Quantitative Profiling of the Detergent-Resistant Membrane

against a nonredundant Homo sapiens proteome database (http://www.ebi.ac.uk/ integr8/EBI-Integr8-HomePage.do (release date 12/08/03). A dynamic ...
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Quantitative Profiling of the Detergent-Resistant Membrane Proteome of Iota-b Toxin Induced Vero Cells Josip Blonder,† Martha L. Hale,‡ King C. Chan,† Li-Rong Yu,† David A. Lucas,† Thomas P. Conrads,† Ming Zhou,† Michel R. Popoff,§ Haleem J. Issaq,† Bradley G. Stiles,‡ and Timothy D. Veenstra*,† Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702-1201, Toxinology Division, Department of Immunology and Molecular Biology, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, 21702-5011, and Anaerobic Bacteria and Toxins Unit, Institut Pasteur, 28 rue Docteur Roux, Paris, 75724 France Received November 17, 2004

Enzyme-mediated 18O/16O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an 18O/16O labeling strategy was optimized for and applied to the solutionbased comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and Iota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the 18O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake. Keywords: membrane proteins • quantitation •

18O/16O

Introduction Changes in gene expression is one of the primary mechanisms by which protein-regulated processes are modulated.1 The capability of simply identifying gene products on a largescale is insufficient to elucidate the physiological or pathological events that take place in a cell or organism. To garner a rudimentary understanding of the physiological effects of various stimuli on a cell system requires measurement of alterations in protein abundances.2 For these reasons, significant developmental efforts have resulted in a number of promising proteomic approaches that enable measurement and comparison of relative abundances of hundreds, if not thousands, of proteins in a proteome.3 * To whom correspondence should be addressed. E-mail: veenstra@ ncifcrf.gov. Phone: (301) 846-7286. Fax: (301) 846-6037. † Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick. ‡ Toxinology Division, Department of Immunology and Molecular Biology, U.S. Army Medical Research Institute of Infectious Diseases. § Anaerobic Bacteria and Toxins Unit, Institut Pasteur. 10.1021/pr049790s CCC: $30.25

 2005 American Chemical Society

labeling • lota-b toxin

A remarkable effort in proteomics has been expended recently at developing methods better suited to investigate membrane proteins using mass spectrometry (MS). Though widely varied in their respective experimental details, these novel membrane proteomic methodologies have allowed unprecedented characterization of many various membrane proteomes by MS.4-13 The membrane proteome represents a dynamic assembly of proteins that act as molecular effectors in accordance to the given cell cycle or ongoing cellular responses to different external and/or internal stimuli. In eucaryotic cells, biological processes are specifically localized within various membranous organelles while their temporal regulation is provided by functionally and morphologically distinct membrane microdomains commonly referred to as lipid rafts.14 Due to a well-known bias of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) against insoluble proteins, this quantitative profiling technique is not well suited for largescale qualitative or quantitative membrane proteomics.10,15 Although advancements have been made in the use of gelJournal of Proteome Research 2005, 4, 523-531

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research articles based techniques for studying membrane proteins, the use of solution-based sample preparation strategies, in conjunction with micro-separations coupled to tandem MS (MS/MS) for characterization of membrane proteins, has been increasingly employed by various laboratories.16 A variety of differential isotopic labeling strategies have been used in combination with these approaches to determine the relative abundances of membrane proteins. These strategies have included isotopecoded affinity tags for measuring changes in microsomal proteins during cell differentiation17 and metabolic labeling of cells in culture to identify lipid raft-specific proteins.18 Differential labeling using normal and heavy isotopic forms of water (H216O and H218O) represents an intriguing method for quantitatively comparing proteome samples due to its inherent simplicity. Trypsin-mediated 18O/16O differential stable isotope labeling is based on enzyme catalyzed exchange of two equivalents of 16O at the C-termini of cleaved peptides for two equivalents of 18O, during or after proteolysis in the presence of H218O. This exchange results in a 4 amu mass difference between the two isotopomeric peptides. This method has recently been used to conduct quantitative proteomic comparison of cytoplasmic proteins isolated from normal and metastatic breast cells19 and control and doxorubicin-resistant MCF-7.20.21 Clostridium perfringens iota-toxin consists of two separate proteins; the cell-binding protein iota b (Ib), which forms highmolecular-weight complexes in cell membranes generating Na(+)/K(+)-permeable pores that presumably allow a second protein, iota a (Ia), an ADP-ribosyltransferase, to enter the cytosol.22 The enzymatic properties of Ia have been well studied, however, there has been little understanding of how Ib targets the cell membrane and facilitates Ia entry into the cytosol. While the receptor(s) for Ib have not been identified, it is known that this protein targets detergent-resistant membrane (DRM) microdomains on the cell surface.23 In this study, a “single tube” approach5 developed for membrane proteomic sample preparation was used in combination with trypsinmediated 18O labeling24 to quantitatively determine changes in the DRM proteome (DRMP) of Vero cells induced with Iota b toxin.25 A buffered methanol solution was utilized to solubilize and tryptically digest proteins from the Triton X-100 resistant membrane proteome followed by trypsin-mediated 18O/16O exchange to enable a quantitative measurement of the changes in the protein complement of the DRMP extracted from Ibinduced (18O) and control (16O) Vero cells. The isotopically labeled samples were combined and the digestate was fractionated using off-line strong cation exchange (SCX) chromatography followed by microcapillary reversed-phase liquid chromatography (µRPLC)-MS/MS analysis of each fraction.

Experimental Procedures Materials. Vero cells were obtained from the American Type Culture Collection (Manassas, VA) and bacteriorhodopsin (BRWT) from MIB GmbH (Marburg, Germany). 18O-water (95% v/v) was purchased from Isotec/Sigma (St. Louis, MO); sequencing grade trypsin was obtained from Promega (Madison, WI); protease inhibitor cocktail (i.e., aprotinin, leupeptin, pepstatin, and Prefabloc SC) was obtained from Roche (Indianapolis, IN); micro BCA protein assay reagents and tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) were purchased from Pierce (Rockford, IL); ammonium bicarbonate (NH4HCO3), Triton X-100 (TX-100), iodoacetamide (IAA), ethylenediamine524

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tetraacetic acid (EDTA), formic acid (HCOOH), trifluoroacetic acid (TFA) and phosphate buffered saline (PBS) were obtained from Sigma (St. Louis, MO); high performance liquid chromatography (HPLC) grade methanol (CH3OH) and acetonitrile (CH3CN) were purchased from EM Science (Darmstadt, Germany); and ultrapure H2O was prepared using a Barnstead purification system (Dubuque, IA). Cell Culture. Cells were detached from culture flasks with 50 mM EDTA in Hank’s balanced salt solution (HBSS) lacking calcium and magnesium. Single-cell suspensions (5 × 107/mL) were incubated with 125 ng Iota b (Ib) toxin for 10 min at 37 °C, as previously described.6 Unbound Ib was removed by three washes with ice-cold (4 °C) HBSS containing 0.2% (w/v) bovine serum albumin (BSA). Control single-cell suspensions were incubated with HBSS + 0.2% (w/v) BSA not containing Ib. Isolation of Detergent Resistant Membrane Proteome (DRMP). The procedure developed by Aman and Ravichandran was used to isolate Vero cell DRMP.26 Briefly, Vero cells were solubilized in 750 µL of cold (4 °C) 25 mM Tris-HCl, pH 7.6, containing 0.5% Triton X-100, 10 mM sodium fluoride, 30 mM sodium pyrophosphate (Na4P2O7), 10 mM β-glycerophosphate, 150 mM sodium chloride, and 0.1% (v/v) phosphatase and protease inhibitor cocktail. After incubating cell suspensions at 4 °C for 60 min, the lysate was mixed with an equal volume of 80% sucrose, placed in a polyallomer centrifuge tube (Beckman-Coulter, Fullerton, CA), and overlaid with 1.5 mL each of 30% and 5% sucrose solutions. After 17 h of centrifugation at 215 000 × g, the Triton X-100 insoluble fraction was collected using a wide-bore pipet tip, and centrifuged for an additional 30 min at 16 000 × g. The pellet was resuspended in 50 µL of PBS, pH 7.4 and the protein concentration determined using the BCA assay. Two 50 µg aliquots of proteins extracted from the Ib incubated and control cells were prepared and stored at -80 °C. All procedures in the DRMP preparation were performed at 4 °C. For these studies, the Ib treated DRMP will be designated as “induced” DRMP because external stimuli have been shown to stimulate sequestering of specific proteins within DRMP.27 Solubilization, Proteolysis, and 18O/16O Labeling of Proteins from DRMP. The Triton X-100 insoluble DRMPs extracted from the control and Ib-induced Vero cells, were solubilized and tryptically digested as previously described.6 Briefly, each pellet was solubilized in 500 µL of 50 mM NH4HCO3, reduced using 1 mM TCEP-HCl (final concentration) and alkylated with 15 mM IAA (final concentration). Triton X-100 removal and buffer exchange was carried out on control and Ib induced samples by ultracentrifugation (Beckman Optima TX Ultracentrifuge, Rotor: TLA 55) using three consecutive washes with 1.5 mL of 25 mM NH4HCO3, pH 7.9. The final pellets were resuspended in 100 µL of 60% CH3OH/40% 25 mM NH4HCO3 (pH 7.9) and the DRMPs solubilized using intermittent sonication and vortexing. The proteins were digested overnight at 37 °C by direct addition of trypsin to the microcentrifuge tube using 1:20 enzyme/protein ratio. Each aliquot was lyophilized and 18O exchange carried out on the Ib induced sample by adding 100 µL of 20% CH3OH /80% 25 mM NH4HCO3 (pH 7.9) prepared in H218O using the same trypsin/protein ratio (1:20). The identical procedure was carried out on the control sample using 20% CH3OH/80% 25 mM NH4HCO3 (pH 7.9) prepared in H216O. The exchange reactions were quenched by boiling the samples for 10 min in a water bath, followed by addition of TFA (0.4%

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Quantitative Proteomics of Lipid Rafts

final concentration). The Ib induced (18O-labeled) and control (16O-labeled) samples were immediately combined and lyophilized. Strong Cation Exchange Liquid Chromatography (SCX-LC) Fractionation of 18O/16O Labeled Peptides. The combined sample was reconstituted in 100 µL of 45% CH3CN containing 0.1% HCOOH immediately prior to SCX-LC. The sample was resolved into 10 fractions using a microcapillary LC system (Model 1100, Agilent Technologies Inc., Palo Alto, CA). Peptide fractions were eluted with an ammonium formate/multistep gradient at a flow rate of 200 µL/min as follows: 1% B/0-2 min, 10% B/62 min, 62% B/82 min, 100% B/85 min. Mobile phase A was 45% (v/v) CH3CN and mobile phase B was 45% (v/v) CH3CN containing 0.5 M ammonium formate, pH 3. The SCX-LC fractions were lyophilized to dryness and reconstituted in 0.1% formic acid immediately prior to µRPLC-MS/MS analysis. Liquid Chromatography-Tandem Mass Spectrometry Analysis of 18O/16O Labeled Peptides. Microcapillary RPLC separations were carried out using a 75 µm i.d. × 10 cm long fused silica capillary (Polymicro Technologies Inc., Phoenix, AZ) column with a flame pulled 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) and µRPLC was performed using an Agilent 1100 LC system. After injecting 8 µL of sample, the column was washed for 30 min with 98% mobile phase A (0.1% v/v HCOOH) and peptides eluted using a linear step gradient from 2 to 60% mobile phase B (0.1% HCOOH in CH3CN) over 100 min and 60-98% mobile phase B over 20 min at a constant flow rate of 0.5 µL/min. The column was washed for 20 min with 98% mobile phase B and reequilibrated with 2% mobile phase B for 30 min prior to subsequent sample loading. The µRPLC column was coupled to an ion-trap (IT) mass spectrometer (LCQ Deca XP, ThermoElectron, San Jose, CA) using a nanoelectrospray ionization source with an applied potential of 1.7 kV, and capillary temperature of 160 °C. The IT mass spectrometer 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 subjected to collision induced dissociation (CID) with a relative energy of 36%. Data Processing. The CID spectra were analyzed using SEQUEST, on a Beowulf 18-node parallel virtual machine cluster computer (ThermoElectron), against a nonredundant Homo sapiens proteome database (http://www.ebi.ac.uk/ integr8/EBI-Integr8-HomePage.do (release date 12/08/03). A dynamic modification of 4 amu was set on the carboxy-terminal of each peptide representing a mass change of 4 amu for all 18 O-labeled peptides. In addition, a dynamic modification of 57 amu was added for heavy and light labeled peptides for cysteinyl (Cys) residues labeled by IAA. Only peptides possessing tryptic termini (allowing for up to two internal missed cleavages) possessing delta-correlation scores (∆Cn) g 0.8 and charge state-dependent cross correlation (Xcorr) criteria as follows were considered as legitimate identifications: g1.8 for [M+H]+1 peptides, g2.2 for [M+H]+2 peptides, and g2.9 for [M+H]+3 peptides. Relative abundances of identified and 18O/ 16 O labeled peptide pairs were quantified using XPRESS (ThermoElectron). The grand average of hydropathy (GRAVY) index for selected proteins and peptides was calculated using ProtParam (http:// us.expasy.org). Species exhibiting positive GRAVY indices are

considered hydrophobic and those with negative indices are deemed hydrophilic. The mapping of R-helical integral membrane proteins was performed using the transmembrane hidden Markov model (TMHMM) algorithm (http://www.cbs.dtu.dk/services/TMHMM). Pathway analysis was performed using PathwayAssist (http://www.stratagene.com) (Strategene, La Jolla, CA).

Results and Discussion Utility of Trypsin-Catalyzed 18O Labeling for Membrane Proteomics. Trypsin-mediated 18O labeling is based on enzymecatalyzed exchange of two equivalents of 16O at the C-terminus of a tryptic peptide for 18O. This exchange results in a 4 amu difference between the two isotopomeric peptides.24 Our group recently developed a single tube-based proteomic approach for isolation and solubilization of DRMP that is compatible with multidimensional fractionation (i.e., SCX followed by µRPLC) prior to MS analysis.8,9 Although this method appears quite promising for simple protein identification, it relies on a high concentration of organic solvent to solubilize membrane proteins, which results in a significant reduction in trypsin activity.5 Because of this diminished tryptic activity, and the observation that 18O labeling historically suffers from incomplete isotope exchange,28,29 refinement of this isotopic labeling protocol on a model integral membrane protein was warranted prior to its application to complex membrane proteome samples. To meet this need, an aliquot of the well-characterized membrane protein bacteriorhodopsin was solubilized in 60% CH3OH/25 mM NH4HCO3 prepared in H216O while a second aliquot was solubilized in 60% CH3OH/25 mM NH4HCO3 prepared in H218O. The samples were digested with trypsin, combined at a 1:1 ratio, and analyzed by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDITOF/TOF) MS. The mass spectra of two selected pairs of 18O/ 16 O labeled bacteriorhodopsin peptides are shown in Figure 1A. The mass spectrum of the differentially labeled doublet corresponding to the membrane spanning peptide VGFGLILLR shows complete 18O incorporation, however, the mass spectrum of the peptide AESMRPEVASTFK revealed a small population of peptides containing a single equivalent of 18O at the C-terminus (marked by an asterisk), indicating incomplete incorporation of the 18O isotope. As mentioned previously, the addition of 60% CH3OH to the buffer solution decreases trypsin activity in comparison to a purely aqueous buffer, however, the same study showed that the addition of 20% CH3OH enhances the activity of trypsin as previously determined using a N-benzoyl-L-arginine ethyl ester assay.5 Therefore a scheme was developed to decouple digestion of the membrane sample and the enzyme-mediated incorporation of 18O. In this procedure both bacteriorhodopsin aliquots were tryptically digested in 60% CH3OH/25 mM NH4HCO3 prepared in H216O. Following lyophilization one aliquot was resuspended in 20% CH3OH/25 mM NH4HCO3 prepared in H216O while another aliquot was resuspended in 20% CH3OH/25 mM NH4HCO3 prepared in H218O. Trypsin was then added to catalyze the 18O-exchange reaction. The MALDI-TOF/TOF MS spectra of the same two peptides showed in Figure 1A revealed complete 18O incorporation using this decoupled labeling scheme, as shown in Figure 1B. This optimized isotopic labeling procedure that separates tryptic digestion of the sample from 18O-incorporation was applied for 18O/16O differential labeling of Ib induced and Journal of Proteome Research • Vol. 4, No. 2, 2005 525

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Figure 1. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS spectrum of a selected pair of bacteriorhodopsin peptides (R.VGFGLILLR and K.AESMRPEVASTFK) differentially labeled with 18O/16O water. (A) After initial digestion in 60% buffered methanol the MALDI-TOF mass spectrum of the 18O/16O labeled membrane spanning peptide (R.VGFGLILLR.S) pair indicates complete incorporation while the mass spectrum of the peptide K.AESMRPEVASTFK suggests incomplete 18O incorporation (as indicated by asterisk). (B) After the second digestion in 20% buffered methanol the present mass spectrum shows complete 18O exchange of both peptides.

control DRMP. To evaluate the efficiency of the 18O-isotope incorporation, an aliquot of the differentially labeled sample was analyzed using MALDI-TOF/TOF MS prior to SCX-LC fractionation. The mass spectra of selected DRMP 18O/16O labeled peptides from the control and induced samples indicate thorough 18O incorporation shown in Figure 2. We hypothesize that complete 18O incorporation, as indicated by the mass spectra in Figures 1B and 2, may be attributed to the observed increase in trypsin activity in 20% buffered methanol.5 It is necessary, however, to conduct the initial digestion of the membrane protein sample in the presence of 60% CH3OH to maintain protein solubility until more soluble peptides are liberated. Our laboratory is presently conducting three other global membrane quantitation studies, not including the one presented below, using this approach and have yet to observe any significant or obvious mis-incorporation of the intended isotope label. While these projects are not analyzed to the point 526

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of whether crucial biological information will be discovered, the analytical methods being used appear quite reproducible and robust. Microcapillary RPLC-ESI-MS/MS Analysis of 18O/16O Labeled Peptides. In this investigation the relative abundances of proteins within DRMPs isolated from Ib-induced Vero cells were compared to DRMPs isolated from control (untreated) cells. A schematic of the methodology employed in this investigation consisting of conventional detergent based isolation of DRMP, simple buffer exchange followed by solubilization, digestion and trypsin-specific 18O exchange for differential isotopic labeling of the peptides is shown in Figure 3. The combined DRMP sample was fractionated by SCX-LC into 10 fractions. Analysis of these fractions by µRPLC-ESI-MS/MS resulted in the identification and quantitation of 1417 peptides, corresponding to 585 unique proteins. A selected list of quantified multipass integral membrane proteins is shown in

research articles

Quantitative Proteomics of Lipid Rafts

Figure 2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrum showing complete extracted from detergent resistant membrane domains from Ib-induced Vero cells.

Figure 3. Outline of the induced Vero cells.

18O/16O

18O

incorporation of peptides

proteolytic labeling procedure used for multidimensional comparative membrane proteomics of Ib-

Table 1. Each of these well-documented membrane proteins were identified by several peptides confirming the utility of the present methodology for conducting quantitative measurements of membrane proteomes. The 18O/16O peptide ratios were calculated from relative abundances of the identified

peptides based on the integrated areas from the extracted ion chromatograms (XICs) of each corresponding peptide molecular ion as calculated by XPRESS. Since differential 18O/16O peptide ratios can be artifactually influenced by many experimental factors such as sample mixing, 18O exchange efficiency, Journal of Proteome Research • Vol. 4, No. 2, 2005 527

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Table 1. Subset of Multipass Integral Membrane Proteins Quantified Based on Ib-Induced Vero Cells acc. no.a

protein name and peptide sequenceb

P28288

ATP-binding cassette, sub-family D R.NSSLAGAAFLLLC*LLHK.R R.NSSLAGAAFLLLC*LLHK**.R Clathrin-coated vesicle proton pump R.GTEHSGSTVPSILNR.M R.GTEHSGSTVPSILNR**.M Facilitated glucose transporter 1 R.GTADVTHDLQEMK.E R.GTADVTHDLQEMK**.E IP3 receptor 3 K.HLATGNYLAAEENPSYK.G K.HLATGNYLAAEENPSYK**.G MDR 1 (P-glycoprotein 1) K.VQSGQTVALVGNSGC*GK.S K.VQSGQTVALVGNSGC*GK**.S Microsomal glutathione S-transferase, GIST III R.IASGLGLAWIVGR.V R.IASGLGLAWIVGR**.V R.AHQNTLEVYPPFLFFLAVGGVYHPR.I R.AHQNTLEVYPPFLFFLAVGGVYHPR**.I R.GALGSIALLGLVGTTVC*SAFQHLGWVK.S R.GALGSIALLGLVGTTVC*SAFQHLGWVK.**S Sodium/potassium-transporting ATPase K.VDNSSLTGESEPQTR.S K.VDNSSLTGESEPQTR**.S Vacuolar proton translocating ATPase K.AAQNEIWQTFFR.G K.AAQNEIWQTFFR**.G

Q93050 P11166 Q14573 P08183 O14880

P05023 Q13488

TMc

3 7 12 7 10 4

10 8

18O/16O

Differential Labeling of Control and

ARd

SDe

T/uf

0.81

0.19

8/4

1.53 0.94 0.82 1.28 1.00

0.95 1.27

0.27 0.29 0.24 0.46 0.25

0.34 0.40

[M+H]+ g

chgh

Xcorri

1827.994 1831.994

2 2

5.2710 5.7361

1554.787 1558.787

2 2

3.5420 3.7010

1444.674 1448.674

2 2

3.9841 4.4170

1877.903 1881.903

2 2

4.8551 4.7840

1661.807 1665.807

2 2

4.4160 4.7560

1312.774 1316.774 2872.493 2876.493 2755.475 2759.475

2 2 3 3 2 2

2.4135 4.5510 5.5925 6.0062 4.2376 4.3649

1619.751 1623.751

2 2

3.9029 4.7483

1510.744 1514.744

2 2

3.2747 3.0718

15/8 20/8 16/5 33/11 13/3

25/11 4/3

* iodoacetamide alkylated cysteinyl residue, **18O labeled C-terminus of corresponding peptide). a ExPASy database accession number. b The peptides identified and quantified for a particular protein with the highest Xcorr value. c Number of mapped transmembrane domains using TMHMM algorithm. d Abundance ratio (i.e., 18O4/16O0 ratio) of protein. e Standard deviation. f A total number vs. number of unique peptides identified from a particular protein. g Calculated mass. h Charge state of parent ion. i Xcorr score.

MS instrument resolution, mass measurement accuracy, and design of MS/MS method, thresholds were statistically determined above which and below which a change in the measured peptide abundance is considered significant.30 A plot of the normalized 18O/16O ratio of the labeled peptides followed by a nonlinear least-squares regression analysis showed that the upper limit above which a measured 18O/16O ratio reflects a significant increase in peptide abundance was determined to be g1.52 with a lower limit e 0.65 (Figure 4). The factors that contribute to the ultimate measurement of the protein’s relative abundance will be unique within a specific study, therefore, this threshold should be calculated for each proteomic quantitative analysis.9 A complete list of the peptides identified in this study along with their related SEQUEST scoring parameters is given in Supplementary Table 1, in the Supporting Information. The complete list of quantified proteins and their relative abundance ratio between Ib-induced and untreated Vero cells is presented within Supplementary Table 2, in the Supporting Information. The utility of the single-tube technique for quantitative proteomics of integral membrane proteins associated with DRMPs is illustrated by the comparative measurement of the relative abundance of microsomal glutathione S-transferase 3 (GST-III) in control and Ib-induced Vero cells. GST-III is a wellcharacterized membrane protein involved in the activation of xenobiotics when conjugated with glutathione.31,32 Three known membrane spanning peptides covering 42% of the sequence of GST-III were identified, as shown in Figure 5. The XICs of the light and heavy isotopomeric peptide ions of R.GALGSIALLGLVGTTVC*SAFQHLGWVK from GST-III are shown in Figure 5 (top). While no significant change was seen in this protein when Vero cells were induced with Ib, the isotopic 528

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Figure 4. Diagram showing the normalized ratio of trypsinspecific labeled peptide identifications as a function of their measured abundance ratios (18O/16O). The colored areas indicate peptide species whose abundance ratios were significantly increased (pink) or decreased (blue) within the proteomes of detergent resistant membrane domains of Vero cells in response to Ib toxin induction.

labeling method used in this study permitted quantitation of three unique peptides that spanned, or were part of, transmembrane sequences within this protein (Figure 5, bottom). This observation that transmembrane peptides are effectively quantitated using this approach, validates the ability of this

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Quantitative Proteomics of Lipid Rafts

Table 2. Subset of Lipid Raft Marker Proteins and Associated Signaling Effectors Quantified from Detergent Resistant Microdomains Isolated from Control and Ib-Induced Vero Cells acc. no.a

protein name and peptide sequenceb

ARc

SDd

T/Ue

P27824

Calnexin precursor K.AEEDEILNR.S K.AEEDEILNR**.S Caveolin-1 K.IDFEDVIAEPEGTHSFDGIWK.A K.IDFEDVIAEPEGTHSFDGIWK**.A CD44 antigen precursor K.SQEMVHLVNK.E K.SQEMVHLVNK**.E Clathrin heavy chain 1 R.RPISADSAIMNPASK.V R.RPISADSAIMNPASK**.V Flotillin-1 K.KAEAFQLYQEAAQLDMLLEK.L K.KAEAFQLYQEAAQLDMLLEK**.L Flotillin-2 K.IGEAEAAVIEAMGK.A K.IGEAEAAVIEAMGK**.A Guanine nucleotide-binding protein, G(i), R-1 K.DSGVQAC*FNR.S K.DSGVQAC*FNR**.S Neuronal axonal membrane protein K.APEQEQAAPGPAAGGEAPK.A K.APEQEQAAPGPAAGGEAPK**.A Stomatin R.YLQTLTTIAAEK.N R.YLQTLTTIAAEK**.N Tyrosine-protein kinase,Yes R.FQIINNTEGDWWEAR.S R.FQIINNTEGDWWEAR**.S

1.83

0.33

11/3

Q03135 P16070 Q00610 O75955 Q14254 P04898 P80723 P27105 P07947

0.98 3.23 1.21

0.17 0.61 0.23

[M+H]+ f

chg.g

Xcorrh

1088.522 1092.522

2 2

2.6494 3.4184

2405.130 2409.130

2 2

4.5668 4.0212

1184.609 1188.609

2 2

3.6161 3.4365

10/2 3/2 30/13 1557.806 1561.806

1.06 0.87 1.20 1.25 0.93 1.15

0.34 0.21 0.34 0.38 0.23 0.35

4.5573 4.4891

56/13 2339.196 2343.196

2 2

5.7278 4.4799

1388.710 1392.710

2 2

3.8718 5.2257

1153.485 1157.485

2 2

3.5335 3.9862

1775.856 1779.856

2 2

4.8877 4.1558

1351.747 1355.747

1 1

2.5659 2.5200

1878.878 1882.878

2 2

4.1798 4.1236

30/10 9/2 18/3 88/10 5/3

a ExPASy database accession number. b The peptides identified and quantified for a particular protein with the highest Xcorr value. c Abundance ratio (i.e.18O/ O ratio) of protein. d Standard deviation. e Total number vs. number of unique peptides identified from a particular protein. f Calculated mass. g Charge state of parent ion. h Xcorr score. 16

technology to provide comprehensive coverage of integral membrane proteins. The overall efficacy of the present approach for the quantitative analysis of DRMPs can be determined by calculating the percentage of integral membrane proteins contained within the dataset. Analysis of the dataset using the TMHMM algorithm33 identified 135 proteins predicted to contain between 1 and 14 R-helical membrane spanning domains. Of these, 73 (54%) were predicted to contain two or more transmembrane domains. In addition, all known caveolar and lipid raft marker proteins (i.e., caveolin, flottilin, etc.)34,35 were identified, as shown in Table 2. A number of proteins commonly associated with cholesterol-enriched plasma membrane microdomains (i.e., folate receptor, stomatin)34,36 were also quantitated in this study. Iota toxin is one of the four dermonecrotic proteins secreted by Clostridium perfringens, a gram-positive, anaerobic bacillus that causes various diseases in humans and animals.37 Iota toxin is composed of two independent proteins that include an enzyme, designated as iota a (Ia) and a cell binding binding/ translocation domain, designated as iota b (Ib) that together form the biologically active toxin.38 Iota a is responsible for cytotoxic effects arising from ADP-ribosylation of monomeric actin that subsequently prevents actin filament formation with eventual disruption of the cytoskeleton.39,40 Iota b facilitates Ia internalization into the cytosol by first binding to an unknown cell-surface protein receptor25 followed by oligomerization within the DRM microdomains.23 A total of 90 proteins from the Vero cell DRMP preparations were significantly upregulated whereas 63 proteins were down-regulated in response to Ib induction, based on the criteria shown in Figure 4. A select

subset of these proteins is shown in Table 3. Using the quantitative measurements obtained in this study, canonical protein pathway analysis software was employed to indicate possible physical or functional interactions within biological networks as a consequence of Ib treatment. Interestingly, CD44 a well-known cell surface receptor found in lipid rafts41 that has been shown to mediate tissue invasion of Streptococcus pyogenes42 and Shigella infection43 was found to be upregulated approximately 3-fold in Vero cells induced with Ib. A recent study has shown that treatment of a renal tubular epithelial carcinoma cell line with Shiga toxin unit 1-B, which acts in an analogous manner as the Ib unit of iota toxin, resulted in the redistribution and increased abundance of CD44 within the plasma membrane (http://jcs.biologists.org/cgi/content/full/ 117/17/3911). The molecular chaperone calnexin was also observed to be upregulated in Vero cells induced with Ib. Calnexin has been shown to bind to rotavirus enterotoxin44 or become hyperphosphorylated in cells treated with Clostridium difficile cytotoxin.45 CD44 expression has been previously shown to be higher in calnexin positive cell lines suggesting that the increased expression of CD44 in Ib-induced Vero cells could be related to calnexin upregulation.46 Several guanine nucleotide-binding regulatory proteins were identified in this comparative study, however, few showed any change in abundance when the cells were induced with Ib. Guanine nucleotide-binding regulatory protein GoR subunit 1 and G-protein-regulated inducer of neurite outgrowth (GRIN; gene product KIAA1893), however, were both found to be upregulated approximately 3-fold in Ib-induced cells. GoR protein has been implicated in the regulation of ion channels47 and its levels are known to be increased by membrane Journal of Proteome Research • Vol. 4, No. 2, 2005 529

research articles

Blonder et al.

Figure 6. Pathway map showing the connectivity between Guanine nucleotide-binding regulatory protein GoR subunit 1 (GoR) G-protein-regulated inducer of neurite outgrowth (GRIN), and CD44.

proteins such as caveolin and flotillin, as shown in Figure 6. It is possible that any of these proteins could function in a concerted manner to propagate the effects of Ib binding onto the surface of Vero cells. Figure 5. Quantitation of the relative abundance of microsomal glutathione-S-transferase 3 (GST-III). Upper. Extracted ion chromatograms of light and heavy isoform molecular ions of the doubly charged peptide R.GALGSIALLGLVGTTVC*SAFQHLGWVK, revealing an 18O/16O ratio of 1.0. Lower left. Table showing the unique peptides identified from GST-III and their relative abundance ratios within the detergent-resistant membrane proteome samples isolated from untreated and Ib-induced Vero cells. Lower right. Sequence of GST-III highlighting this protein’s transmembrane domains (red) and the peptides quantitated in this study (underlined). Table 3. Subset of over Expressed and under Expressed Proteins Quantified from Vero Cell Detergent Resistant Microdomains Employing 18O/16O Differential Labeling of Control and Ib-Induced Samples acc. no.a

protein description/gene name

ARb

SDc

NPId

SCe

P05111 P16070 P27824 P29777 Q96PZ4 P11233 Q9Y2A7 P01121 P26006

Inhibin R chain (INHA) CD44 Calnexin Guanine-protein GoR (subunit 1) GRIN Ras-related protein Ral-AC (RALA) Nck-associated protein 1 (NAP 1) Transforming protein (RhoB) Integrin R-3 precursor (ITGA3)

6.27 3.23 1.83 3.04 2.70 0.35 0.47 0.54 0.58

2.61 0.61 0.33 3.57 n.a. 0.11 0.01 0.11 0.03

7 3 11 3 1 3 2 5 3

XCLf IMPg IMPg IMPg IMPg MAPh MAPh MAPh IMPg

a ExPASy database accession number. b Average 18O/16O ratio for particular protein. c standard deviation. d number of peptides identified. e subcellular localization. f extracellular. g integral membrane protein. h membrane associated protein.

depolarization.48 Little functional information is known about GRIN, however, this protein has been shown to interact with GoR subunits as well as subunits from other G-proteins.48 Since GRIN interacts directly with GoR, this suggests that this protein may function physiologically as a downstream target of this and other G-protein coupled receptors. Interestingly as it pertains to the analysis of differential protein abundances between untreated and Ib-induced Vero cells, GoR, GRIN, and CD44 all interact through an assembly of known lipid raft 530

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The results presented demonstrate the utility of trypsinmediated18O/16O stable isotope labeling when optimized for quantitative membrane proteomics in conjunction with an organic solvent-based technique for the solubilization and tryptic digestion of membrane proteins. The use of enzymecatalyzed stable isotope labeling in combination with the buffered methanol-based solubilization and proteolysis of membrane proteins offers a number of benefits. First, the incorporation of the isotopic tag is trypsin-mediated. This process fits seamlessly within the common high-throughput proteomics workflow that relies on the peptide termini constraint generated by this enzyme as an aid to identify peptides via their MS/MS spectra. Second, this method requires minimal sample manipulation when compared to others that incorporate isotopic tags via chemical modification of the peptides.19 Third, when compared to quantitative methods that target specific residues within protein structure (e.g., cysteinyl residues) this approach allows broader quantitation of membrane proteins, including those that contain no cysteinyl residues (e.g., bacteriorhodopsin). This comprehensive labeling of all tryptic peptides has another significant advantage over techniques such as isotope-coded affinity tags that targets only those peptides that contain cysteinyl residues. Protein modifications, especially glycosylation and phosphorylation, play a major role in the function of membrane proteins. Using an 18O/ 16 O labeling approach, modified peptides are retained in the sample preparation and are available for analysis by LC-MS. While outside the scope of this project, modified peptides could be identified and quantitated using a suitable bioinformatic process to search for such the desired modifications. Although quantitation is possible using this global labeling approach, a method that incorporates steps to enrich for specific classes of modified peptides would most likely have a greater chance of success as the scheme presented in this study is designed primarily for simple protein quantitation.

research articles

Quantitative Proteomics of Lipid Rafts

Acknowledgment. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1CO-12400. Supporting Information Available: A complete list of the peptides identified in this study along with their related SEQUEST scoring parameters (Supplemental Table 1) and the complete list of quantified proteins and their relative abundance ratio between Ib-induced and untreated Vero cells (Supplemental Table 2). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Simons, K.; Toomre, D. Nat. Rev. Mol. Cell Biol. 2000, 1, 31-39. (2) Rappsilber, J.; Mann, M. Genome Biol. 2002, 3, 2008.1-2008.5. (3) Griffin, T. J.; Han, D. K. M.; Gygi, S. P.; Rist, B.; Lee, H.; Aebersold, R.; Parker, K. C. J. Am. Soc. Mass Spectrom. 2001, 12, 1238-1246. (4) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351360. (5) Blonder, J.; Conrads, T. P.; Yu, L. R.; Terunuma, A.; Janini, G. M.; Issaq, H. J.; Vogel, J.; Veenstra, T. D. Proteomics 2004, 4, 31-45. (6) Blonder, J.; Hale, M. L.; Lucas, D. A.; Schaefer, C. F.; Yu, L. R.; Conrads, T. P.; Issaq, H. J.; Stiles, B. G.; Veenstra, T. D. Electrophoresis 2004, 25, 1307-1318. (7) Blonder, J.; Goshe, M. B.; Xiao, W.; Camp, D. G., 2nd; Wingerd, M.; Davis, R. W.; Smith, R. D.; Hale, M. L.; Lucas, D. A.; Schaefer, C. F.; Yu, L. R.; Conrads, T. P.; Issaq, H. J.; Stiles, B. G.; Veenstra, T. D. J. Proteome Res. 2004, 3, 434-444. (8) Blonder, J.; Rodriguez-Galan, M. C.; Chan, K. C.; Lucas, D. A.; Yu, L. R.; Conrads, T. P.; Issaq, H. J.; Young, H. A.; Veenstra, T. D. J. Proteome Res. 2004, 3, 862-870. (9) Blonder, J.; Terunuma, A.; Conrads, T. P.; Chan, K. D.; Yee, C.; Lucas, D. A.; Issaq, H. J.; Schaefer, C. F.; Buetow, K. H.; Veenstra, T. D.; Vogel, J. C. J. Invest. Dermatol. 2004, 133, 691-699. (10) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R., 3rd Nat. Biotechnol. 2003, 21, 532-538. (11) Pedersen, S. K.; Harry, J. L.; Sebastian, L.; Baker, J.; Traini, M. D.; McCarthy, J. T.; Manoharan, A.; Wilkins, M. R.; Gooley, A. A.; Righetti, P. G.; Packer, N. H.; Williams, K. L.; Herbert, B. R. J. Proteome Res. 2003, 2, 303-311. (12) Bouchal, P.; Kucera, I. J. Basic Microbiol. 2004, 44, 17-22. (13) Wu, C. C.; MacCoss, M. J.; Mardones, G.; Finnigan, C.; Mogelsvang, S.; Yates, J. R., 3rd; Howell, K. E. Mol. Biol. Cell 2004, 15, 2907-2919. (14) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (15) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (16) Wu, C. C.; Yates, J. R., 3rd Nat. Biotechnol. 2003, 21, 262-267. (17) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946-951. (18) Foster, L. J.; de Hoog, C. L.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5813-5818.

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