Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for

Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis. Michael B. Goshe,† Josip Blonder,‡ and Richard D. S...
0 downloads 0 Views 146KB Size
Download PDF
Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis Michael B. Goshe,† Josip Blonder,‡ and Richard D. Smith* Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, MSIN K8-98, Richland, Washington 99352 Received August 12, 2002

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses. Keywords: affinity labeling • biotinylation • membrane proteins • hydrophobic proteins • proteomics • mass spectrometry

Introduction Integral membrane proteins are important biological and pharmacological targets involved in intercellular communication, cellular development, cell migration, and drug resistance.1-9 Consequently, the expression of these proteins can have a profound effect on cell activation and tolerance. Contemporary genomic analyses indicate that 20-30% of all open reading frames (ORFs) encode for integral membrane proteins.10 However, proteomic efforts to determine the identity and function of membrane proteins have been experimentally challenging. The difficulties associated with maintaining the solubility of these proteins once they have been extracted from the lipid bilayer has been the main difficulty encountered in most analytical approaches. Proteomics using two-dimensional polyacrylamide gel electrophoresis based mass spectrometry (2DPAGE-MS) has produced some satisfactory results for outer membrane protein identification;11,12 however, integral mem* To whom correspondence should be addressed. † Current address: Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Campus Box 7622, Raleigh, North Carolina 27695. ‡ Current address: SAIC-Frederick Inc., National Cancer Institute at Frederick, Analytical Chemistry Laboratory, Mass Spectrometry Center, P.O. Box B, Frederick, Maryland 21702. 10.1021/pr0255607 CCC: $25.00

 2003 American Chemical Society

brane proteins tend to be more hydrophobic and are frequently under-represented13-15 primarily due to precipitation of these proteins near their isoelectric points. As a result one-dimensional (1D) electrophoretic separations have been used to separate membrane protein fractions, and subsequently gel slices containing several proteins are in-gel digested and the proteins identified by peptide mass fingerprinting using matrixassisted laser desorption/ionization time-of-flight mass spectrometry16,17 or tandem mass spectrometry.18 Sample preparation techniques for proteomics involving microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (µLC-MS/MS) can circumvent the problems encountered with 2D gel separations by chemically or enzymatically cleaving the surface exposed portions of integral membrane proteins belonging to isolated membrane fractions.19,20 Washburn et al. have been successful in using this approach in conduction with multidimensional liquid chromatography to identify integral membrane proteins;21 however, only those proteins that have a significant fraction of their structure exposed and are soluble to the conditions used are detected. Proteins containing a large number of transmembrane domains (TMDs) or are extremely hydrophobic may not yield peptides that are detected as effectively as those from Journal of Proteome Research 2003, 2, 153-161

153

Published on Web 01/14/2003

research articles membrane proteins composed primarily of hydrophilic solventexposed domains. Recently, we have developed the use of methanol for extracting integral membrane proteins from the lipid bilayer and demonstrated more effective detection of these hydrophobic proteins using µLC-MS/MS.22 Using this method, extremely hydrophobic peptides originating from membrane spanning domains of membrane proteins, not previously detected using any 2D-PAGE-MS or µLC-MS/MS method, were identified. Although successful, other labeling and affinity isolation techniques were desired to reduce sample complexity, enhance identification of low abundance membrane proteins, and provide a means for chemical isotope labeling. In this study, a method of specific and quantitative labeling of highly hydrophobic membrane proteins has been developed. The labeling and affinity chromatography methods can be used with a variety of commercially available stable-isotope and/or biotinylation reagents and provides a viable method for comparative membrane proteomics.

Experimental Procedures Materials. Deinococcus radiodurans strain R1 cells were obtained from American Type Culture Collection N0.13939 (Manassas, VA). (+)-Biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) and Coomassie assay reagents were obtained from Pierce (Rockford, IL). The peptides VTCG and GRGDSC were purchased from Bachem (Torrance, CA). Sequencing grade-modified trypsin used for in-solution digestion was from Promega (Madison, WI). The peptide TCVEWLRRYLKN and phenylmethylsulfonyl fluoride (PMSF) were from Sigma (St. Louis, MO). Acetonitrile (HPLC grade) and formic acid (ACS regent grade) were from Aldrich (Milwaukee, WI). Water was purified using a Barnstead Nanopure Infinity water purification system (Dubuque, IA). Bacterial Growth. D. radiodurans strain R1 cells were grown acerbically at 30 °C with shaking at 225 rpm in tryptoneglucose-yeast extract culture medium (2.5 g tryptone, 0.5 g glucose, and 0.5 g yeast extract in 500 mL of water) then harvested in late exponential-early stationary phase by centrifugation at 3200 × g for 10 min at 4 °C. The cells were washed once with 50 mM Tris, pH 7.3, then collected by centrifugation at 3200 × g for 10 min at 4 °C. Membrane Protein Enrichment. The extraction, solubilization, and enrichment of membrane proteins were performed as described elsewhere.22 Briefly, cells were lysed using a French press and the debris removed by centrifugation at 3200 × g for 10 min at 4 °C. The supernatant was collected and treated with ice-cold sodium carbonate. The membranes were pelleted by ultracentrifugation and then resuspended and washed in 50 mM NH4HCO3, pH 8.0, and again pelleted using ultracentrifugation. The supernatant was discarded, and the membrane proteins were extracted and solubilized using 60% methanol/ 40% 50 mM NH4HCO3, pH 8.0. The Coomassie assay was used to determine membrane protein concentration. A standard curve for BSA protein standards prepared in the organic solution was generated between 0 and 0.5 µg/µL and used to determine the membrane protein sample concentration. Biotinylation of Cysteinyl-Membrane Proteins. The membrane protein sample in methanol/50 mM NH4HCO3, pH 8.0, (60/40) was incubated in airtight tubes in a boiling water bath for 5 min. After cooling to room temperature, a thirty molar excess of tributylphosphine to protein was added to reduce disulfide bonds and incubated at 37 °C for 30 min. After 154

Journal of Proteome Research • Vol. 2, No. 2, 2003

Goshe et al.

reduction, the cysteinyl residues were biotinylated by adding a thirty molar excess of iodoacetyl-PEO-biotin to protein. The solution was slowly agitated using a shaker/rotisserie (Labquake, Conroe, TX) for 1.5 h at room temperature in the dark. After alkylation, the reaction buffer was exchanged for methanol/ 50 mM NH4HCO3, pH 8.0, (60/40) by size exclusion chromatography using prepacked columns containing Excellulose (Pierce, Rockford, IL). Proteolysis was immediately performed using a 1:20 (w/w) trypsin-to-protein ratio for 5 h at 37 °C and then was quenched by rapid freezing by immersing the tube into liquid nitrogen. The sample was stored at -80 °C until avidin affinity chromatography was performed. Avidin Affinity Purification of PEO-Biotin-Labeled Peptides. The PEO-biotin-labeled peptides were purified by affinity chromatography using Ultralink immobilized monomeric avidin from Pierce (Rockford, IL). Irreversible biotin binding sites were blocked as per the manufacturer’s instructions. The avidin slurry was packed in a glass Pasteur pipet containing a glass wool plug. Tryptic activity in the proteolyzed sample was quenched by adding PMSF to a final concentration of 1 mM and incubating for 30 min at 37 °C. The sample was then incubated in a boiling water bath for 5 min, cooled to room temperature, the then diluted by 50 mM NH4HCO3, pH 8.0, until a concentration of 10% methanol was achieved. After the sample was loaded onto the avidin column, the column was then washed with 10 column volumes of 2X PBS (0.2 M sodium phosphate, 0.3 M NaCl, pH 7.2), 1X PBS, and then five column volumes of 20% acetonitrile in 50 mM NH4HCO3, pH 8.0. Elution of the PEO-biotin-labeled peptides was performed using 30% acetonitrile and 0.4% formic acid in water. After lyophilization, the PEO-biotin labeled peptides were stored at -20 °C until LC-MS/MS analysis. LC-MS/MS Analysis. Microcapillary reversed-phase HPLC was performed using an Agilent 1100 Series capillary LC system (Agilent Technologies, Inc., Palo Alto CA) coupled to a LCQ Deca XP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) using an in-house manufactured ESI interface. The reversed-phase separation was performed using a 360 µm o.d. × 150 µm i.d. × 60 cm length capillary column (Polymicro Technologies Inc., Phoenix, AZ) containing 5 µm Jupiter C18 stationary phase (Phenomenex, Torrence, CA) that was slurry packed in-house. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The lyophilized peptide sample was solubilized in 5% B plus 95% A to produce 0.4 µg/µL. After injecting a sample volume of 8 µL onto the reversed-phase microcapillary HPLC column, one of two gradient programs was used for the separation at a flow rate of 1.8 µL/min. One program held the mobile phase at 5% B for 20 min, followed by a linear gradient to 70% B over 80 min, then a linear gradient to 85% B over 45 min. The other gradient program held the mobile phase at 5% B for 20 min, followed by a series of linear gradients: from 5% to 25% B over 10 min, from 25% to 70% B over 120 min, and from 70% to 85% B over 30 min, and at 85% B for 10 min. Each gradient program was followed by re-equilibration of the column at 5% B for 60 min prior to the next injection. The LCQ Deca XP was operated in the data-dependent MS/ MS mode where a full-MS scan was followed by three MS/MS scans. The precursor ions were dynamically selected based on their intensity in the MS scan and subjected to collisioninduced dissociation (CID) using the normalized collision energy setting of 45%. Samples were analyzed over the follow-

Affinity Labeling of Integral Membrane Proteins

research articles

ing m/z ranges: 400-2000, 400-700, 700-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1600, and 16002000. Peptide Identification. Both labeled and unlabeled peptides were identified by searching the MS/MS spectra against the complete annotated D. radiodurans database using SEQUEST23 (Thermo Finnigan, San Jose, CA). All of the spectra were analyzed using a dynamic mass modification on cysteinyl residues (i.e., both modified and unmodified forms of the residues) equal to the additional mass of the acetyl-PEO-biotin label (414.194 Da). Only tryptic peptides displaying a crosscorrelation score (Xcorr) of at least 2.0 and a delta-correlation score (∆Cn) of at least 0.1 were accepted for protein identification. The Xcorr of the peptide is based on the “fit” of the MS/ MS data to the theoretical distribution of ions produced for the peptide, and the ∆Cn is the “difference” between the top two Xcorrs for a given MS/MS spectrum. Acceptable scores for all labeled peptides were manually inspected to determine the presence of at least two or more label-specific fragment ions to verify labeling.24 Hydropathy Calculations and Transmembrane Mapping. All identified proteins and the entire genome sequence of D. radiodurans strain R1 were analyzed using the ProtParam program (available at http://www.expasy.ch/sprot/sprot-top. html) to calculate the grand average of hydropathicity (GRAVY) for each protein.25 PSORT (available at http://psort.nibb.ac.jp/ form.html) was used to map TMDs and predict the subcellular location for each protein encoding ORF to recognize the proteins of the membrane subproteome.26

Results and Discussion Specific and Quantitative Labeling of Cys-Residues. Because the integral membrane proteins were solubilized in methanol/50 mM NH4HCO3, pH 8.0, (60/40) the ability to specifically and quantitatively label Cys-residues must be demonstrated. Because the reaction was an SN2 displacement of iodide ion from iodoacetyl-PEO-biotin, the organic composition of 60% methanol could affect the specificity of the nucleophile or alter the kinetics of S-alkylation. To establish the labeling reaction conditions, several peptides were used. As shown in Figure 1A and B, the [M + 2H]2+ peak of the unlabeled 12 residue peptide TCVEWLRRYLKN at m/z 791.3 decreased in abundance with a concomitant increase in the formation of the labeled peptide [M + 2H]2+ peak at m/z 998.1. This is the direct result of modifying the peptide with acetylPEO-biotin. On the basis of the average integrated intensity of the corresponding peaks for the labeled and unlabeled peptide ions, the reaction time course for each test peptide appears in Figure 1C. These data indicate that >95% labeling efficiency is achieved within 90 min. This is similar to other Cys-alkylation incubation times and labeling efficiencies using other iodinated reagents, e.g., iodoacetamide and the ICAT reagents.27,28 Although the data presented in Figure 1 indicates effective alkylation, the specific site of the modification needed to be ascertained. To elucidate the modification site, precursor ions corresponding to the labeled peptides were subjected to collision-induced dissociation (CID) and the fragment ions examined to access the residue specificity of alkylation. As shown in Figure 2, CID of labeled peptide precursor ions produces b and y product ions that correspond to the modification occurring at the Cys residue. Both infusion ESI-MS/ MS and µLC-MS/MS indicated that during the course of the reaction, labeling only occurred at the Cys residue of VTCG,

Figure 1. Cysteinyl-alkylation of peptides. The MS spectrum of the peptide TCVEWLRRYLKN produced at (A) at 0 min and (B) at 90 min during labeling with iodoacetyl-PEO-biotin. During the time course of the reaction (C) the [M + 2H]2+ ion of the unlabeled peptide at m/z 791.3 is converted to the labeled species at m/z 998.1. The time courses of VTCG and GRGDSC labeling are also plotted as well.

GRGDSC, and TCVEWLRRYLKN with no detectable evidence of labeling (1) the -nitrogen of Lys, (2) the N-terminal nitrogen, or (3) the para oxygen of Tyr. In fact, the extracted ion chromatograms of the [M + 2H]2+ and [M + 3H]3+ precursor ions of the labeled peptide TCVEWLRRYLKN indicted that it eluted in a single peak. If an alternative site were modified, then this labeled peptide would most likely exhibit a different retention time during µLC-MS/MS. Journal of Proteome Research • Vol. 2, No. 2, 2003 155

research articles

Goshe et al. Table 1. SEQUEST Results of µLC-MS/MS Analysis of Biotinylated Cys-Peptides Obtained Using the Developed Method to Label Highly Hydrophobic Membrane Proteins no. of peptides identified

all peptides non-Cys-peptides Cys-peptides Cys-labeled peptides % of total peptides labeled unlabeled Cys-peptides % of labeling Cys-peptides

no. of proteins identified

totala

uniqueb

uniquec

931 229 702 673 72% 29 96%

380 159 221 220 58% 1 > 99%

179 89 90 89 50% 1 99%

a The number reported includes multiple identifications of the same peptide. b The number reported is the unique peptides identified, regardless of the number of times the peptide was detected. c The number reported is the unique proteins identified, regardless of the number of peptides detected for each protein.

Figure 2. Elucidating the alkylation site of acetyl-PEO-biotin labeled peptides. The MS/MS spectrum for each labeled peptide precursor peak of (A) VTCG, (B) GRGDSC, and (C) TCVEWLRRYLKN is shown. For both A and B the [M + 2H]2+ precursor ion was subjected to CID, whereas in C, the [M + 3H]3+ precursor ion was selected. The y and b ions produced during CID of each labeled peptide indicate that the Cys residue, and thus the thiolate of the side chain, is the site of modification. The presence of labelspecific product ions (designated by [) is also indicative of peptide labeling and can be used as a constraint during peptide identification.

The peptides of 4 and 6 amino acid residues in length, although much smaller than typically observed for peptides produced from tryptic digestions, were chosen to access the labeling reaction for two reasons. First, the lack of steric hindrance for these peptides would not preclude labeling at kinetically slower sites and thus provide a better control to access potential alternative site reactivity. Second, CID of the doubly charged precursor ions of smaller peptides produces 156

Journal of Proteome Research • Vol. 2, No. 2, 2003

readily interpretable b and y ions (Figure 2A and B), as opposed to longer peptides that are triply charged (Figure 2C) or whose doubly charged ions do not produce observable b and y ions (data not shown). For protein labeling, secondary and tertiary structure can preclude Cys alkylation. However, the presence of 60% methanol in the reaction solution acts as a denaturant to facilitate exposure of disulfide bonds to reduction and subsequent labeling. In fact, using the developed affinity labeling method on membranes isolated from D. radiodurans, only one unlabeled Cys-peptide was identified by µLC-MS/MS (Table 1). SEQUEST analysis of the resulting MS/MS spectra using a dynamic mass modification of 414 Da for cysteinyl residues in which both labeled and unlabeled forms of the residues are searched, 220 out of 221 (>99%) unique Cys-peptides were identified as containing the acetyl-PEO-biotin label. This labeling efficiency is consistent with the data presented in Figure 1. Thus, depending on whether (1) the proteins are labeled before tryptic digestion or (2) the peptides are labeled after protein digestion, the affinity alkylation reaction in the methanol/ 50 mM NH4HCO3, pH 8.0, (60/40) buffer can be quantitatively performed. Affinity Isolation of Biotinylated Peptides using Immobilized Avidin. After the determination of the labeling conditions for specific and quantitative labeling of cysteinyl groups, the next step required enrichment of these biotinylated peptides using immobilized avidin. Directly applying the 60% methanol reaction mixture after tryptic digestion to an avidin affinity column could result in low recovery of biotinylated peptides because the high organic content of the solution may disrupt the avidin-biotin interaction and may even partially denature the avidin. Analyzing the flow-through for samples containing 60% methanol in the reaction buffer using µLC-MS/MS indicated that some biotinylated peptides were not retained during column loading (data not shown). On the basis of evaluating a series of dilutions of the 60% methanol sample using additions of 50 mM NH4HCO3, pH 8.0, it was determined that a dilution to 10% methanol prior to loading the avidin affinity column is sufficient to permit binding of the biotinylated peptides. The flow-through from each washing step was also evaluated by µLC-MS/MS. The steps using 1X PBS, 2X PBS, and 20% acetonitrile in 50 mM NH4HCO3, pH 8.0, did not elute detectable biotinylated peptides; however, many unlabeled peptides from these washes were detected (data not shown). More

research articles

Affinity Labeling of Integral Membrane Proteins

Table 2. Cys-Labeled Peptides of Iron-Sulfur Binding Reductase (DR2565) Detected by µLC-MS/MS Using SEQUEST

peptidea

observed massb

calculated massb

charge state

Xcorrc

∆Cnd

ionse

labelspecific ionsf

R.C*QDVC*PANATGK.A K.KEAC*TGDSAR.R K.VPTIDENPEPDVIYWVGC*AASYDPGAQK.V K.LIVATC*PHC*MNAIGNEYR.Q R.TIHHTEYLEQLVAAGKLPTAQLHDNVVYHDPC*YLGR.H R.ENSFC*C*GAGGAQFWKEEEEGRER.V R.LFGFWGALGSILLFLSYFPFSK.H

2035.7 1452.9 3450.3 2836.3 4517.8 3449.6 2512.6

2035.3 1452.3 3450.5 2834.8 4518.8 3449.2 2512.0

2 2 3 3 3 3 2

2.805 2.301 3.218 2.816 3.204 2.272 2.999

0.377 0.447 0.482 0.260 0.494 0.218 0.635

14/22 11/18 22/108 21/68 30/140 20/88 16/42

2 1 1 2 1 2 0

a The amino acid residues appearing before and after the periods correspond to the residues proceeding and following the peptide in the protein sequence. Average peptide mass. c Cross-correlation score (Xcorr) of the peptide is based on the “fit” of the MS/MS data to the theoretical distribution of ions produced for the peptide. d A calculated “difference” between the top two Xcorr values for the given peptide. e The total number of b and y ions identified/theoretical. f The number of detected acetyl-PEO-biotin label-specific ions at m/z of 227.1, 270.1, 332.2, 375.2, or 449.2 formed during collision-induced dissociation of the given Cys-labeled peptide. b

Figure 3. Identification of putative undecaprenol kinase by µLC-MS/MS. The MS/MS spectrum produced by CID of the peptide peak at m/z 791.7 was identified by database searching the product ions using SEQUEST. The tryptic peptide identified, ALAIGAAQC*LALLWPGFSR, completely covers one mapped TMD of the putative undecaprenol kinase (as determined by PSORT) and is labeled with acetyl-PEO-biotin at the Cys residue (C*). Characteristic label-specific peaks were also detected and are shown in the inset. As shown in the protein sequence, the region corresponding to the identified peptide is bold and underlined and the six mapped TMDs are boxed.

aggressive washing steps (e.g., using a higher percentage of acetonitrile or methanol during the final washing step) to reduce the number of unlabeled peptides binding to the column were examined, but these washes resulted in the elution of some biotinylated peptides and were not incorporated into the protocol. When the affinity labeling procedure was applied to the membrane subproteome of D. radiodurans, a total of 931 peptides were identified from the µLC-MS/MS analysis with 72% being Cys-labeled peptides (Table 1). On the basis of the

number of unique peptides identified, 58% are Cys-labeled. Although not typically reported in the literature, our experience with immobilized monomeric avidin using various biotinylation approaches in aqueous buffers for proteome-wide analysis (e.g., ICAT28 and PhIAT29,30) indicates that as high as 30% of the eluted peptides do not contain a cysteinyl residue. These peptides tend to be hydrophobic and (1) contain aliphatic and aromatic residues near the N-terminus (an example is listed in Table 2 for DR2565) and (2) sometimes contain a proline residue near the N-terminus. These findings are consistent with Journal of Proteome Research • Vol. 2, No. 2, 2003 157

research articles the role of hydrophobic interactions in the binding of biotin to avidin.31,32 Because the organic-aqueous buffer is used to solubilize highly hydrophobic integral membrane proteins for labeling, it is not surprising that the percentage of hydrophobic non-Cys peptides has increased by approximately 10%. Although the presence of some unlabeled peptides increases sample complexity, the affinity enrichment step enhances the identification of unique Cys-labeled peptides by a factor of 10 when compared to detecting Cys-labeled peptides prior to avidin affinity enrichment. Improvements in affinity isolation techniques using immobilization on solid-phase supports, such as the immobilized ICAT reagent,33 provide promising alternatives to reduce nonspecific binding of peptides, while increasing the enrichment of labeled peptides. Because of the chemical differences between solid phase and solution phase labeling and enrichment, experiments regarding labeling and enriching highly hydrophobic proteins/peptides from complex mixtures using solid-phase reagents remain to be developed. CID of Cys-Labeled Peptides. An additional constraint used in the identification of Cys-labeled peptides was the presence of label-specific product ions produced during CID. As shown previously,24 the presence of [M + H]+ ions at m/z values of 227.1, 270.1, 332.2, 375.2, and 449.2 is indicative of acetyl-PEObiotin labeling of Cys residues. All identified Cys-labeled peptides reported in this study displayed at least two-label specific peaks in the MS/MS spectrum as shown in Figures 2 and 3. In the previous report24 a normalized collision energy setting of 35% was found to produce sufficient fragmentation of Cys-labeled peptides for confident identification by SEQUEST. In the previous experiments with unlabeled peptides obtained from the membrane protein enrichment protocol,22 it was determined that increasing the collision energy setting to 45% produced an increase in the number of SEQUEST identified peptides. This is probably due to enhanced fragmentation efficiencies at the higher collision energy amplitude, especially for the larger tryptic peptides that seem to be produced from the tryptic digestion of integral membrane proteins. Therefore, a normalized collision energy setting of 45% was used on the biotinylated-peptide mixture obtained with the affinity labeling procedure. However, additional experiments are required to verify whether a setting of 35% or 45% is optimal for fragmentation of Cys-labeled peptides isolated using the affinity labeling technique. Labeling and Identification of Integral Membrane Proteins of D. radiodurans. For the affinity labeling procedure to be effective, cysteinyl residues of hydrophobic integral membrane proteins must be solvent exposed and reduced to produce reactive thiolates for the biotinylation reagent. The ability to promote cysteinyl residue labeling is illustrated by the labeled peptides identified by MS/MS for the iron-sulfur binding protein (DR2565) of D. radiodurans. As shown in Table 2, this protein was identified by six Cys-labeled peptides that produced reasonable Xcorr values and contained at least two or more label-specific product peaks in their MS/MS spectra. The identification of these peptides demonstrates the ability of the affinity labeling method to label multiple cysteinyl residues of a given protein, including those residues in close proximity in primary sequence. The detection of the unlabeled membrane spanning peptide listed in Table 2 is further evidence of the enhanced solubility achieved by the methanol-aqueous buffer used in the affinity labeling. The efficacy of the developed affinity labeling and enrichment procedure is exemplified by identifying Cys-labeled 158

Journal of Proteome Research • Vol. 2, No. 2, 2003

Goshe et al. Table 3. Unique Proteins Identified by Using the Post-Extraction Affinity Labeling and Enrichment Protocol ORF

protein identity

DR0161 DR0235 DR0333 DR0481 DR0540 DR0562 DR0692 DR0790 DR0804 DR0807 DR0924 DR0951 DR1219 DR1262 DR1415 DR1429 DR1567 DR1663 DR1682 DR1692 DR1699 DR1708 DR1748 DR1838 DR1940 DR2150 DR2220 DR2235 DR2271 DR2295 DR2316 DR2329 DR2405 DR2463 DR2549 DRA0005 DRA0037 DRA0046 DRA0142 DRA0233 DRB0003 DRB0054 DRB0066

hypothetical protein 6-aminohexanoate-cyclic-dimer hydrolase hypothetical protein hypothetical protein hypothetical protein maltose abc transporter, permease protein nitrogen regulatory protein p-ii hypothetical protein sigma factor, putative prolipoprotein diacylglyceryl transferase hypothetical protein succinate dehydrogenase, iron-sulfur subunit ferrous iron transport protein b 60-kda ss-a/ro ribonucleoprotein homolog (ro sixty related) ornithine aminotransferase, putative hypothetical protein peptide abc transporter, atp-binding protein hypothetical protein hypothetical protein long-chain fatty acid- -coa ligase hypothetical protein hypothetical protein hypothetical protein gtp pyrophosphokinase hypothetical protein hypothetical protein tellurium resistance protein terb (terb) putative hypothetical protein hypothetical protein hypothetical protein abc transporter, atp-binding protein hypothetical protein ubiquinone/menaquinone biosynthesis methyltransferase transport protein, putative epoxide hydrolase-related protein alcohol dehydrogenase, zinc-containing glycosyltransferase hypothetical protein transcriptional regulator, tetr family oxidoreductase, iron-sulfur subunit hypothetical protein hypothetical protein hypothetical protein

peptides from two highly hydrophobic integral membrane proteins. The MS/MS spectrum of the Cys labeled peptide ALAIGAAQC*LALLWPGFSR shown in Figure 3 was identified as originating from a putative undecaprenol kinase (bacitracin resistance protein) (DR0454) which contains six TMDs as mapped using the PSORT algorithm. The peptide identified contains one TMD with a labeled Cys residue within this membrane-spanning region. Further evidence for labeling is the presence of label-specific fragment ions characterizing the acetyl-PEO-biotin tag. The identification of another Cys-labeled membrane spanning peptide AFIPLILGFGC*NVPAVSATR is attributed to the ferrous ion transport protein B (DR1219). This protein from the D. radiodurans membrane subproteome was one of several not identified previously using the other membrane enrichment protocol.22 In fact, out of the 89 proteins identified by Cys-labeled peptides, 43 were unique to the labeling method and are listed in Table 3. Thus, 50% of the proteins identified by the affinity labeling approach produced about a 10% increase over the 509 proteins previously identified without labeling and affinity capture. This fact demonstrates the utility of the method to enable identification of proteins not detected previously using the same µLC-MS/MS analysis. Further examination of the 179 unique proteins identified revealed an increased coverage of the membrane subproteome. A total of 44 proteins had positive GRAVY values ranging from

research articles

Affinity Labeling of Integral Membrane Proteins

Table 4. Integral Inner Membrane Proteins Identified by Cys-Labeled Peptides Using µLC-MS/MS Analysis ORFa

protein identity

DR0454 DRA0046 DR0959

bacitracin resistance protein hypothetical protein peptide abc transporter, permease protein maltose abc transporter, permease protein. peptide abc transporter, permease protein pts system, fructose-specific iibc component ferrous iron transport protein b peptide abc transporter, permease protein prolipoprotein diacylglyceryl transferase. hypothetical protein hypothetical 22.9 kDa protein acid tolerance protein act206-related protein osteoblast specific factor 2-related protein hypothetical protein cytochrome C6, putative hypothetical protein hypothetical 27.2 kDa protein transport protein, putative iron abc transporter, periplasmic substrate-binding protein hypothetical protein hypothetical 18.5 kDa protein hypothetical protein hypothetical 31.1 kDa protein long-chain fatty acid- -coa ligase hypothetical 50.5 kDa protein hypothetical 18.1 kDa protein peptide abc transporter, atp-binding protein hypothetical protein pilin, type iv, putative hypothetical protein conserved hypothetical protein tellurium resistance protein terb (terb) putative. signal peptidase i transcriptional regulator, tetr family. iron-sulfur binding reductase, putative hypothetical 86.6 kDa protein gtp pyrophosphokinase hypothetical protein conserved hypothetical protein hypothetical 84.4 kDa protein

DR0562 DR0365 DRB0073 DR1219 DR1569 DR0807 DR0790 DR2342 DR1353 DR0399 DR0333 DR0434 DR2271 DR0769 DR2463 DR2588 DR0161 DR0486 DR1748 DR0115 DR1692 DR2528 DR1212 DR1568 DR2295 DR0548 DR0540 DR0753 DR2220 DR1427 DRA0142 DR2565 DR1231 DR1838 DR1682 DR2596 DR2310

gravy valueb

no. of TMDsc

Xcorrd

0.79 0.64 0.61

6 8 6

2.61 2.19 2.80

K.ALAIGAAQC*LALLWPGFSR.S K.DGC*QLTPTLQR.N R.GTPESIAAC*NHDR.G

0.59

5

5.13

R.TPSC*VGDTLVLYDEDAATPHEVK.I

0.58

5

2.33

K.AIFASPPEC*YKMER.V

0.56

5

3.47

K.YIVGITSC*PTGIAHTFMAAEGLEGGAK.S

0.43 0.36

8 6

2.49 3.65

R.AFIPLILGFGC*NVPAVSATR.T K.LMGVDEPC*KLYLLGSDNFGR.D

0.30

5

3.28

R.AFHEGMC*RPNPNPDMDLSK.Y

0.27 0.25

4 3

2.08 2.34

R.C*YLLSAPLLPDR.F R.GLADALITDPC*R.G

0.22

7

2.78

K.YGTYVC*YDSVFPWVAR.Q

0.20

1

2.12

K.PAAKPAASC*K.S

0.16 0.11 0.10 0.09 0.06 0.02

1 4 6 1 2 1

2.17 2.21 3.64 2.00 4.39 2.31

R.GRLWLAAPELGLLAC*PYDSR.G R.NDAVMPGVAIFC*AAVMWIVLLFLFNKETAPK.P R.AGDDAC*ALNNLGAIAQSRDDLPQAR.T R.VPSFQPSC*PR.L R.SIGGKLTVTDHYMDGC*WIDAAAPTTEELAR.V R.LISVLPSTSETLC*AIGAC*NK.L

-0.02 -0.03 -0.04 -0.07 -0.12 -0.14 -0.15 -0.17

1 1 1 1 2 2 1 1

2.45 3.09 2.34 2.94 3.46 2.16 3.11 3.84

K.VQGFC*DDAGAPVFPVPPDAADLTAHAYER.A R.LLLSGSEFGGC*TAQLR.R.C*TIDGDKISGR.A K.GLFIGYPDGSFDWC*SAITR.Q R.AC*ISGSAPLMQDTAR.T K.EIPLSSGC*TWSYSSDPGGR.S K.DTAGAVRPLVPVNMQC*TYDK.S R.C*KFAVPQC*SQAVPALEDTGGGHMAR.C

-0.18 -0.19 -0.19 -0.21

1 1 2 1

3.14 4.19 2.19 4.20

R.VGETVVVASGAPSTPPETC*DSR.R R.LPAAGNC*DSTANLGTNAVATPGAITAAGAYSLDGTGSK.F R.DMPNGRPFC*VIAGPGYSEEGVAVVPVQGLPAER.I R.C*VYAQGKNDAFNTYKEILFR.A

-0.22

1

3.02

R.FNNAAFADATMAAC*ALIGAADGQIDSQER.S

-0.25 -0.26

1 1

3.92 2.11

R.KSNLKYDC*AAETAGELSGENELNWR.V K.YGSVLVIFMVGFC*YAELNGNFAPSAFAQRR.A

-0.27

3

3.93

K.VPTIDENPEPDVIYWVGC*AASYDPGAQK.V

-0.36 -0.42 -0.43 -0.47 -0.52

1 1 1 1 1

4.14 2.20 2.17 3.64 3.64

R.TVTC*QNTASNTVITDEYR.Y R.GDQIMGYLTRGRGVSIHRIDC*PNMVR.L R.ATNSAGGASYEC*ILSRPK.L R.LELQADC*FAGVWGNSVK.G K.VLDRKPGDLTPLYAC*PEPAGNIAR.E

peptide identifiede

a Open reading frame. b The grand average of hydropathicity (GRAVY) for each protein was determined using the ProtParam program (available at http:// www.expasy.ch/sprot/sprot-top.html). c The transmembrane domains (TMDs) were mapped using PSORT (available at http://psort.nibb.ac.jp/form.html).d Crosscorrelation score (Xcorr) of the peptide is based on the “fit” of the MS/MS data to the theoretical distribution of ions produced for the peptide. e The amino acid residues appearing before and after the periods correspond to the residues proceeding and following the peptide in the protein sequence. C* is the cysteinyl residue labeled with acetyl-PEO-biotin.

0.01 to 1.09 and, thus, were categorized as hydrophobic. Using the PSORT algorithm, 103 (58%) were classified as membrane proteins with 73 inner membrane, 18 outer membrane, and 12 periplasmic. When examining only Cys-labeled peptides, 89 were Cys labeled (50%) in which 24 (27%) had a positive GRAVY value between 0.02 and 1.09. A total of 62 proteins (70%) was classified as integral membrane proteins with 40 inner membrane, 15 outer membrane, and 7 periplasmic. Those compris-

ing only the inner integral membrane proteins are listed in Table 4. Members from several important membrane protein families were identified, including six ATP-binding cassette (ABC) transporters. Because most biochemical analyses suggest that a considerable percentage of transporters belong to low abundant proteins,34 the ability to detect them using this labeling method represents a beginning to proteomic analysis of this protein class. Overall, these data indicate that the affinity Journal of Proteome Research • Vol. 2, No. 2, 2003 159

research articles

Goshe et al.

abundances for a comparative proteome analysis of the virulent wild type and nonvirulent strains of Pseudomonas aeruginosa to elucidate infection related pathways. The differences in membrane subproteomes will be compared to gene expression levels measured using DNA microarrays and will represent a multidisciplinary approach to study P. aeruginosa pathogenesis.

Conclusions The integral membrane proteins identified by the developed affinity labeling procedure enabled the identification of proteins not previously detected using only the methanol extraction procedure. The ability to reduce the complexity of the peptide mixture by avidin affinity chromatography and the potential ability to enrich for low-level membrane proteins make this membrane subproteome even more accessible to µLC-MS/ MS analysis. The membrane proteins identified using the developed protocol encompassed several important membrane protein families, including the ABC transporters, which have been implicated in multidrug resistance of tumors and pathogenic bacteria.36-38 The developed affinity labeling approach is amenable with stable isotope labeling reagents, such as ICAT, and membrane proteins metabolically labeled using 14Nminimal and 15N-enriched media or isotopically encoded amino acids. The ability to quantify differences in membrane protein abundance will potentially provide insights into the mechanism of antibiotic resistance of pathogenic bacteria and contribute to the development of new strategies to curtail efflux-mediated resistance.

Figure 4. Hydropathy comparison of the predicted and identified D. radiodurans membrane subproteome. The hydropathy plots for the (A) theoretically expressed ORFs of D. radiodurans membrane subproteome which includes inner membrane, outer membrane, and periplasmic proteins and (B) proteins identified by Cys-labeled peptides obtained from the affinity labeling membrane protein approach. Each histogram was generated by plotting the number of proteins per 0.1 GRAVY value increment.

labeling method was able to target highly hydrophobic integral membrane proteins as opposed to only enriching for labeled peptides corresponding to outer membrane proteins or cytosolic/membrane associated proteins. The effectiveness of the developed procedure to label highly hydrophobic membrane proteins of D. radiodurans becomes evident when examining the hydropathy plots of the distribution of GRAVY values for the identified and theoretically expressed membrane proteins (Figure 4). The hydrophobic portion of the proteome (GRAVY values > 0) is well represented by the proteins identified using Cys-labeled peptides. This coverage is consistent with that observed for proteins characterized by unlabeled peptides using the methanol-assisted extraction procedure22 and exceeds that previously achieved in MS analyses using 1D or 2D-PAGE separations11-18 or methods to chemically or enzymatically cleave the surface exposed portions of integral membrane proteins.21 Attempts to label membrane proteins using ICAT reagents have been reported;35 however, highly hydrophobic integral membrane proteins were not identified by this technique. The ability to identify highly hydrophobic integral membrane proteins demonstrates the efficacy of the method to identify these proteins by Cys-labeled peptides using µLC-MS/MS. Currently, the developed method is being used with ICAT reagents to measure the differences in membrane protein 160

Journal of Proteome Research • Vol. 2, No. 2, 2003

Acknowledgment. The authors would like to thank the United States Department of Energy Office of Biological and Environmental Research for support of this research. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy under Contract No. DE-AC06-76RLO-1830. Supporting Information Available: A comprehensive list of all identified proteins from affinity labeling the membrane protein enriched sample. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Alessandro, R.; Kohn, E. C. Clin. Exp. Metastasis 2002, 19, 26573. (2) Goette, A.; Lendeckel, U.; Klein, H. U. Cardiovasc. Res. 2002, 54, 247-58. (3) He, Z.; Wang, K. C.; Koprivica, V.; Ming, G.; Song, H. J. Sci. STKE 2002, 2002, RE1. (4) Krummel, M. F.; Davis, M. M. Curr. Opin. Immunol. 2002, 14, 66-74. (5) Sharpe, A. H.; Freeman, G. J. Nature Rev. Immunol. 2002, 2, 11626. (6) Blackmore, C. G.; McNaughton, P. A.; van Veen, H. W. Mol. Membr. Biol. 2001, 18, 97-103. (7) Markham, P. N.; Neyfakh, A. A. Curr. Opin. Microbiol. 2001, 4, 509-14. (8) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nature Rev. Cancer 2002, 2, 48-58. (9) Loscher, W.; Potschka, H. J. Pharmacol. Exp. Ther. 2002, 301, 7-14. (10) Wallin, E.; von Heijne, G. Protein Sci. 1998, 7, 1029-38. (11) Phadke, N. D.; Molloy, M. P.; Steinhoff, S. A.; Ulintz, P. J.; Andrews, P. C.; Maddock, J. R. Proteomics 2001, 1, 705-720. (12) Molloy, M. P.; Phadke, N. D.; Maddock, J. R.; Andrews, P. C. Electrophoresis 2001, 22, 1686-1696. (13) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (14) Nouwens, A. S.; Cordwell, S. J.; Larsen, M. R.; Molloy, M. P.; Gillings, M.; Willcox, M. D. P.; Walsh, B. J. Electrophoresis 2000, 21, 3797-3809.

research articles

Affinity Labeling of Integral Membrane Proteins (15) Molloy, M. P.; Herbert, B. R.; Slade, M. B.; Rabilloud, T.; Nouwens, A. S.; Williams, K. L.; Gooley, A. A. Eur. J. Biochem. 2000, 267, 2871-2881. (16) Galeva, N.; Altermann, M. Proteomics 2002, 2, 713-22. (17) Maltman, D. J.; Simon, W. J.; Wheeler, C. H.; Dunn, M. J.; Wait, R.; Slabas, A. R. Electrophoresis 2002, 23, 626-39. (18) Pflieger, D.; Le Caer, J. P.; Lemaire, C.; Bernard, B. A.; Dujardin, G.; Rossier, J. Anal. Chem. 2002, 74, 2400-6. (19) Gelasco, A.; Crouch, R. K.; Knapp, D. R. Biochemistry 2000, 39, 4907-14. (20) Kraft, P.; Mills, J.; Dratz, E. Anal. Biochem. 2001, 292, 76-86. (21) Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd Nat. Biotechnol. 2001, 19, 242-7. (22) 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. (23) Eng, J. K.; McMormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. (24) Borisov, O. V.; Goshe, M. B.; Conrads, T. P.; Rakov, V. S.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2002, 74, 2284-92. (25) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-32. (26) Nakai, K.; Horton, P. Trends Biochem. Sci. 1999, 24, 34-35. (27) Gurd, F. R. N. In Methods in Enzymology; Hirs, C. H. W., Timasheff, S. N., Eds.; Academic Press: New York, 1972; Vol. 25,

424-438. (28) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-9. (29) Goshe, M. B.; Veenstra, T. D.; Panisko, E. A.; Conrads, T. P.; Angell, N. H.; Smith, R. D. Anal. Chem. 2002, 74, 607-16. (30) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-86. (31) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5076-80. (32) Kurzban, G. P.; Gitlin, G.; Bayer, E. A.; Wilchek, M.; Horowitz, P. M. Biochemistry 1989, 28, 8537-42. (33) Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 20, 512-5. (34) Paulsen, I. T.; Nguyen, L.; Sliwinski, M. K.; Rabus, R.; Saier, M. H., Jr. J. Mol. Biol. 2000, 301, 75-100. (35) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946-51. (36) Wolfger, H.; Mamnun, Y. M.; Kuchler, K. Res. Microbiol. 2001, 152, 375-89. (37) Gottesman, M. M.; Ambudkar, S. V. J. Bioenerg. Biomembr. 2001, 33, 453-8. (38) Mendez, C.; Salas, J. A. Res. Microbiol. 2001, 152, 341-50.

PR0255607

Journal of Proteome Research • Vol. 2, No. 2, 2003 161