction, Solubilization and Proteolysis of Membrane Proteins. The lipid microdomain fraction was mixed with 1 mL of water and pelleted at 100 000 × g. The detergent-insoluble pellet containing ∼50 µg of LPS-activated HUVEC protein from lipid microdomains was resuspended in 50 mM NH4HCO3, pH 7.9 and pelleted at 100 000 × g for 30 min at 4 °C. The supernatant was discarded and the pellet washed two more times with NH4HCO3 to remove detergent. The final pellet was resuspended in 60% CH3OH/40% 50 mM NH4HCO3 buffer to solubilize integral and membrane-bound proteins.19 The pro-

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Activated Endothelial Lipid Microdomains

teins were digested by direct addition of trypsin to the ultracentrifuge tube at a final ratio of 1:20 (enzyme:protein). Following lyophilization, the peptides were resuspended in 0.1% F3CCOOH and desalted using ZipTip C-18 as recommended by the manufacturer (Millipore, Bedford, MA). The desalted peptides were lyophilized and reconstituted in 50 µL of 0.1% F3CCOOH/5% CH3OH. Microcapillary Liquid Chromatography Reversed-Phase (µRPLC)-Tandem Mass Spectrometry (MS/MS). Chromatographic separations were conducted using a 75 µm inner diameter × 60 µm outer diameter × 10 cm long fused silica capillary column (Polymicro Technologies Inc., Phoenix, AZ) with one end flame-pulled to a fine tip (∼5-7 µm orifice). The column was slurry packed in-house with 3 µm, 300 Å pore size C-18 stationary phase (Vydac, Hercules, CA). Microcapillary RPLC was performed using an Agilent 1100 capillary LC system (Agilent Technologies, Palo Alto, CA) coupled online to an iontrap (IT) mass spectrometer (LCQ Deca XP, ThermoElectron, San Jose, CA). Reversed-phase separations were conducted after injecting 5 µL of sample for each analysis. The reversed-phase µRPLC column was washed for 15 min with 98% mobile phase A (0.1% v/v HCOOH in water) and peptides eluted using a linear gradient from 2% to 98% mobile phase B (0.1% HCOOH in CH3CN) over 110 min with a constant flow rate of 0.5 µL/min. The column was washed for 15 min with 98% mobile phase B and reequilibrated with 98% mobile phase A prior to subsequent sample loading. The µRPLC column was coupled online to an IT-MS using the nanoelectrospray source with an applied electrospray potential of 1.7 kV and capillary temperature of 160 °C. The IT-MS was operated in a data-dependent mode where each full MS scan was followed by three MS/MS scans, in which the three most abundant peptide molecular ions detected from the MS scan were dynamically selected for three subsequent MS/ MS scans using a collisional-induced dissociation (CID) energy of 38%. To conduct the GPFm/z experiments,20,21 eight sequential µRPLC-MS/MS analyses of the sample were sequentially performed where the MS was operated such that only the following limited segmented m/z ranges were employed for data-dependent precursor ion selection for CID for each sample injection: 400-600, 600-800, 800-1000, 1000-1200, 12001400, 1400-1600, 1600-1800, and 1800-2000. Data Processing and Analysis. The CID spectra were analyzed using SEQUEST operating on a Beowulf 18-node parallel virtual machine cluster computer (ThermoElectron) using the nonredundant Homo sapiens proteome database (http://www. ebi.ac.uk). Only peptides with conventional tryptic termini (allowing for up to two internal missed cleavages) possessing delta-correlation scores (∆Cn) g 0.08 and charge state-dependent cross-correlation (Xcorr) criteria as follows were considered as legitimate identifications: >1.9 for +1 charged peptides, >2.2 for +2 charged peptides, >3.1 for +3 charged peptides. The results in this study represent data collected from one µLC-MS/MS analysis with a broad precursor ion selection scan range of m/z 475-2000 and one complete GPFm/z analysis as described above. The identified proteins were classified by Gene Ontology using terms created by the Gene Ontology Consortium (http://www.geneontology.org/). The mapping of R-helical integral membrane proteins identified in this work was performed using the transmembrane hidden Markov model (TMHMM) algorithm, available at http://www.cbs.dtu.dk/services/ TMHMM.22,23 Mapping of identified proteins onto canonical

pathways was carried out using Ingenuity Pathways Analysis software (Ingenuity, Mountain View, CA). Immunoblotting. Cells were lysed in 1% Brij35 and fractionated on a sucrose gradient as described above. The sucrose gradient fractions were run as individual fractions, or pooled into detergent-resistant fractions (1-6) and the remaining fractions (7-11) separately, precipitated in 10% Cl3CCOOH and resolubilized in SDS-PAGE gel loading buffer containing 1% n-octyl-β-D-glucopyranoside (Sigma). Protein was separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and immunoblotted using standard methods. Membranes were developed by ECL chemiluminescence (PerkinElmer Life Science, Boston, MA) and visualized using Bioflex MSI X-ray film (Clonex Corporation, Markham, Ontario, Canada).

Results Optimization of Endothelial Lipid Detergent-Resistant Membrane Preparations. It has previously been shown that lipid rafts facilitate LPS-induced cell signaling.8,24 However, the complex of proteins that accumulate within lipid microdomains in response to LPS stimulation has not been determined. To identify endothelial proteins that localize within the cholesterolrich domains required for LPS signaling, we optimized a protocol to enrich lipid raft microdomains. Isolation of lipid rafts relies upon the detergent insolubility of these lipid-rich microdomains, with the most widely used detergent being Triton X-100, usually at 1% concentration.25 However, the heterogeneous composition of membrane microdomains may require different detergents or concentrations for raft isolation in different cell types.26 We thus tested the ability of Triton X-100 and Brij 35 to purify lipid microdomains on sucrose density-gradients. The sucrose density-gradient fractions were immunoblotted with an antibody against Caveolin-1 to identify those that contained lipid-rich microdomains. As seen in Figure 1B, 1% Triton X-100 resulted in complete disruption of lipid microdomains as Caveolin-1 did not partition into low-density fractions (fractions 1-6). Although lowering the Triton X-100 concentration to 0.05% did result in purification of lipid microdomains as determined by the presence of Caveolin-1 in fractions 1-6, the purification was not complete as significant amounts of Caveolin-1 remained in the higher-density fractions (7-11) (Figure 1B). In contrast, the use of 1% Brij 35 (Figure 1B) or 0.1% Triton X-100 (data not shown) resulted in almost complete partitioning of Caveolin-1 into the low-density fractions. Previous results and our own data suggest significant contamination of lipid raft preparations with nuclear material27,28 (and data not shown). To reduce nuclear contamination the 1% Brij 35-lysed HUVEC were centrifuged at 3000 × g for 5 min at 4 °C to pellet nuclei. Figure 1C demonstrates that this additional centrifugation step removes the majority of nuclear material present in the lysate as determined by immunoblotting for the nuclear enzyme, PARP. µRPLC-MS/MS Analysis of LPS-Activated Endothelial Detergent-Resistant Membranes Demonstrates Enrichment of Lipid Microdomain-Associated Proteins. On the basis of the above experiments, the protocol outlined in Figure 2 was used to conduct a global proteomic analysis of lipid microdomains from primary human endothelial cells. Following detergent removal and buffer exchange of the low-density fraction of the sucrose density-gradient (fractions 1-6), the pellet was sonicated in 60% methanol in 50 mM (NH4)2HCO3 and digested Journal of Proteome Research • Vol. 4, No. 2, 2005 351

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Karsan et al. Table 1. Selected Subset of Identified Lipid Raft-Associated Proteins protein

unique peptidesa

5′-nucleotidase Aminopeptidase N Annexin II Calnexin CD44 CD59 EPCR Ephrin A2 Ephrin B4 Flotillin-1 Flotillin-2 Galectin-1 HSP 70 HSP 90-beta Heme oxygenase 1 Integrin alpha-V Integrin beta-1 Integrin beta-3 Stomatin SNAP 23

1 11 9 20 1 1 1 2 1 4 2 2 1 2 1 10 4 5 7 4

Swiss-Prot accession no.

P21589 P15144 P07355 P27824 P16070 P13987 Q9UNN8 P29317 P54760 O75955 Q14254 P09382 P17066 P08238 P09601 P06756 P05556 P05106 P27105 O00161

a Number of unique peptides identified from the listed protein. EPCR, Endothelial cell protein C receptor; HSP, Heat shock protein; SNAP 23, Synaptosomal-associated protein 23

Figure 2. Schema of experimental protocol. Outline of the protocol to isolate LPS-activated HUVEC for microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (µRPLC-MS/MS) analysis. LMD, lipid microdomain, LC.

directly using trypsin. The resulting peptides were subjected to µRPLC-MS/MS utilizing GPFm/z as follows: 400-600, 600800, 800-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800, 1800-2000. Compilation of the results of all the µRPLC-MS/ MS analyses resulted in the identification of a total of 894 unique peptides corresponding to 358 unique proteins (Supplementary Table 1 in Supporting Information). In addition to Caveolin-1, a large number of other raft/ caveolae-associated proteins were identified in the isolated detergent-resistant membrane fractions. Table 1 shows a selected subset of those previously identified lipid microdomain-associated proteins. Numerous other raft-associated proteins and putative and potential lipid microdomain proteins were also identified (Supplementary Table 1 in Supporting Information). For example, a number of small GTP-binding proteins and heterotrimeric G proteins, which are known to localize to lipid microdomains, were also identified (Supplementary Table 1 in Supporting Information). The strategy employed in the current proteome analysis significantly enriched the analyzed cellular lysate for proteins that are classified by Gene Ontology (www.geneontology.org) as localizing to membranes (Figure 3A). In a global proteomic analysis of HUVEC, whole cell lysates (unpublished data) in which over 1000 proteins were identified only 23% were classified as being localized to the membrane (Figure 3B). By utilizing the strategy outlined in this manuscript, however, the 352


Figure 3. Gene ontology analysis of cellular localization of lipid microdomain proteins. Subcellular localization of proteins isolated from HUVEC lipid microdomains (A) compared to total cell lysates (B) as classified by Gene Ontology.

proportion of proteins classified as being localized to the membrane increased to 49% of the total identified. Analyzed in an alternate way, 157 of the 358 proteins (∼44%) identified in the detergent-resistant membrane fractions contain at least one R-helical transmembrane domain as predicted by TMHMM.22,23 This algorithm has been shown in independent analyses to have the greatest accuracy for the identification of transmembrane sequences and generates the fewest number of false positives and false negatives.29,30 The proteins predicted to contain 2 or more R-helical transmembrane domains are listed in Table 2, while a distribution of the number of predicted

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Activated Endothelial Lipid Microdomains Table 2. Proteins Containing Multiple Transmembrane Domains protein

5′-nucleotidase AD158 ADP/ATP translocase 1 ADP/ATP translocase 2 Amiloride-sensitive sodium channel γ-subunit Amino acid transporter system A2 Peptide transporter TAP2 ARL-6 interacting protein-1 ATP-binding cassette, sub-family D, member 3 Basigin C-4 methyl sterol oxidase CD9 Chromosome 9 open reading frame 5 Cleft lip and palate-associated transmembrane protein 1 Cop-coated vesicle membrane protein p24 Defender against cell death 1


protein

TMDa


2 4 3 2 2

P21589 Q8TDW0 P12235 P05141 P51170

Minor histocompatibility antigen H13 Mitochondrial carrier homologue 1 isoform b Monocarboxylate transporter 1 Monocarboxylate transporter 4 Multidrug resistance-associated protein 1

7 2 11 12 16

Q8TCT9 Q9NZJ7 P53985 O15427 P33527

9

Q9HAV3

Myeloid-associated differentiation marker

8

Q96S97

6 4 3

Q03519 Q15041 P28288

NADH-ubiquinone oxidoreductase chain 4 RER1 protein Neurite outgrowth inhibitor (Nogo protein)

10 3 2

P03905 O15258 Q9NQC3

2 3

P35613 Q15800

9 13

Q15758 P46977

4 14

P21926 Q9H330

2 7

Q00325 P20020

5

Q9BSS5

8

P23634

2

Q15363

Neutral amino acid transporter B(0) Oligosaccharyl transferase STT3 subunit homologue (B5) Phosphate carrier protein (PTP) Plasma membrane calcium-transporting ATPase 1 Plasma membrane calciumtransporting ATPase 4 Probable cation-transporting ATPase 2

7

Q9HD20

3

P46966

10

P38378

2

Q9P2 × 0

Protein transport protein Sec61 R subunit isoform 1 Retinol dehydrogenase 10

2

Q8IZV5

2

P39656

Translocase of outer membrane TOM70

2

O94826

4

P04844

Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 Secretory carrier-associated membrane protein 3 Sideroflexin 1 Similar to hypothetical protein FLJ10856 Similar to membrane bound C2 domain containing protein Similar to RIKEN cDNA 0610010I12 Sodium/potassium-transporting ATPase R-1 chain Sodium/potassium-transporting ATPase R-3 chain Solute carrier family 2, facilitated glucose transporter, member 1 Source of immunodominant MHCassociated peptides Steroid dehydrogenase homolog Synaptic glycoprotein SC2 T-cell surface glycoprotein E2 Translocation protein SEC63 homolog Translocon-associated protein, γ subunit Transmembrane protein PT27 Transmembrane protein Tmp21

7

O14983

8

P16615

4

O14828

3 2 2

Q9H9B4 Q96H09 Q9BSJ8

4 10

Q8N6L1 P05023

8

P13637

12

P11166

10

Q8TCJ2

3 4 2 3 4

Q9Y6G8 Q9NZ01 P14209 Q9UGP8 Q9UNL2

7 2

Q9HC07 P49755

TMDa

Dolichol-phosphate mannosyltransferase subunit 3 Dolichyl-diphosphooligosaccharides protein glycosyltransferase 48 kDa subunit Dolichyl-diphosphooligosaccharides protein glycosyltransferase 63 kDa subunit Elongation of very long chain fatty acids protein 1 Equilibrative nucleoside transporter 1

7

Q9BW60

11

Q99808

Gap junction alpha-1 protein HSPC121 Hypothetical protein

4 5 12

P17302 Q9P035 Q8N3V4

Hypothetical protein Hypothetical protein

9 2

Q8TCS5 Q9BQH9

Hypothetical protein FLJ14971

4

Q9BTV4


5

Q96N66


2

Q8N7W5

Hypothetical protein KIAA0062 (Fragment) Hypothetical protein KIAA1400 (Fragment) Hypothetical protein KIAA1691 (Fragment) Interferon-induced transmembrane protein 1 Intestinal membrane A4 protein

6 3 6 2 4

Q15043 Q9P2E7 Q9C0H2 P13164 Q04941

Microsomal glutathione S-transferase 3 Microsomal signal peptidase 12 kDa subunit Microsomal signal peptidase 25 kDa subunit

4 2 2

O14880 Q9Y6A9 Q15005

a

Number of R-helical transmembrane domains predicted by TMHMM (www.cbs.dtu.dk/services/TMHMM).

R-helical transmembrane domains found within these proteins is shown in Figure 4. Despite the high proportion of predicted transmembrane domains, it should be noted that this algorithm does not predict other types of transmembrane domains such as β-barrels. More importantly GPI-linked proteins and other lipid modifications that target cytosolic proteins such as small G proteins to lipid rafts, are also not identified by this algorithm. If previously identified lipid raft proteins are included, then 235 of the 358 identified proteins (∼66%) contain at least one

transmembrane domain or have previously been identified to localize to lipid rafts.27,28,31 These results suggest that the majority of the remaining proteins, although not previously encountered in lipid rafts, are likely bona fide lipid microdomain-associated proteins. To confirm that proteins identified herein that had not previously been identified in lipid microdomains were in fact present in the detergent-resistant fractions, we performed immunoblotting for two proteins. CD31, a well-studied endoJournal of Proteome Research • Vol. 4, No. 2, 2005 353

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Figure 4. Number of transmembrane domains of identified proteins. Distribution of the number of predicted transmembranespanning R-helices of the proteins identified in this study.

Figure 5. Validation of lipid microdomain proteins identified by mass spectrometry. Pooled fractions of the low- (Fr. 1-6, containing lipid microdomains) and high-density (Fr. 7-11) portions of sucrose density-gradient separated, LPS-stimulated HUVEC lysates were immunoblotted to determine the expression of CD31, Dysferlin and Caveolin-1.

thelial protein that is also known as PECAM-1, was identified by multiple peptides.32 In contrast, Dysferlin, a protein that is mutated in certain types of muscular dystrophy syndromes was identified by only a single peptide, and has not previously been reported to be expressed in the endothelium or lipid microdomains.33 Immunoblotting with antibodies against each of these proteins demonstrates that both are present in the detergent-resistant fraction (Figure 5). Organic Solvent Based Detergent-Resistant Membrane Solubilization Permits Trypsin Digestion of Intramembranous Portions of Membrane Proteins. One of the major difficulties with proteomic analysis of membrane proteins is the relatively high hydrophobicity of these proteins making them less tractable to assay by two-dimensional gel electophoresis. The embedding of these proteins within the lipid bilayer compounds the problem of analysis since this limits the overall accessibility of the protein, particularly the transmembrane domains, to tryptic digestion. To overcome this limitation a miscible organic/aqueous solvent (60% methanol in 50 mM NH4HCO3) was used, which not only solubilized the detergent-resistant membrane proteins, but permitted their direct proteolytic digestion using trypsin.19 As outlined above, the methodology used allowed for the identification of a significant number of transmembrane proteins (Table 2 and Supplementary Table 1 in Supporting Information). Use of buffered methanol also permitted tryptic cleavage within transmembrane domains. A hydropathy plot depicting the predicted transmembrane domains of Amino acid transporter system A2, a protein with 9 predicted transmembrane domains, is shown in Figure 6A. Both peptides from this protein that were identified by tandem MS confirmed that the sites within a predicted transmembrane domain were cleaved by trypsin (Figure 6A,B). The MS spectrum obtained from the 354


Figure 6. Secondary structure prediction of Amino acid transporter system A2. (A) Display of the TMHMM analysis of Amino acid transporter system A2 showing predicted transmembrane domains in red (probability > 0.8). Green bars under the x-axis display the location of the two peptides identified in this study. (B) Amino acid sequence of Amino acid transporter system A2. The italicized, bolded sequence depicts the tryptic peptides identified in this study, and the underlined portion represents the two predicted transmembrane domains that within which tryptic cleavage occurred.

1600-1800 m/z gas-phase fraction from which one of these peptides was selected, is shown in Figure 7A. The tandem MS spectrum generated by CID of the selected parent ion (m/z 1742.9) is shown in Figure 7B. A second peptide from Amino acid transporter system A2 was isolated in the 800-1000 m/z gas-phase fraction and identified through the tandem MS spectrum shown in Figure 7C. Analysis of Lipid Microdomain Protein Interactions. Given that the proteins identified in this study were isolated from microdomains following activation of a specific receptor, one would predict that many of the proteins should interact either physically or functionally. To assess potential interactions between the proteins identified within lipid microdomains, Ingenuity Pathways Analysis software was used to interrogate a structured database of biological networks. Forty-eight networks (with a maximum of 35 genes per network) were generated containing proteins identified in this study. Of the networks generated, 9 utilized 10 or more genes identified in LPS-activated endothelial lipid microdomains. An example of one network generated along with the subcellular localization of the proteins identified is shown in Figure 8. Arrows represent a functional interaction between proteins and lines connecting nodes indicate a physical interaction identified in a mammalian system. As seen in Figure 8 multiple protein interactions are identified that impinge on β-catenin (CTNNB1). β-catenin is localized to adherens junctions or the cytosol, but following activation by various stimuli translocates to the nucleus where

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Figure 7. Mass spectrometric identification of peptides from Amino acid transporter system A2. (A) Truncated mass spectrum of the 1600-1800 m/z gas-phase fraction highlighting the Amino acid transporter system A2 peptide molecular ion selected for CID (m/z ) 1742.9, arrow) and its 13C isotopes (m/z ) 1743.8 and 1744.9). (B) Tandem mass spectrum of the [M+H]1+ peptide molecular ion selected in (A) depicting the b- and y-ions identified. (C) Tandem mass spectrum of the second [M+2H]2+ peptide molecular ion from Amino acid transporter system A2 selected for CID.

it modulates transcription.34 Recently, it has been shown that LPS activates the protein kinase Akt, which results in nuclear accumulation and transcriptional activation of β-catenin.35 HSP90 (HSPCA) has been shown to interact with Akt in endothelial cells, and HSP90 also clusters in lipid rafts with CD14 in response to LPS.36,37 Thus, global proteomic analyses such as presented here can assist in generating hypotheses regarding mechanisms of activation of downstream signals, as

well as providing insights into potential cross-talk between identified proteins and canonical pathways.

Discussion We describe a relatively rapid method to analyze detergentresistant lipid microdomain fractions from small samples in a global fashion using MS-based proteomic technologies. Several elements of the experimental design presently described have Journal of Proteome Research • Vol. 4, No. 2, 2005 355

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Figure 8. Interaction network of a subset of identified proteins in LPS-stimulated HUVEC lipid microdomains. Interconnecting lines between nodes identify physical interactions and arrows represent physical and functional interactions. Proteins are identified by their gene names. + indicates that the protein interacts in additional networks generated in this analysis (not shown).

resulted in, to our knowledge, the most comprehensive analysis of lipid microdomains in endothelial cells. First, the choice of detergent, and its appropriate concentration, used to lyse endothelial cells by monitoring the partitioning of Caveolin-1 ensures optimal lipid microdomain purification. Removal of nuclear material greatly reduces contaminating proteins from intact nuclei that may separate in the low-density fraction in which lipid microdomains are found.27 While nuclear pelleting may have reduced the total number of raft and raft-associated proteins identified, the identifications that remain are much more likely to constitute bona fide lipid microdomain proteins, as significantly fewer nuclear proteins are observed compared to previous studies.27,28 The use of a buffered organic solvent facilitates the miscible extraction, solubilization and tryptic digestion of integral membrane proteins in a fashion compatible with downstream separations and µRPLC-MS/MS.19 Finally, GPFm/z permits the identification of low-abundant peptide molecular ions when sample quantity is limited and multidimensional LC separations are less feasible.20,21 The results of this study are validated, both by our own immunoblotting data and by specific results published by others. For instance the heat shock proteins, HSP70 and HSP90, have previously been shown to be involved in a CD14independent LPS receptor cluster in epithelial cells and monocytes.38 These proteins have been demonstrated to be present in lipid rafts of macrophages.8 Our results demonstrate that LPS-stimulated endothelial cells also contain HSP70 and HSP90 (R and β) within lipid raft fractions, and permit hypotheses to be generated relating to their function in endothelial signaling based on their interaction with other identified lipid micro356


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domain proteins. Further we demonstrate that HSP60 and HSC71 are also present within lipid microdomain fractions. The exact role of these various heat shock proteins in LPS signaling in endothelial cells remains to be determined, but they may be involved in trafficking LPS to the Golgi apparatus.39 Additional supporting evidence that the proteins identified in this study are, in the majority, true lipid microdomain proteins comes from a recent study demonstrating a role for the membrane receptor, Nogo (Neurite outgrowth inhibitor), in vascular remodeling.40 Nogo was found to be expressed in endothelial and vascular smooth muscle lipid microdomains, and our findings agree with this subcellular localization. Interestingly, the LPS receptor has also recently been shown to be necessary for outward vascular remodeling, and it is tempting to speculate that the above receptors interact functionally in promoting vascular remodeling.41 Multiple other proteins known to reside in lipid microdomains were found in the current study (Table 1), and we established the localization of two additional proteins, CD31 and Dysferlin, not previously identified as components of lipid microdomains. Preliminary data indicate that qualitative proteomic analysis of detergentresistant membrane domains can be extended to quantitative profiling when coupled with 18O stable isotope labeling.16 Our current studies are designed to exploit this technology to further understand the lipid microdomain function in endothelial cells. While we have focused on proteins containing transmembrane domains in this study, it is clear that proteins can also partition to lipid rafts through glycolipid interactions.42 For instance, proteins can associate with lipid rafts in the outer leaflet of lipid bilayers by glycosylphosphatidylinositol anchors, or in the inner leaflet by acylation, palmitolyation, or direct interaction with cholesterol.43-45 Unfortunately, algorithms that predict these types of interactions are in their infancy, and cannot reliably be used to assess the data in this report. However, a recent study outlines a novel strategy to target glycosylphosphatidylinositol-anchored membrane proteins for proteomic analysis, and may be a useful strategy toward defining the endothelial lipid microdomain proteome.46 By pelleting nuclei, we have eliminated a large source of contamination of lipid raft preparations seen in many studies. However, this maneuver has its own repercussions, as it is clear that the plasma membrane directly or indirectly connects with cytoplasmic membranes.47 Thus, physical removal of one subfraction i.e., nuclei, may in turn remove other membrane fractions containing lipid raft proteins. Conversely, it is clear that lipid rafts are also present in intracellular membranes, and intracellular proteins present in the low-density fractions may represent contamination or true lipid raft proteins from the cell interior.10 An example of this “contamination” in our study is the identification of NADH-ubiquinone oxidoreductases which are constituents of the mitochondrial respiratory chain. Finally, membrane disruption during lipid raft isolation is not homogeneous, and undoubtedly lipid microdomain fractions contain adjacent membrane fractions that are not functional constituents of the lipid raft, and thereby provide another potential source of cross-contamination. The best way to isolate uncontaminated lipid microdomains remains a controversial issue, and it is axiomatic that various independent methods will be required to confirm protein localization and function. However, it has been suggested that the pH/carbonate-resistant method of lipid microdomain isolation results in a much greater proportion of contaminating proteins than the detergent-resistant method.27 Recently, an

research articles


elegant and elaborate study mapped proteins on the luminal surface of endothelial cells in vivo.48 The method used in this study however, utilized cationic silica which results in effective isolation of membrane constituents, but there is significant contamination with endoplasmic reticulum proteins.31 Ultimately, direct visualization using high-resolution imaging methods to demonstrate discrete spatial and temporal localization of specific proteins will be required to achieve a comprehensive and dynamic view of lipid microdomains and the specifics of their contribution to signal transduction.

Acknowledgment. This work was supported by grants to A.K. from the Heart and Stroke Foundation of British Columbia and the Yukon and the Canadian Institutes of Health Research and in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400.A.K. is supported by a personnel award from the Heart and Stroke Foundation of Canada and a Scholarship from the Michael Smith Foundation for Health Research. Supporting Information Available: Identification of the 894 unique peptides corresponding to 358 unique proteins, the raft-associated proteins and putative and potential lipid microdomain proteins, and a number of small GTP-binding proteins and heterotrimeric G proteins, which are known to localize to lipid microdomains (Supplementary Table 1, pdf). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Janeway, C. A., Jr.; Medzhitov, R. Annu. Rev. Immunol. 2002, 20, 197-216. (2) Lei, M. G.; Gao, J. J.; Morrison, D. C.; Qureshi, N. Mo. Med. 2003, 100, 524-529. (3) Glauser, M. P. Crit. Care Med. 2000, 28, S4-8. (4) Reinhart, K.; Bayer, O.; Brunkhorst, F.; Meisner, M. Crit. Care Med. 2002, 30, S302-312. (5) Levi, M.; ten Cate, H.; van der Poll, T. Crit. Care Med. 2002, 30, S220-224. (6) Wong, P. M.; Chugn, S. W.; Sultzer, B. M. Scand. J. Immunol. 2000, 51, 123-127. (7) Beutler, B. Curr. Top. Microbiol. Immunol. 2002, 270, 109-120. (8) Triantafilou, M.; Miyake, K.; Golenbock, D. T.; Triantafilou, K. J. Cell. Sci. 2002, 115, 2603-2611. (9) Pugin, J.; Schurer-Maly, C. C.; Leturcq, D.; Moriarty, A.; Ulevitch, R. J.; Tobias, P. S. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 27442748. (10) Pike, L. J. J. Lipid Res. 2003, 44, 655-667. (11) Simons, K.; Toomre, D. Nat. Rev. Mol. Cell Biol. 2000, 1, 31-39. (12) Pike, L. J. Biochem. J. 2004, 378, 281-292. (13) Carver, L. A.; Schnitzer, J. E. Nat. Rev. Cancer 2003, 3, 571-581. (14) Sowa, G.; Pypaert, M.; Sessa, W. C. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 14072-14077. (15) Triantafilou, K.; Fradelizi, D.; Wilson, K.; Triantafilou, M. J. Virol. 2002, 76, 633-643. (16) Blonder, J.; Conrads, T. P.; Veenstra, T. D. Expert Rev. Proteomics 2004, 2, 153-163. (17) Karsan, A.; Yee, E.; Kaushansky, K.; Harlan, J. M. Blood 1996, 87, 3089-3096. (18) Noseda, M.; McLean, G.; Niessen, K.; Chang, L.; Pollet, I.; Montpetit, R.; Shahidi, R.; Dorovini-Zis, K.; Li, L.; Beckstead, B.; Durand, R. E.; Hoodless, P. A.; Karsan, A. Circ. Res. 2004, 94, 910917.

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Journal of Proteome Research • Vol. 4, No. 2, 2005 357

Proteomic Analysis of Lipid Microdomains from Lipopolysaccharide

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