Enrichment of Integral Membrane Proteins for Proteomic Analysis

Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry Josip Blonder, Michael B. Goshe, Ronald J. Moore, Ljiljana Pasa-Tolic, Christophe D. Masselon, Mary S. Lipton, and Richard D. Smith* Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, PO. Box 999, MSIN K8-98, Richland, Washington 99352 Received March 29, 2002

An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures. Keywords: membrane proteins • sample preparation • hydrophobic proteins • proteomics • mass spectrometry

Introduction Membranes are critical components of cellular structure and function involving the partitioning of organelles, protecting the integrity of genome and proteome, and providing defense from foreign molecules and external conditions that may damage or destroy the cell. Because prokaryotes lack intracellular organelles, the cell membrane is the most important structure of the bacterial cell. Membrane proteins that are inserted into the phospholipid bilayer are defined as integral membrane proteins and are important biological and pharmacological targets. Contemporary genomic analyses indicate that 20-30% of all open reading frames (ORFs) encode for integral membrane proteins.1 Integral membrane proteins display two membrane spanning tertiary structural motifs: (1) R-helix bundles that are omnipresent in the cytoplasmic membrane and (2) β-barrels that are typically found in the outer membrane of Gram-negative bacteria.2 Integral cytoplasmic membrane proteins are composed of R-helices containing predominantly nonpolar amino acids that are inserted into the phospholipid bilayer. The composition and number of trans* To whom correspondence should be addressed. Phone: 509-376-0723. Fax: 509-376-7722. 10.1021/pr0255248 CCC: $22.00

 2002 American Chemical Society

membrane spanning helices make these proteins particularly difficult to characterize primarily due to their water insolubility. However, integral outer membrane proteins typically have a β-sheet secondary structure that usually contains significantly fewer nonpolar amino acid residues, which decreases their hydrophobicity and makes them more amenable to study.3-5 Despite their biological importance and natural abundance, a large-scale proteomic analysis of this protein class has been difficult. Techniques using two-dimensional polyacrylamide gel electrophoresis based mass spectrometry (2D-PAGE-MS) often result in under-representation of these hydrophobic proteins.2,5,6 This is probably due to the inability of detergents to effectively solubilize hydrophobic proteins in the aqueous medium used for isoelectric focusing and the tendency of these proteins to precipitate at their isoelectric point.7-10 Attempts to extract hydrophobic integral membrane proteins using organic solvent precipitation prior to 2-D electrophoresis did not show significant improvement compared to detergentbased protocols.10,11 An alternative technique to 2D-PAGE-MS analysis is insolution solubilization of the membrane proteins using surfactants12 followed by enzymatic or chemical fragmentation of membrane proteins, separation, and analysis of the complex Journal of Proteome Research 2002, 1, 351-360

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Published on Web 05/18/2002

research articles mixture of peptides using liquid chromatography (LC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). However, the presence of surfactants can suppress analyte ionization and interfere with chromatographic separations, requiring additional manipulations to remove surfactants and potentially leading to significant sample loses, particularly for low abundance proteins.13,14 Thus, the main obstacle for a large-scale mass spectrometric analysis of integral membrane proteins is the inability to achieve the dissolution of these hydrophobic proteins from the phospholipid bilayer while maintaining their solubility throughout the entire isolation and separation process and avoiding the reagents which may interfere with ESI.2,8,15 Consequently, there is considerable interest in having a sample preparation method that is more compatible to LC-MS/MS analysis and improves mass spectrometric accessibility to the membrane subproteome. To address this need, we have developed a surfactant-free sample preparation method using miscible extraction with organic solvent designed for large-scale LC-ESI-MS/MS analysis of integral membrane proteins in prokaryotes. The effectiveness of our approach was evaluated by comparing the experimentally identified integral membrane proteins from D. radiodurans to those predicted using calculated hydropathy values,16 transmembrane mapping,17 and sequence similarity with known membrane proteins whose function has been demonstrated and annotated for other organisms. To our knowledge, this is the first report of a large-scale isolation and identification of highly hydrophobic integral membrane proteins containing multiple TMDs from a single sample preparation. The efficacy and flexibility of the method make it amenable to study other types of prokaryotic organisms and different cell types in a high-throughput manner not previously possible.

Experimental Procedures Materials. D. radiodurans strain R1 cells were obtained from American Type Culture Collection No. 13939 (Manassas, VA). Yeast extract, tryptone, glucose, Tris hydrochloride, sodium carbonate, ammonium bicarbonate, and sequencing grade methanol were obtained from Sigma (St. Louis, MO). Bicinchoninic acid (BCA) assay reagents were purchased from Pierce (Rockford, IL). Sequencing grade-modified trypsin used for insolution digestion was from Promega (Madison, WI). Water was purified using a Barnstead Nanopure Infinity water purification system (Dubuque, IA). Bacterial Growth Conditions. D. radiodurans strain R1 cells were grown aerobically at 30 °C with shaking at 225 rpm in TGY culture medium (2.5 g of tryptone, 0.5 g of glucose, and 0.5 g of yeast extract in 500 mL of water). Cells were harvested in the late exponential-early stationary phase by centrifugation at 3200g for 10 min at 4 °C, resuspended, washed in 50 mM Tris, pH 7.3, and collected by centrifugation at 3200g for 10 min at 4 °C. Membrane Protein Preparation. Pelleted cells were diluted in 50 mM Tris, pH 7.3, using a 1:3 cells/buffer (mg/mL) ratio. The cells were lysed using a French press (Amnico Rochester, NY) with three presses at 16 000 psi. Unbroken cells and debris were removed by centrifugation at 3200g for 10 min at 4 °C. The supernatant was collected, and the protein concentration was determined by BCA assay.18 The membrane proteins were isolated using a slightly modified carbonate fractionation procedure.5,19 The lysate (containing approximately 20 mg of cellular proteins) was 352

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diluted with ice-cold 100 mM sodium carbonate, pH 11, to produce a final volume of 72 mL. The solution was slowly agitated using a shaker/rotisserie (Labquake, Conroe, TX) for 1.5 h at 4 °C and then centrifuged at 115000g for 1 h at 4 °C. The supernatant was discarded, and the membrane pellet was rinsed with deionized water, washed in 8 mL of 50 mM Tris, pH 7.3, and then pelleted by centrifugation at 115000g for 20 min. The membranes were resuspended in 50 mM ammonium bicarbonate, pH 8.0, via intermittent vortexing and sonication using a sonicating bath (Bronson model 1510, Danbury CT). The proteins were thermally denatured using a previously described in-solution technique.20 Briefly, the sample was incubated in airtight tubes at 90 °C for 20 min and transferred to ice-cold water, and the membrane protein concentration was determined by the BCA assay. The sample was then diluted with methanol to produce a composition of 60% organic solvent, resulting in a final protein concentration of 0.5 mg/mL. The membrane proteins were extracted and solubilized by performing intermittent vortexing and sonication. Tryptic digestion was immediately carried out using a slightly modified mixed organic-aqueous solvent proteolysis technique previously described.21 Briefly, sequencing grade-modified trypsin was prepared by adding 20 µL of resuspension buffer (provided by the manufacturer) to a vial containing 20 µg of enzyme, and after 15 min at room temperature the trypsin was added to the sample. The proteolysis was performed for 5 h at 37 °C using a 1:20 (w/w) trypsin-to-protein ratio, then quenched by rapid freezing using liquid nitrogen. The sample was stored at -80 °C until LCMS/MS analysis. For the control experiment, the membrane sample was prepared as described above. However, the protein solubilization step (prior to proteolysis) was performed using only 50 mM ammonium bicarbonate, pH 8.0, to produce final concentration of 0.5 mg/mL. Tryptic digestion was immediately carried out for 12 h at 37 °C using a 1:50 (w/w) trypsin/protein ratio and then quenched by rapid freezing using liquid nitrogen. The sample was stored at -80 °C until LC-MS/MS analysis. Whole Cell Lysate Preparation. Bacterial growth and cell lysis were the same as described above. Unbroken cells and debris were removed by centrifugation at 3200g for 10 min at 4 °C. The supernatant was collected and the protein content quantified using the BCA assay. Approximately 2 mg of protein was diluted up to 1 mL by the addition of 50 mM Tris, pH 7.3. The proteins were denatured and reduced by the addition of guanidine hydrochloride (6 M) and dithiothreitol (1 mM) and then incubated in a boiling water bath for 3 min. The sample was dialyzed using Spectra/Por-Float-A-Lyzer dialysis tubing (Spectrum Laboratories Inc., Rancho Dominguez, CA) against 50 mM ammonium bicarbonate (pH 8.0), and then the sample volume was adjusted to produce a final protein concentration of 0.5 mg/mL. Tryptic digestion was performed using sequencing grade-modified trypsin prepared as previously described. Trypsin was added to the protein mixture in 1:50 (w/w) trypsinto-protein ratio and the sample digested overnight at 37 °C. Tryptic activity was quenched by rapid freezing using liquid nitrogen and then the sample was stored at -80 °C until LCMS/MS analysis. Capillary LC-MS/MS Analysis. The high-pressure liquid chromatography (HPLC) system used in this study has been previously described.22 Briefly, a reversed-phase capillary column was made by packing 5 µm Jupiter C18 stationary phase

Membrane Protein Enrichment for LC-MS/MS Analysis

(Phenomenex, Torrence, CA) into a 65 cm, 150 µm i.d. × 360 µm o.d. capillary (Polymicro Technologies Inc., Phoenix, AZ) incorporating a 2 µm retaining mesh in a HPLC union (Valco Instruments Co., Houston, TX). The mobile phases consisted of (A) 0.05% trifluoroacetic acid in water and (B) 0.1% trifluoroacetic acid in 90% acetonitrile/10%water and were degassed off-line with helium prior to filling the pumps. The HPLC system was equilibrated at 5000 psi with 100% mobile phase A prior to sample injection. Each peptide sample (stored at -80 °C at a concentration of 0.5 mg/mL) was thawed to room temperature, and a 10 µL injection was loaded onto the HPLC column for each analysis. The exponential gradient was initiated 10 min after sample loading. Flow through the capillary HPLC column was ∼1.8 µL/min when equilibrated with 100% mobile phase A. The capillary LC system was coupled to a LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) using an inhouse manufactured ESI interface in which no sheath gas or makeup liquid was used. The temperature of heated capillary and electrospray voltage was 200 °C and 2.2 kV, respectively. Samples were analyzed using the data-dependent MS/MS mode over the following m/z ranges: 400-2000, 500-700, 700900, 900-1100, 1100-1300, 1300-1500, 1500-1700, and 17002000. The three most abundant ions detected in each MS scan were selected for collision-induced dissociation using a normalized collision energy setting of 35%. The results reported in this study include LC-MS/MS analyses for sample aliquots originating from either a single membrane sample preparation or a single whole cell lysate sample preparation. Data Processing and Analysis. The tandem mass spectra were analyzed against the genome database of D. radiodurans strain R1 using SEQUEST (Thermo Finnigan, San Jose, CA).23 After SEQUEST analysis was performed, the results were tabulated for each identified protein incorporating the protein loci and corresponding number of peptides assigned to each ORF. Only conventional tryptic peptide cleavages (containing up to two missed cleavages) displaying a cross-correlation score (Xcorr) of at least 2.0 and delta-correlation score (∆Cn) of at least 0.1 were considered for protein identification. Hydropathy Calculations. 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), which allows the calculation of the grand average of hydropathicity (GRAVY) value for a given protein.16 The proteins exhibiting positive GRAVY values were recognized as a hydrophobic. Transmembrane Mapping. All identified proteins and the entire genome sequence of D. radiodurans strain R1 were examined by the PSORT prediction algorithm using the version for bacteria (available at http://psort.nibb.ac.jp/form.html.). The analysis was performed to map TMDs and predict the subcellular location for each protein encoding ORF and recognize the proteins of the membrane subproteome.17,24-26

Results Proteins were isolated from D. radiodurans using either the membrane subproteome targeted technique or the whole cell lysate approach as shown in Figure 1. In all sample preparations the cells were grown, harvested, and disrupted prior to further processing. The membrane protocol uses a combination of carbonate extraction, ultracentrifugation, and methanolassisted solubilization to enrich for integral membrane proteins. Following proteolytic digestion in the mixed organic aqueous

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Figure 1. Membrane enrichment and whole cell lysate protocols used for proteomic analysis of D. radiodurans by LC-MS/MS.

system, the peptide sample was analyzed using LC-MS/MS with the product ions generated from each MS/MS scan searched against the genomic database of D. radiodurans using SEQUEST. The control membrane sample preparation was performed similarly, excluding only the organic solvent solubilization step, and thus contained membrane proteins that were solubilized and digested in aqueous solution. The whole lysate approach involves proteins released during cell lysis into the aqueous buffer at neutral pH. Isolated proteins were chemically denaturated and then dialyzed against the proteolysis buffer. Following proteolytic digestion in the aqueous medium, analysis was performed using the same LC-MS/MS method. Identification of Membrane Proteins. A summary of the results for each preparation appears in Table 1. Although the membrane protein preparation using methanol-assisted solubilization permitted the identification of 1134 unique peptides corresponding to 503 unique proteins in the annotated genome sequence of D. radiodurans, the total number of proteins identified from the whole cell lysate preparation and the membrane protein preparation are comparable. The total percentage of all putative proteins identified were 16% and 14% for the membrane protein and whole cell lysate preparations, respectively, while that of the control membrane protein preparation (no methanol) produced a 2% yield. However, to assess the efficacy of the developed protocol for the enrichment of integral membrane proteins, it was necessary to determine the hydropathy character of the identified proteins, recognize their subcellular location, map putative TMDs and compare the results. Partitioning of the proteins theoretically expressed by all ORFs of D. radiodurans and those identified by each preparation using LC-MS/MS was performed using hydrophobicity calculations,16 algorithmic prediction of protein subcellular location, and transmembrane mapping.17 In addition, the predicted membrane proteins were compared to known membrane proteins of other organisms having sequence similarity and whose subcellular location has been previously characterized. The positive GRAVY value is considered as a reliable marker for indicating the hydrophobic nature of a protein and a valid indicator of its membrane involvement.6-8,16 To obtain the Journal of Proteome Research • Vol. 1, No. 4, 2002 353

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Table 1. Proteins Identified from Membrane and Cell Lysate Preparations of D. radiodurans Using LC-MS/MS protein subcellular locationa

no. of proteinsa

identified from membrane preparation using MeOHb,c

identified from membrane preparation without MeOHb,c

identified from whole cell lysate preparationb,c

integral inner membrane proteins integral outer membrane proteins periplasmic proteins cytoplasmic proteins total

997 122 212 1781 3116

215 (21%) 53 (43%) 35 (17%) 200 (11%) 503 (16%)

16 (2%) 3 (2%) 5 (2%) 43 (2%) 67 (2%)

89 (9%) 12 (10%) 15 (7%) 341 (19%) 457 (14%)

a Predicted by PSORT. b Proteins were identified by database searching the product ions using SEQUEST. The results reported are for proteins that had at least one tryptic peptide having an Xcorr of greater that 2.0 and a ∆Cn score greater than 0.1. c The value in parentheses is the percentage of proteins identified relative to the total of each predicted subcellular class.

Table 2. Predicted and Identified Hydrophobic Proteins of D. radiodurans no. of hydrophobic positive no. of hydrophobic subproteome GRAVY proteins proteinsa coverage (%) range

theoretically expressed ORFs by the genome identified from the membrane sample using methanol identified from the membrane sample without methanol identified from the whole cell lysate sample

3316

896

100

0-1.58

503

135

15

0-1.30

67

10

1

0-0.58

457

19

2

0-0.32

a All identified proteins and the entire genome sequence of D. radiodurans were analyzed using the ProtParam program, which allows the calculation of the grand average of hydropathicity (GRAVY) for a given protein. The proteins exhibiting positive GRAVY values were recognized as a hydrophobic.

hydrophobic content and coverage of the D. radiodurans proteome, GRAVY values were calculated for each predicted ORF and experimentally identified protein as shown in Table 2. The results indicated that D. radiodurans has the potential to express 896 proteins (28% of its genome) containing positive GRAVY values ranging from +0.0005 to +1.58, which is in agreement with previous observations.1 The same hydropathy computational analysis of the 503 experimentally identified proteins from the membrane preparation indicated that 135 of them were hydrophobic with positive GRAVY values ranging from +0.005 to +1.3, corresponding to 15% coverage of the theoretically predicted hydrophobic proteome. The same analysis of the 457 experimentally identified proteins from the whole cell lysate preparation resulted in only 19 of them being hydrophobic, ranging from +0.005 to +0.32. This corresponds to 2% coverage and agrees with the GRAVY cut off of contemporary 2-D-PAGE separated hydrophobic proteins.7,8 Although the increased coverage of hydrophobic proteins afforded by the membrane protein protocol suggested the enrichment of membrane proteins, additional confirmation was required. Hydropathy plots are proven remarkably accurate for localization of transmembrane regions due to the nonpolar character of the amino acid residues and the length of TMDs17,27,28 and are particularly suited for prokaryotes because of the simplicity of their cellular structure and the considerable number of sequenced genomes. To better identify integral membrane proteins, all protein sequences were subjected to TMD mapping using the PSORT algorithm.17 The PSORT program predicts bacterial protein cellular location, their membrane position, and the number of TMDs. The current version is able to correctly classify 83% of 106 proteins with 354


Table 3. Comparison of the Number of Predicted and Identified Integral Cytoplasmic Membrane Proteins Based on the Number of Predicteda Transmembrane Domains (TMDs) no. of predicted TMDs

no. of proteins predicted in each class

no. of proteins identified in each class

proteins identified (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 total

526 120 74 61 48 46 26 23 23 15 21 8 1 3 1 1 997

123 24 10 12 8 12 3 4 6 2 6 1 1 1 1 1 215

23 20 13 19 16 26 11 17 26 13 28 12 100 33 100 100 21

a The number of predicted transmembrane domains (TMDs) for each protein-encoding ORF of the membrane subproteome of D. radiodurans strain R1 was determined using the PSORT algorithm (available at http:// psort.nibb.ac.jp/form.html). The number of TMDs for each experimentally identified membrane protein was determined using the same algorithm.

known localization from Gram-negative bacteria and is appropriate for the cell envelope structure of D. radiodurans.29 The analysis revealed that 997 ORFs (30% of the genome) are predicted to encode for integral cytoplasmic membrane proteins that contain from one to sixteen predicted TMDs with an additional 122 ORFs encoding for integral outer membrane proteins and 212 ORFs for periplasmic proteins (Table 1). Subjecting the data set of 503 identified proteins from the membrane-targeted sample preparation indicated that 215 (21% of all predicted) are integral cytoplasmic membrane proteins that contain from 1 to 16 predicted TMDs (Table 3), 53 (43% of all predicted) are integral outer membrane proteins, and 35 (17% of all predicted) are periplasmic proteins (Table 1). The same analysis of 457 proteins identified from the whole cell lysate preparation showed a significant decrease in number of identified integral membrane proteins particularly those with multiple TMDs in which 89 integral cytoplasmic proteins were identified (9% of all predicted) containing from one to five predicted TMDs (Table 1). The hydrophobicity profile calculated for the 1331 theoretically expressed proteins of the predicted membrane subproteome, which includes the cytoplasmic membrane, the outer membrane, and the periplasmic proteins of D. radiodurans, is shown in Figure 2A. The proteins identified for each sample preparation are shown in parts B-D of Figure 2. The remarkable similarity in the hydrophobicity frequency distribution


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Figure 2. Hydropathy comparison of the identified and predicted D. radiodurans membrane subproteome. The hydopathy plots for the (A) theoretically expressed ORFs of D. radiodurans membrane subproteome, which includes inner membrane, outer membrane, and periplasmic proteins, (B) proteins identified by using the membrane protein protocol, (C) proteins identified by using the whole cell lysis protocol, and (D) proteins identified by the membrane protocol without using methanol are shown. Each histogram was generated by plotting the number of proteins per 0.1 GRAVY value increment.

between the predicted proteins of the membrane subproteome and those identified from the membrane enrichment preparation indicate that our technique is efficient in solubilizing integral membrane proteins regardless of their hydropathy. However, the hydropathy profiles of the proteins identified from the control and the whole cell lysate preparations show a significant under representation of hydrophobic proteins. The correlation between the number of predicted TMDs and the hydropathy character of integral cytoplasmic membrane proteins remains an open issue.2 It has been determined that the majority of integral cytoplasmic membrane proteins (Rhelix bundles) are hydrophobic while the majority of outer integral membrane proteins (β-barrels) are hydrophilic.2,6 To investigate this further, the number of mapped TMDs in 215 identified integral cytoplasmic membrane proteins from the membrane preparation (Table 3) was correlated with their hydropathy (GRAVY index). It was observed that all proteins containing four or more predicted TMDs elicited positive GRAVY values, and thus were hydrophobic. Interestingly, the two most hydrophobic proteins identified had only one or two predicted TMDs. Only 54 (36%) of the identified integral cytoplasmic membrane proteins having from one to three predicted TMDs showed positive GRAVY values. These findings suggest that hydropathy calculation alone is not sufficient to recognize integral membrane proteins. Likewise, the number of mapped TMDs does not indicate the hydropathy character of proteins with three or less TMDs. Nevertheless, the complementary application of both computation analyses resulted in a more reliable characterization and partitioning of the theoretical and identified proteins of the D. radiodurans membrane subproteome. Out of 268 integral membrane proteins identified in this study, 22% (60 proteins) were annotated as hypothetical and having no significant similarity to any other proteins in the

current protein databases accessible at SWISS-PROT, and 10% (28 proteins) were annotated as conserved hypothetical and had limited similarity to other proteins. All identified integral membrane proteins except those characterized as hypothetical were examined in order to compare them with known membrane proteins of other organisms whose subcellular location has been characterized. The databases interrogated for this study were SWIS-PROT, TIGR, and ITNERPRO (accessible at http://www.ebi.ac.uk/swissprot/members.html). After comparison, 173 (83%) were annotated as integral membrane proteins by similarity, 22 (11%) as cytoplasmic, and 13 (6%) with unknown subcellular location. These findings lend additional evidence for preferential isolation of integral membrane proteins by the membrane protein enrichment method. Identification of TMD-Spanning Peptides of Integral Cytoplasmic Membrane Proteins. The data appearing in Table 3 reveal that 68 highly hydrophobic integral cytoplasmic membrane proteins having three or more predicted TMDs were detected in the enriched membrane protein sample by LCMS/MS. Detection and identification of this class of integral cytoplasmic membrane proteins with multiple TMDs using 2-D gel methods are rare. To our knowledge, the data presented in Table 3 represents the highest number of identified integral cytoplasmic membrane proteins in prokaryotes reported to date. The ability to detect peptides spanning TMDs using our approach is exemplified by the identification of the integral membrane proteins presented in Table 4, and in particular, to those of the cytochrome superfamily listed in Table 5. These proteins were identified by peptides eliciting a high Xcorr based on their MS/MS spectra. Among those identified is the succinate dehydrogenase cytochrome subunit (DR0954), a protein whose structure has been determined from several prokaryotes.30,31 It is a mono-heme transmembrane component of the Journal of Proteome Research • Vol. 1, No. 4, 2002 355

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Table 4. Partial List of Identified Integral Membrane Proteins Containing Multiple Transmembrane Domains (TMDs) Only Detected Using the Membrane Protein Protocol accession no.

gene name

description

GRAVY index

no. of mapped TMDs

protein identified by a TM peptidea

Q9RUR0 Q9RT37 Q9RXE0 Q9RVJ1 Q9RRU0 Q9RYZ4 Q9RX61 Q9RSC8 Q9RRG7 Q9RUH5 Q9RUS5 Q9RVZ0 Q9RTP8 Q9RZJ4 Q9RR77

DR1322 DR1929 DR0373 DR1037 DR2395 DRA0159 DR0454 DR2197 DR2525 DR1411 DR1307 DR0880 DR1709 DRB0133 DR2620

putative sugar efflux transporter glycerol uptake facilitator protein cation exchanger, putative major facilitator family protein Na+/H+ antiporter, putative phosphate ABC transporter, permease protein bacitracin resistance protein ABC transporter, permease protein, CYSTW family C4-dicarboxylate transport protein transporter, sodium/sulfate symporter family major facilitator family protein NADH-ubiquinone/plastoquinone subunit, putative probable manganese transport protein MNTH Na (+)-linked D-alanine glycine permease cytochrome c oxidase, subunit I

0.97 0.93 0.87 0.85 0.83 0.79 0.78 0.73 0.71 0.71 0.71 0.69 0.69 0.69 0.67

9 5 9 8 11 6 6 5 8 10 6 15 9 11 16

yes yes no yes yes yes yes no yes no no yes no yes yes

a

Each protein was identified by at least one peptide that completely spans one mapped TMD.

Table 5. Proteins of the Cytochrome Superfamily Identified by Using the Membrane Protein Protocol and Subsequent Analysis by LC-MS/MS GRAVY index

accession no.

gene name

description

Q9RVR8 Q9RVR9

DR0954 DR0953

Q9RR77 Q9RX79 Q9RX81 Q9RSM9

DR2620 DR0436 DR0434 DR2095

succinate dehydrogenase, cytochrome subunit succinate dehydrogenase, hydrophobic subunit SDHD, putative cytochrome c oxidase, subunit I cytochrome b6 cytochrome c6, putative c-type cytochrome, putative

respiratory chain of all aerobic organisms. As shown in Figure 3, two hydrophobic transmembrane spanning peptides, which cover 49% of its sequence, have been detected by LC-MS/MS. The MS/MS spectrum for one of these transmembrane peptides produced an Xcorr of 6.9. Another cytochrome of known structure,32 cytochrome c oxidase, subunit I (DR2620), contains 16 predicted TMDs and was identified by LC-MS/MS as shown in Figure 4. It is a key enzyme in aerobic metabolism which reduces dioxygen to water and involves four redox-metal sites comprised of two protohemes and two copper centers.33 Three unique peptides were detected by LC-MS/MS in which two of them completely cover two distinct TMDs, producing Xcorr’s of 3 and 4. The ability to identify 37 (65%) out of 58 identified integral cytoplasmic membrane proteins with four and more predicted TMDs by detecting at least one hydrophobic peptide spanning a TMD, demonstrates the efficacy of the developed method in isolating and solubilizing hydrophobic integral membrane proteins for LC-MS/MS analysis. Together these data indicate that it is possible using the membrane enrichment protocol to solubilize and identify integral cytoplasmic membrane proteins regardless of the number of predicted TMDs.

Discussion D. radiodurans is a pigmented, non-spore-forming, nonmotile, spherical Gram-positive bacterium and was chosen to develop our integral membrane protein protocol for several reasons. Its genome has been sequenced,34 and its proteome has been extensively studied in our laboratory using various LC-MS methods.35 Although classified as Gram-positive, D. radiodurans possess a distinctive cell wall with a structure similar to that of a Gram-negative organism.36 Its cell envelope is composed of the S layer, outer membrane, compartmental356


Xcorr

no. of mapped TMDs by PSORT

membrane location by PSORT

0.81 0.43

6.9127 3.4894

2 2

inner inner

0.67 0.55 0.11 -0.66

4.2448 4.1064 3.4518 3.6204

16 8 4 0

inner inner inner outer

ized layer, thick peptidoglycan layer, and cytoplasmic membrane.29 It has a high resistance to genotoxic chemicals, oxidative damage, dehydration, and high levels of ionizing and ultraviolet radiation. Hence, there is an interest in elucidating the DNA repair mechanism of D. radiodurans due to its potential application for bioremediation.37 Since these environmental perturbations occur extracellularly, the membranes and integral membrane proteins may play an important role in resistance of D. radiodurans to extreme environmental conditions. Subproteomic Approach. The identification of an entire proteome, regardless of its origin, is a daunting task for several reasons. The dynamic range of current instrumentation is limited by fluctuating protein expression levels, which may span more than 6 orders of magnitude. In addition, the limited sensitivity and ability of contemporary proteomics to characterize proteins with high molecular masses, extreme isoelectric points, or extremes in hydrophobicity precludes complete coverage of a given proteome.6,38 One approach to circumvent these difficulties is to reduce sample complexity by subcellular fractionation. The enrichment of proteins from a selected part of a proteome, referred to as the subproteome, is expected to facilitate the identification of low abundance proteins and assist in deducing protein or organelle function under a given set of experimental conditions or well-defined environmental circumstances.6,35,39 Hydrophobic Character of Membrane Proteins. Although the membrane subproteome of many organisms is of great interest, the difficulties associated with this class of proteins have seriously impaired proteomic analysis. The hydrophobicity of the majority of integral membrane proteins makes them difficult to solubilize, particularly in a complex protein mixtures.


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Figure 3. Identification of the cytochrome b-556 subunit of succinate dehydrogenase by LC-MS/MS. (A) The base peak chromatogram obtained during the separation of peptides from a 10 µL injection of a 0.5 µg/µL solution of the D. radiodurans membrane protein sample was analyzed using the m/z range of 400-2000. (B) The MS/MS spectrum of EGQWAFLLHRLSGLAILLYLMLHVFSIGSVILGEEFYMR. This tryptic peptide was identified by database searching the productions using SEQUEST and produced an Xcorr of 6.9. This peptide completely covers one TMD of the cytochrome b-556 subunit of succinate dehydrogenase. The tryptic peptide containing part of the other TMD (spectrum not shown) was identified with an Xcorr of 3.5. The mapped TMDs are in italics, and the peptides identified are boxed.

It has been inferred from previous studies that initial protein solubilization is the critical step where hydrophobic proteins are lost.7 Thus, various detergent-based techniques have been used to solubilize hydrophobic integral membrane proteins5,12 and used extensively in 2D-PAGE membrane sample preparations.8-10 The role of the detergents is 2-fold: (1) to inhibit intraor inter-protein hydrophobic interactions and (2) to prevent the loss of hydrophobic integral membrane proteins through aggregation or adsorption. Although significant progress has been made in 2D-PAGE sample preparation for investigations of integral membrane proteins of prokaryotes,5,6,40-46 highly hydrophobic membrane proteins are still missing from 2D gel analyses.2,7,8 In fact, one of the largest 2D-PAGE studies of the membrane subproteome of P. aeruginosa identified 189 proteins from the membrane fraction; however, approximately 95% of the identified proteins had negative GRAVY values with the highest positive value being +0.27.6 These data suggest hydrophobic proteins are not solubilized to permit electrophoretic separation and subsequent in-gel tryptic digestion and LCMS/MS analysis. This is in contrast to the 268 integral membrane proteins identified by LC-MS/MS using our membrane enrichment protocol. In this preparation 135 (50%) of the identified integral membrane proteins were determined to be hydrophobic (Table 2 and Figure 2) with the highest GRAVY value being +1.302. In-solution surfactant-based sample preparation techniques may be developed for large-scale LC-ESI-MS/MS analysis of

integral membrane proteins and provide a less labor-intensive approach than possible with 2D-PAGE-MS. However, the adverse effects of detergents on chromatographic separation and the quality of mass spectra could diminish LC-MS measurements13,14 unless other separation or cleanup steps are employed. For example, in the large-scale multidimensional LC-MS/MS analysis of the membrane proteome of human myeloid leukemia (HL-60) cells, the microsomal fraction was dissolved in a labeling buffer containing SDS.47 A total of 491 proteins was identified and quantified using the isotope-coded affinity tag (ICAT) approach and multidimensional chromatography coupled to tandem mass spectrometry. Although a considerable fraction of membrane proteins was identified, the highly hydrophobic integral membrane proteins were not amenable to this technique. An efficient surfactant-free sample preparation was also reported.48 Formic acid and cyanogen bromide were used to cleave solvent-accessible portions of integral membrane proteins, resulting in one of the largest number of identified yeast membrane proteins using twodimensional LC-ESI-MS/MS analysis. Of the proteins identified, 131 were assigned as integral membrane proteins with three or more predicted TMDs, of which 58 were assigned as cytoplasmic integral membrane proteins. Although this study was not specifically designed for the analysis of the membrane subproteome, the detection of hydrophobic peptides spanning TMDs was notably underrepresented. The large-scale identification of hydrophobic integral membrane proteins (Tables Journal of Proteome Research • Vol. 1, No. 4, 2002 357

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Figure 4. Identification of cytochrome c oxidase subunit I by LC-MS/MS. (A) The base peak chromatogram obtained during the separation of peptides from a 10 µL injection of a 0.5 µg/µL solution of the D. radiodurans membrane protein sample was analyzed using the m/z range of 400-2000. Three tryptic peptides, whose MS/MS spectra are shown in B-D, were identified by database searching their product ions using SEQUEST and produced Xcorr values of 4.0, 2.5, and 3.0, respectively. The regions mapped by each peptide are shown in E, where the mapped TMDs are in italics and the peptides identified are boxed. The region of the MS/MS spectrum for the peptide shown in D emphasizes the identified b and y ions that span the TMD.

1-3) by our membrane enrichment protocol demonstrates a significant improvement over previously reported 2D-PAGE or LC-MS/MS analyses. These results are even more impressive when examining the extent of identified peptides that span TMDs. Identification of Hydrophobic Transmembrane Proteins. Water-insoluble membrane proteins with multiple TMDs are rarely identified on 2-D gels, significantly limiting 2D-PAGE based analysis of integral membrane proteins.2,8,12 Using our membrane enrichment method outlined in Figure 1, a broad range of hydrophobic integral membrane proteins was identified. The most hydrophobic protein identified was a hypothetical 9.7 kDa protein (DR1825) having a GRAVY value of +1.30 and is the fourth most hydrophobic protein of the genome of D. radiodurans. The least hydrophobic protein identified in the study was a hypothetical 68.6 kDa protein (DR0885) having a positive hydropathy value of +0.005. In fact, the coverage obtained using the membrane enrichment protocol exhibits approximately the same hydropathy profile as the entire membrane subproteome (Figure 2), a result not achieved with 358


the control or whole cell lysate preparation or any 2-D-PAGE analysis previously reported. The ability to detect peptides spanning TMDs using our approach is exemplified in the MS/MS spectra presented in Figures 3 and 4. The MS/MS spectrum in Figure 3 is the peptide encoding residues 6 to 44 of the first TMD of the cytochrome b-556 subunit of succinate dehydrogenase (DR0954), and the two spectra shown in Figure 4 are assigned as peptides spanning two separate TMDs of cytochrome c oxidase subunit I (DR2620). Collision-induced dissociation of each TMD spanning peptide produced a significant number of y and b ions of sufficient intensity, enabling unambiguous identification the integral membrane protein by database searching the product ions using SEQUEST. For all integral membrane proteins containing four or more TMDs, 65% were identified by at least one hydrophobic peptide spanning the TMD. These unprecedented results support the effectiveness of the method to enrich and solubilize integral membrane proteins containing multiple TMDs, allowing large-scale detection and identification of this protein class not previously attainable.

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In addition to MS detection, effective separation of these hydrophobic TMD peptides needs to be achieved. Both chromatograms shown in Figures 3 and 4 are similar, indicating the reproducibility of the reversed-phase separation of the same membrane protein sample preparation. The peptides spanning the TMDs of these proteins possess a high retention time. This is consistent with the C18 chain length used in the stationary phase and the hydrophobicity of the peptides identified. Work to enhance the detection of these hydrophobic TMD spanning peptides is currently being conducted, including the use of different mobile phases and alternative stationary phases employing shorter-alkyl chain lengths (e.g., C8). Increasing separation efficiency of these hydrophobic peptides will potentially increase the number of identified proteins and expand the coverage of a given membrane subproteome using LCMS/MS. In addition, multidimensional separations at the protein or peptide level prior to LC-MS/MS analysis would significantly enhance the detection of low abundance membrane proteins. Maintaining Membrane Protein Solubility. The inability of current methodologies to achieve efficient large-scale LC-MS/ MS analysis of highly hydrophobic integral membrane proteins is the direct consequence of their insolubility. To perform a high-throughput analysis, the solubility of these proteins must be maintained during the course of sample processing, including proteolytic digestion and reversed-phase column loading. The interactions between the solvent or solutes and the cell membrane should promote protein dissolution and subsequent solubilization. When examining the interaction between alcohols and cell membranes, it was observed that alcohol partitioning is strongly dependent on its chain length, suggesting that shorter-chain alcohols easily disrupt hydrophobic interactions taking place on the aqueous-membrane interface.49 Methanol was chosen as the solvent for our membrane protein protocol because it is a short-chain, polar, water miscible, and ESI-MS compatible solvent. It was anticipated that methanol would preferentially partition into the membrane and induce swelling and dissolution of the phospholipd bilayer.50 Since the membrane bilayer was suspended in an aqueous buffer solution, an optimal concentration of methanol would reduce the phospholipidwater interfacial tension to zero, resulting in a single-phase solution and miscible extraction of the membrane proteins. The analysis and results of the control (performed without methanol) explicitly indicate the significance of methanol-assisted solubilization for the enrichment of hydrophobic integral membrane proteins. The use of the methanol to provide for membrane protein solubilization did not interfere or impede tryptic digestion and agrees with various reports indicating that organic solvents have the potential to accelerate tryptic digestion without significant loss of enzyme activity.21,51-54 On the basis of the large-scale detection of hydrophobic, transmembrane spanning peptides, and results from the control membrane preparation, it is evident that 60% methanol assisted by sonication was able to achieve miscible extraction and solubilization of the membrane proteins from D. radiodurans and promote complete proteolysis. Significant protein precipitation was avoided due to sufficient hydration and polarity of the methanol-aqueous solution, allowing the largescale identification of hydrophilic integral membrane proteins as well. However, based on the nature of the phospholipid bilayer derived from different bacteria and cell-types, other alcohols such as ethanol, propanol, or various combinations

of these shorter chain alcohols or other organics (e.g., acetonitrile or dimethyl sulfoxide) may be alternative solvents. Additional experimental work at optimizing the isolation and solubilization conditions is currently in progress. Further work at optimizing the tryptic digestion conditions is ongoing and will be required when employing different organic-aqueous systems. Quantitating Membrane Protein Expression. Although the membrane protein enrichment protocol was used for protein identification by LC-MS/MS, it can be implemented to quantify membrane protein expression as well. For comparative quantitation of membrane proteins from cell cultures, proteins can be metabolically labeled by growing cells in either 14N or 15 N enriched media.55,56 The cells are then combined and the membrane proteins enriched using the developed protocol. In this manner quantitation of membrane protein expression can be performed by measuring the 14N/15N abundance ratios for peptide pairs using high-resolution mass spectrometry. In a similar manner, 16O/18O proteolytic labeling57 could be accomplished using light and heavy water when performing tryptic digestion. In addition, quantitation and enrichment of cysteinyl-peptides may be performed using ICAT labeling.58 Depending on the organism and the membrane proteins identified, quantitation and identification of phosphorylation and O-linked glycosylation sites could be performed using the phosphoprotein isotope-coded affinity tag approach in which these sites are modified by β-elimination and Michael addition of an isotopic linker with subsequent biotinylation.59,60

Conclusions The results from our study demonstrate that it is possible to achieve efficient enrichment and solubilization of hydrophobic integral membrane proteins using organic-aqueous miscible extraction. In the described method, efficient extraction of hydrophobic membrane proteins was achieved using high pH fractionation, thermal denaturation, and organic solvent-assisted solubilization. Avoiding chaotropic denaturants and surfactants enhanced ESI-MS analysis significantly, facilitating a large-scale LC-MS/MS analysis of the membrane subproteome. The identification of 268 integral membrane proteins of D. radiodurans in a single experiment (24% coverage of the predicted integral membrane proteins) represents a significant improvement for LC-MS/MS analysis for this protein class. The detection of hydrophobic peptides spanning the TMDs of 65% of the identified integral cytoplasmic membrane proteins support the effective enrichment and efficient solubilization of these hydrophobic proteins. These results further indicate that hydrophobic peptides are readily detected when separated using single-dimension high-pressure reversedphase capillary LC coupled with ESI-MS/MS analysis. The membrane enrichment protocol constitutes a directed subproteomic approach for the analysis of integral membrane proteins that should be amenable to other prokaryotic organisms and similarly useful for eukaryotic membrane subproteome analysis.

Acknowledgment. We thank Dr. Kristina P. Taylor, Dr. David G. Camp, Deanna L. Auberry, and Kim K. Hixson for their technical assistance during the completion of this work. We also thank the United States Department of Energy Office of Biological and Environmental Research, Life Sciences Division, for support of this research. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy under contract DE-AC06-76RLO 1830. Journal of Proteome Research • Vol. 1, No. 4, 2002 359

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PR0255248

Enrichment of Integral Membrane Proteins for Proteomic Analysis

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