An Efficient Organic Solvent Based Extraction Method for the

An Efficient Organic Solvent Based Extraction Method for the Proteomic Analysis of Arabidopsis Plasma Membranes Srijeet K. Mitra,† Benjamin T. Walters,‡,# Steven D. Clouse,† and Michael B. Goshe*,‡ Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695-7609, and Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622 Received December 4, 2008

Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work (Mitra, S. K.et al. J. Proteome Res. 2007, 6, (5), 1933-50), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis. Keywords: Proteomics • mass spectrometry • liquid chromatography • strong cation exchange • phase partitioning • membrane proteins • transporters • Arabidopsis • methanol

Introduction The plasma membrane is a complex structure that separates the cell from its external environment while providing a means for cellular communication. It consists of a phospholipid bilayer and associated proteins which fall into two major classes: the integral or intrinsic proteins which traverse the membrane and the peripheral or extrinsic proteins which associate with components residing in the membrane, but do not themselves reside within the membrane. The integral membrane proteins are crucially involved in many physiological processes and constitute 20-30% of the cellular proteome.1 They are typically characterized by having either a β-barrel porin type fold or * Address correspondence to: Dr. Michael B. Goshe, Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Campus Box 7622, Raleigh NC 27695-7622. Phone: 919.513.7740. Fax: 919.515.2047. E-mail: [email protected]. † Department of Horticultural Science, North Carolina State University. ‡ Department of Molecular and Structural Biochemistry, North Carolina State University. # Current address: Department of Biochemistry and Biophysics, 1005 Stellar-Chance Laboratories, University of Pennsylvania, Philadelphia PA 19104-6059.

2752 Journal of Proteome Research 2009, 8, 2752–2767 Published on Web 04/01/2009

hydrophobic R-helical segments that span the membrane. Because of their participation in cell signaling mechanisms, detailed analysis of membrane proteins has been a subject of intense investigation, including many plant proteomic studies.2-5 Our interest in plant membrane proteins centers around proteins involved in signaling pathways in Arabidopsis, particularly the family of leucine-rich repeat receptor-like kinases (LRR RLKs). Several members of this family have proven functional roles in the regulation of plant growth, morphogenesis, disease resistance, and responses to the environment, but the functions of most members of the very large family of LRR RLKs remain unknown.6 Although much progress has been made recently on the study of plant proteomes,2 one of the challenges is to establish an effective protocol for the identification of these highly hydrophobic membrane proteins that is ideally suited for both discovery and quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Integral membrane proteins containing multiple transmembrane domains (TMDs) tend to aggregate and precipitate upon removal from the lipid bilayer and this appears to be exacerbated by the presence of reactive thiolates 10.1021/pr801044y CCC: $40.75

 2009 American Chemical Society

Multiple Organic Solvent Method for Membrane Proteomic Analysis 7

of reduced cysteinyl residues. In addition, the varying sizes of the cytoplasmic and extracellular loops and the presence of post-translational modifications (e.g., glycosylation and phosphorylation) provide integral membrane proteins with diverse physicochemical properties that can result in sample losses depending upon the sample preparation method used. Typically, detergents are used to promote membrane protein solubility, but these reagents require multiple LC processing steps to remove them prior to LC/MS/MS analysis, which can result in sample losses.8 These losses can complicate quantitative proteomic analysis when they are performed prior to the isotope-coded labeling step using reagents such as ICAT9 and iTRAQ.10 Modifications to bottom-up protocols have been required to address proteomic analysis of integral membrane proteins.11 Because of the hydrophobicity of TMDs, peptides spanning these regions are difficult to identify; thus, most membrane proteins are often identified by a single peptide which is more easily recovered, making the determination of false discovery rates a significant issue.12 In an effort to increase the detection of Arabidopsis membrane proteins from plant tissue, we previously developed both a detergent- and methanol-assisted microsomal solubilization and tryptic digestion approach using solid-phase extraction (SPE), offline strong cation exchange (SCX), and reversed-phase LC/MS/MS.13 Although the methanolassisted solubilization was superior to the detergent-based method, losses occurring during sample workup after tryptic digestion and the interference of highly abundant proteins, such as Rubisco and other photosynthetic proteins, and phytochemicals undermined the ability to characterize membrane proteins. To address this problem in regard to plasma membrane proteins, we developed an efficient chloroform-based extraction procedure used in conjunction with two-phase partitioning14 and methanol solubilization13 to more effectively identify plasma membrane proteins from plant tissue, including LRR RLKs, while eliminating additional sample preparation steps and removing phytochemical contaminants or lipid-based components. The developed procedure should also be readily applicable to other plasma membrane protein systems to facilitate bottom-up proteomic analysis and identification.

Materials and Methods Materials. After seed sterilization, Arabidopsis thaliana (ecotype Columbia-0) seedlings were grown for 11-days under constant light in liquid shaking culture as previously described.13 Methanol (HPLC grade), chloroform (pesticide grade), BCA Assay kit, sodium carbonate, Tris · HCl, tris(2-carboxyethyl)phosphine hydrochloride (TCEP · HCl), and iodoacetamide were from Thermo Fisher Scientific (http://www.thermofisher. com). Ammonium bicarbonate and ammonium formate were from Fluka (Milwaukee, WI). N-benzoyl L-arginine ethyl ester (BAEE), polyethylene glycol 3350, dextran (450 000-500 000 g/mol polymer mixture), phenylmethanesulfonyl fluoride (PMSF), and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO). Acetonitrile (HPLC grade) and formic acid (ACS reagent grade) were purchased from Aldrich (Milwaukee, WI). Sequencing grade-modified trypsin was from Promega (Madison, WI). Microcystin was from EMD Biosciences, Inc. (San Diego, CA) and protease inhibitor cocktail tablets were from Roche Diagnostics (Indianapolis, IN). The peptide Ac-SYSMEHFRWGKPV-OH was from Bachem Americas, Inc. (Torrance CA). All other chemicals were obtained from Thermo Fisher or Sigma-Aldrich unless otherwise noted. Water

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(18 MΩ) was distilled and purified using a High-Q 103S water purification system (Wilmette, IL). UV-Vis Spectroscopy Analysis of the Chloroform Extraction. The effectiveness of chloroform in extracting phytochemicals from protein samples prepared from plant tissue was assessed using UV-vis spectroscopy. A sample of 0.5 g of Arabidopsis seedlings was homogenized in 1 mL of 50 mM Tris · HCl (pH 7.4) and centrifuged at 6 000g for 15 min to remove cellular debris and the supernatant was transferred to a new 1.5 mL siliconized microcentrifuge tube. Then 100 µL of chloroform (a volume equivalent of 10%) was added to the sample followed by vortexing at medium speed for 15 s. The initial sample, the upper aqueous layer, and the lower chloroform layer were analyzed by a UV-vis diode array detector (Agilent Technologies, Inc., Palo Alto, CA) to generate a spectrum from 400 to 700 nm. A 1:5 dilution was used for all spectrophotometer measurements. Chloroform Extraction on a Model System. The use of chloroform extraction to facilitate the removal of trypsin from the aqueous phase while allowing peptides to remain was established using a model system containing 10 µg of a test peptide with a single trypsin cleavage site (Ac-SYSMEHFRWGKPV-OH) in 100 µL of 50 mM ammonium bicarbonate, pH 8.3, in a 1.5 mL siliconized microcentrifuge tube. To this sample, 10 µL of chloroform was added, the sample was then vortexed on med/high speed for 15 s, and the phases were separated by centrifuging at 14 000 rpm for 2 min. For tryptic digestion, 0.5 µg trypsin was added to another sample and was incubated overnight at 37 °C prior to chloroform extraction. In a third sample, the solution contained all of the above components plus 10% methanol. It was observed that chloroform was miscible when added to the 60% methanol/40% 50 mM ammonium bicarbonate buffer (pH 8.3) used in our previous work.13 With the use of a series of samples containing a reduced amount of methanol, it was determined that a composition of 10% methanol would facilitate a distinct twophase system upon chloroform addition as observed for the aqueous samples. Once all the samples were subjected to the chloroform extraction, the upper and lower layers were removed and transferred into separate siliconized 1.5 mL microcentrifuge tubes and lyophilized. As a control, one sample was treated with 10 µL of buffer instead of chloroform and the entire sample was lyophilized. For mass spectrometry analysis, each residue was dissolved in 100 µL of methanol/0.1% formic acid (1/1, v/v) and then diluted 1:10 to produce a final peptide concentration of 10 ng/ µL. The samples were infused at a flow rate of 1.5 µL/min into a custom built electrospray (ESI) interface coupled with an LCQ Deca ion trap mass spectrometer (Thermo Scientific, San Jose, CA) operating in the positive ion mode at an ESI voltage of 2.2 kV. Data were acquired in the profile mode for the m/z range 400-2000 for 1 min with the integrated mass spectrum used in post-acquisition analysis. Measuring Trypsin Depletion and Activity after Chloroform Extraction. To determine the extent of trypsin removal from the aqueous sample to the phase interface of the chloroform/aqueous phase system, both the quantity and its activity were measured. In one experiment, 10 µg of BSA was treated with 0.5 µg of trypsin in either 100 µL of 50 mM ammonium bicarbonate (pH 8.3) or 60% methanol/40% 50 mM ammonium bicarbonate (pH 8.3) and incubated overnight at 37 °C. Both samples were chloroform extracted; the sample containing 60% methanol was reduced to 10% methanol by Journal of Proteome Research • Vol. 8, No. 6, 2009 2753

research articles using a stream of nitrogen, and each sample was analyzed by SDS-PAGE and visualized by Coomassie staining. To determine the remaining trypsin activity, the hydrolysis of N-benzoyl L-arginine ethyl ester (BAEE) was measured using a modified version of a previously described assay.15 For these experiments, 10 µg of trypsin was added to 500 µL of either 50 mM ammonium bicarbonate (pH 8.3) or 10% methanol/90% 50 mM ammonium bicarbonate (pH 8.3) (v/v) and the trypsin activity of the solution was measured before and after chloroform extraction using an Agilent 8453X UV-vis diode array spectrophotometer (Agilent Technologies, Inc., Palo Alto CA) to monitor the absorbance at 253 nm every 20 s for 10 min. For the BAEE assay, a quartz cuvette containing 3.0 mL of 100 mM Tris (pH 7.8) and 1.0 mM BAEE was equilibrated to 24 ( 2 °C along with each sample. After subtraction from a reference measurement for the buffer, the data acquisition was started to establish the initial baseline. After 2 min, 50 µL of each sample was added to the reaction cuvette and mixed using a stirrer during the interval between measurements. The rate of the reaction was obtained from the linear regression analysis of the absorbance versus time data using the kinetics software of the UV-visible ChemStation Program (Rev. A.10.01) (Agilent Technologies, Inc., Palo Alto CA). All experiments were performed in triplicate. Preparation of Purified Plasma Membranes. The microsomal fractions were isolated from plant tissue as previously described13 and then reconstituted in 2 mL of resuspension buffer (330 mM sucrose, 4 mM potassium phosphate (pH 7.8), 2 mM potassium chloride, 2 mM EDTA and inhibitors). To enrich for plasma membranes, a PEG/dextran phase separation using a 6.5% polymer composition was implemented.14 The resuspended membranes (1.0 mL) were added to 4.0 g of an aqueous polymer two-phase system containing a final composition of 6.5% (w/w) dextran 500, 6.5% (w/w) PEG 3350, 5 mM potassium phosphate, pH 7.8, and 3 mM potassium chloride. Plasma membranes were then purified by aqueous polymer two-phase partitioning as described previously.14 The final upper phases were diluted at least 2-fold with 330 mM sucrose, 5 mM potassium phosphate, pH 7.8, and 0.1 mM EDTA. The plasma membranes were isolated by ultracentrifugation at 100 000g for 1 h. The entire procedure was performed at 4 °C. Tryptic Digestion of Membrane Proteins Using MethanolBicarbonate Buffer System. The proteins contained in the enriched plasma membranes were solubilized in a methanolbicarbonate buffer and digested with trypsin as previously described.13 Chloroform Extraction. After complete digestion in the methanol-bicarbonate buffer, chloroform extraction was performed. Prior to extraction, the methanol concentration was lowered to e10% (v/v) using a stream of nitrogen which did not promote any peptide precipitation. Once the sample contained e10% methanol, chloroform was added to the digested sample using a volume equivalent of 10%. The sample was then vortexed for at least 30 s and the phases were separated by centrifuging at 14 000 rpm for 2 min. The upper aqueous layer containing the peptides was removed, lyophilized, and stored at -80 °C. Solid-Phase Extraction (SPE). Following digestion and the evaporation of methanol to 10%, peptides were purified via SPE using a Prevail C18 Extract-Clean column (Alltech Associates, Inc., Deerfield, IL) connected to a vacuum manifold (Thermo Fisher) as previously described.13 Briefly, the column was 2754

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Mitra et al. equilibrated with 1 mL of water and 1 mL of 10% methanol/ 90% 50 mM ammonium bicarbonate, pH 8.0, (v/v) followed by sample loading, then washing with 2 mL of 10% acetonitrile/ 90% 50 mM ammonium bicarbonate, pH 8.0, (v/v). Peptides were eluted with 1 mL of 90% acetonitrile/10% of 0.1% formic acid (v/v), lyophilized, and then stored at -80 °C. Peptide Fractionation and Microcapillary Reversed-Phase LC/MS/MS Analysis. SCXC was performed as previously described13 using a 4.6 mm × 200 mm, 5 µm polySULFOETHYL aspartamide SCX column (PolyLC, Inc., Columbia, MD) connected to an Agilent 1100 Analytical HPLC system equipped with a UV-vis diode array detector. Peptides collected during SCXC were analyzed by microcapillary reversed-phase LC/MS/ MS using an Agilent 1100 Series high-performance capillary LC system coupled with a LCQ Deca ion trap mass spectrometer containing an in-house slurry packed 55 cm × 360 µm o.d. × 150 µm i.d. capillary (Polymicro Technologies, Inc., Phoenix, AZ) containing 5 µm, 300 Å Jupiter C18 stationary phase (Phenomenex, Torrance, CA) connected to a custom ESI interface as previously described.13 The mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The peptides in each fraction were solubilized in 20 µL of 5% B, and 8 µL injections were analyzed using two different reversed-phase gradients. For the first gradient program, the sample was loaded onto the reversed-phase column and the gradient program held the flow rate of 1.5 µL/min at 5% B for 20 min, and then initiated a linear gradient to 95% B over 90 min. For the second gradient, after sample loading, the gradient program held the flow rate of 1.5 µL/min at 5% B for 20 min, and then initiated a linear gradient to 20% B over 7.5 min, a linear gradient to 75% B over 110 min, and then a linear gradient to 95% B over 6.5 min. After each separation, the column was washed with 95% B for 20 min and then equilibrated with 5% B for 60 min prior to the next injection. The LCQ Deca was operated in the data-dependent acquisition (DDA) MS/MS mode in which the four most intense ions detected in the precursor MS scan were selected for collisioninduced dissociation (CID). A 2 min dynamic m/z exclusion list for selected precursor ions was utilized to increase the detection of lower abundant peptides during gradient elution. Data were acquired in the centroid mode for the m/z range 400-2000 using a normalized collision energy setting during CID of 45%. Peptide and Protein Identification. Peptides were identified by searching the product ion spectra against the A. thaliana database obtained from The Arabidopsis Information Resource (TAIR) Web site (http://www.arabidopsis.org) using TurboSEQUEST (BioWorks 3.3, Thermo Electron, San Jose, CA). An indexed decoy Arabidopsis database was also constructed using the reversed amino acid sequence for each protein. Both databases were then indexed by TurboSEQUEST using our searching criteria and used for interrogating all product ion spectra. Peptide identifications were extracted from the output files using in-house developed software. All searches included a static carboxamidomethyl modification of 57.0 u on Cys residues due to alkylation and a variable phosphorylation modification of 80.0 u on Ser, Thr, and Tyr residues. For DTA file generation and precursor ion tolerance, a value of 1.5 u was used for all searches. The peptides listed in all tables are unique sequences; however, these peptides may not be unique to a single protein and thus may also exactly match several other protein isoforms which are not considered in the post-acquisition analysis unless otherwise noted.

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Multiple Organic Solvent Method for Membrane Proteomic Analysis Determination of Xcorr and ∆Cn Threshold Values. The product ion spectra acquired with each method was searched using the same forward and decoy Arabidopsis database and search parameters. However, since each data set was produced from two different extraction methods and each contained a DDA-based LC/MS/MS analysis of 60 fractions using two different reversed-phase gradients, a false discovery rate (FDR) was determined using a more comprehensive approach in order to generate data sets that allow for a more direct qualitative comparison. For our analysis, a 5% FDR was obtained based on statistical analysis of each precursor charge state and used to determine the threshold values for cross-correlation score (Xcorr) and delta-correlation score (∆Cn) for each data set. The initial data set for each sample had a ∆Cn of at least 0.08 and an Xcorr of 2.1, 2.5, and 3.1 for +1, +2, and +3 charged precursor ions, respectively. Changes in the FDR based on only the ∆Cn were calculated using increments of 0.1 units to determine an upper limit to yield the lowest FDR across all charge states while maximizing the number of peptides identified in the forward database. The optimum ∆Cn value for each data set was determined to be 0.12. Next, the Xcorr thresholds were determined at a 5% FDR for each charge state using the ∆Cn value of 0.12 or greater. Data were plotted and statistical analysis was performed using EasyFit (MathWave Technologies, http://www.mathwave.com). Probability density distributions of Xcorrs of each charge state (+1, +2, and +3) were tested for normality using Kolmogorov-Smirnov and Anderson-Darling goodness of fit analysis. Although each of the Xcorr distributions for product ions generated from each charge state followed several probability distributions with similar goodness of fit, a General Pareto distribution was chosen as the best fitting distribution since it was the highest scoring distribution for each charge state. An Xcorr score probability distribution was generated for the forward and reverse database search results for each charge state. The Xcorr threshold was determined for a 5% FDR using eq 1 nr

FDR ) nf

∫

φf

x

∫

φr

x

p(xr) dx

p(xf) dx + nr

∫

φr

x

× 100

(1)

p(xr) dx

where FDR is the false discovery rate, n is the number of Xcorrs in a probability distribution, φ is the upper bound of the probability distribution, x is the Xcorr threshold, p(x) is the probability distribution function for all Xcorrs in a data set, r denotes the reverse (decoy) database, and f denotes the forward (real) database. Bioinformatic Analysis. The list of AGI numbers for the proteins identified by the chloroform and SPE methods were submitted to the Arabidopsis Subcellular Proteomic Database (SUBA) (http://www.plantenergy.uwa.edu.au/applications/ suba/index.php) to obtain protein localization as determined by WoLF PSORT (http://wolfpsort.seq.cbrc.jp/aboutWoLF_ PSORT.html.en) and the grand average of hydropathicity (GRAVY) values. Peptide GRAVY values were determined by the ProtParam sequence analysis tool (available at http://us.expasy.org/tools/). Putative TMDs were determined by ARAMEMNON Arabidopsis integral membrane protein database (http:// aramemnon.botanik.uni-koeln.de/).

Results and Discussion Plasma Membrane Protein Identification. Comprehensive identification of plasma membrane proteins using bottom-up methodologies is an important step for cataloging the location and levels of critical proteins necessary for cellular function. Because of the complexities and variation of plants and their tissues, alternatives in sample preparation are needed, especially if certain classes of proteins are to be targeted. Along these lines, we previously developed a methanol-based procedure for extracting and solubilizing membrane proteins from Arabidopsis seedlings which was particularly effective for identifying hydrophobic integral membrane proteins, and LRR RLKs in particular, as compared to a nonionic detergent approach.13 Although the samples were enriched in total membranes, they still were quite complex, prompting the need for further isolating the plasma membrane. Even this additional enrichment produced a sample containing a significant number of chloroplast proteins, chlorophyll and other phytochemicals that could not be removed with SPE prior to SCXC fractionation. To enhance our approach, we developed an organic extraction procedure using chloroform to provide for more effective SCXC-LC/MS/MS analysis of plasma membrane proteins. Phytochemical Removal. After tissue homogenization and removal of cellular debris, a high level of chlorophyll, other phytochemicals, and lipid-based components were present in the sample and remained after tryptic digestion of the proteins. Often, many of these contaminants are removed during SCXC but may lead to alterations in SCXC fractionation from one sample to another due to varying levels that may be present in different tissue samples or plants. Furthermore, these components typically require removal if chemical isotope labeling is to be used for quantitative measurements. To eliminate these contaminants, we have determined that the addition of chloroform at a level of 10% of the initial sample volume can be used to effectively extract chlorophyll from the aqueous layer as shown in Figure 1. Initially, the sample was very green in color indicating the presence of chlorophylls. However, after extraction with chloroform, the aqueous layer appeared clear, whereas the lower layer was green and produced a spectrum featuring relative maxima at 433 and 655 nm, characteristic of chlorophyll a, and at 458 nm, indicative of chlorophyll b. Other features of the spectrum also suggest the presence of other organic molecules such as other chlorophyll derivatives, phytochemicals, and lipid-based components. To achieve a more effective extraction, the sample was vortexed, leading to a white precipitant at the interface, which was presumed to be proteins that have precipitated under the shearing forces generated by vortexing the sample containing the two immiscible phases. This phenomenon can be used to selectively remove the remaining protease and other undigested protein components from the sample after proteolytic digestion. To test this hypothesis, the chloroform extraction procedure was performed on a peptide sample containing trypsin. Trypsin Removal. After tryptic digestion using a methanolbased buffer, the proteome sample contains about 5% trypsin (relative to the starting amount of protein) which can become a problem for reproducible SCXC or reversed-phase LC due to its high abundance. By the addition of a volume corresponding to 10% chloroform and vortexing the sample, Journal of Proteome Research • Vol. 8, No. 6, 2009 2755

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Mitra et al.

Figure 1. Chlorophyll removal using chloroform extraction. After tissue homogenization and removal of debris, the aqueous solution containing the protein is green and produces the spectrum (440-800 nm) shown in black. Upon addition of 10% chloroform (by volume) and performing the extraction as described in Materials and Methods, the upper aqueous layer becomes clear (red spectrum) and the lower chloroform layer becomes green (green spectrum). The green spectrum is characteristic for a mixture of chlorophyll a and b but contains other features that indicate other extracted components are also present. Each measurement was based on an equivalent dilution of the initial sample and extraction layers.

trypsin can be effectively removed. This is experimentally demonstrated for a sample containing BSA in 50 mM ammonium bicarbonate buffer, pH 8.3, in which trypsin was added (1:20 trypsin-to-protein ration, w/w). As shown in Figure 2A, SDS-PAGE analysis and Coomassie staining indicated that the chloroform extraction was effective for removing trypsin from proteolytic digests in aqueous buffers. However, the use of chloroform to remove trypsin from our 60% methanol/40% 50 mM ammonium bicarbonate buffer system did not work using a direct addition of 10% chloroform since it was miscible with the methanol-based buffer. To enable the use of chloroform, the methanol concentration was lowered to e10%, which upon the addition of 10% chloroform and vortexing allowed efficient removal of trypsin from the aqueous phase (Figure 2A, lane 3). This confirmed that most, if not all, of the trypsin used to digest BSA had been removed and remained in the white interface region (which was not present in the control). However, because of the limits of detection of protein staining, there could still be some undetectable trypsin present in the solution. To determine if this was indeed the case, we measured the trypsin activity remaining in the aqueous layer using the BAEE assay in which the carboxylic acid methyl ester is hydrolyzed by trypsin to produce a chromophoric carboxylate that can be monitored at 253 nm.15 The results of these measurements are shown in Figure 2B. Regardless of the buffer used, the use of the chloroform extraction eliminates all trypsin activity and agrees with the SDS-PAGE analysis. This is an analytical advantage since protease activity can be immediately quenched without the addition of inhibitors that can interfere in downstream analysis. In addition, our procedure effectively removed the protease from the aqueous layer, enabling direct analysis of the peptides without the 2756

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Figure 2. Protease removal after tryptic digestion using chloroform extraction. (A) SDS-PAGE analysis of a BSA digest using trypsin and subsequent chloroform extraction. BSA (10 µg) in 60% methanol/40% 50 mM ammonium bicarbonate (pH 8.3) buffer was digested with a 1:20 trypsin-to-protein ratio and subjected to chloroform extraction. Each lane contains the following: lane M, molecular weight marker; lane 1, BSA; lane 2, BSA and trypsin after proteolytic digestion; and lane 3, aqueous phase of a BSA tryptic digest after chloroform extraction. The gel was visualized by Coomassie staining. A peptide smear is barely detectable in lane 3. (B) The effect of chloroform extraction on trypsin activity. Trypsin activity was measured by the rate of hydrolysis of BAEE in 100 mM Tris (pH 7.8), 50 mM ammonium bicarbonate (pH 8.3), and 10% methanol/90% 50 mM ammonium bicarbonate (pH 8.3) (v/v) before and after chloroform extraction. Each assay was performed in triplicate. The error bars (() are based on the calculated standard deviation of the average normalized rate.

concerns of having a highly abundant protease react with isotope-coded reagents such as iTRAQ (http://www. appliedbiosystems.com) or digest immobilized antibodies used in affinity chromatography. Effect of Chloroform Extraction on Mass Spectrometry Analysis of the Test Peptide. After demonstrating that the chloroform extraction method was effective in removing phytochemicals (chlorophyll) as well as undigested protein (trypsin), the next step was to determine whether peptides in the aqueous

Multiple Organic Solvent Method for Membrane Proteomic Analysis

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Figure 3. MS analysis of peptides before and after chloroform extraction. Samples containing the Ac-SYSMEHFRWGKPV-OH peptide in (A) 50 mM ammonium bicarbonate (pH 8.3) or (B) 10% methanol/90% 50 mM ammonium bicarbonate (pH 8.3) were analyzed by ESI-MS. The generated mass spectra are nearly identical and contain the same predominant peptide ion [M + 2H]2+ at m/z 833.4 with various salt adducts. After digestion with trypsin, each sample was subjected to chloroform extraction and the aqueous layer was analyzed by ESI-MS. The spectra for the peptides in (C) 50 mM ammonium bicarbonate (pH 8.3) and (D) 10% methanol/90% 50 mM ammonium bicarbonate (pH 8.3) are nearly identical, each containing the same peptide ions Ac-SYSMEHFR-OH (M1) and H2N-WGKPVOH (M2) at m/z 1098.3 and 586.3 for the [M1 + H]+ and [M2 + H]+ species, respectively, as well as the same salt adducts.

layer were extracted into the chloroform layer. The experiment conducted used the test peptide Ac-SYSMEHFRWGKPV-OH (monoisotopic m/z 1664.8 for the [M + H]+ ion) which contains a single tryptic cleavage site and represents a typical tryptic peptide from a proteolytic digestion used in a bottom-up approach. Solutions containing the test peptide in 50 mM ammonium bicarbonate (Figure 3A,C) and 10% methanol/90% 50 mM ammonium bicarbonate (Figure 3B,D) were treated with or without trypsin and then subjected to the chloroform extraction procedure. The aqueous layers were analyzed by ESIMS. In each buffer system before trypsin addition, the intact peptide was detected at m/z 833.4 for the [M + 2H]2+ ion and m/z 1665.5 for the [M + H]+ ion along with various potassium adducts as shown in Figure 3A,B. After addition of trypsin, the test peptide was cleaved into two distinctive peptides AcSYSMEHFR-OH (M1) and H2N-WGKPV-OH (M2) at m/z 1098.3 and 586.3 for the [M1 + H]+ and [M2 + H]+ ion, respectively, along with other charge states and adducts as shown in Figure 3C,D. Both buffered solutions produced essentially identical

mass spectra indicating that the chloroform extraction can be effectively applied to the ammonium bicarbonate buffered solution that contains 10% methanol. The chloroform layer from each extraction was also analyzed by ESI-MS, but no peptides were detected (data not shown), indicating that charged peptides do not partition from the bicarbonate buffered solution at pH 8.3 into the chloroform layer. Application of the Chloroform Extraction to Plasma Membrane Proteomic Analysis. To demonstrate that the chloroform extraction procedure can be used to remove neutral organic components and undigested proteins from the aqueous buffer to enhance peptide identification and increase proteome coverage, we applied this method to the analysis of plasma membrane proteins from Arabidopsis seedlings using our previously developed approach of 60% methanol extraction and solubilization of microsomal membrane protein fractions.13 The overall method used in our current study also employed phase partitioning14 to enrich for plasma membranes from the microsomal fraction as outlined in Figure 4. After preparation Journal of Proteome Research • Vol. 8, No. 6, 2009 2757

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Mitra et al. Table 1. SEQUEST Xcorr Threshold Values Obtained at a 5% FDRa According to Precursor Charge Stateb for the Chloroform Extraction and SPE Methods precursor ion

forward matches

[M + H]+ [M + 2H]2+ [M + 3H]3+ Total

538 4610 208 5356

[M + H]+ [M + 2H]2+ [M + 3H]3+ Total

129 4130 154 4413

reverse matches

threshold Xcorr

Chloroform Extraction 29 2.31 240 2.54 11 3.51 280

average Xcorr ( SD

CV (%)

2.67 ( 0.31 3.39 ( 0.66 4.32 ( 0.58

11.6 19.3 13.3

2.62 ( 0.27 3.14 ( 0.55 3.97 ( 0.50

10.4 17.5 12.6

SPE 7 222 8 237

2.35 2.45 3.49

a The FDR was determined according to eq 1 as described in Materials and Methods using a minimal ∆Cn value of 0.12 for all calculated threshold Xcorr values. b Each charge state is indicated as [M + nH]n+ were n is 1, 2, and 3 for the singly, doubly, and triply changed peptide precursor ions, respectively.

Figure 4. Chloroform extraction and SPE methods for multidimensional LC/MS/MS analysis. Each extraction method was applied to the sample after methanol-bicarbonate solubilization and digestion of proteins enriched in plasma membranes via twophase partitioning of microsomal preparations.

of the membrane protein fraction using the 60% methanol approach, the sample was divided into equivalent aliquots with one processed by the chloroform extraction procedure and the other with SPE. Each peptide sample was fractionated by SCXC and each collected fraction was analyzed twice by LC/MS/MS using two different reversed-phase gradients. Minimizing the Effects of DDA-Based Protein Identification. A 5% FDR was used in our previous study to compare the differences between methanol and Brij-58 membrane protein solubilization of microsomal fractions from Arabidopsis seedlings.13 In the current study, two independent LC/MS/MS analyses of every SCXC fraction were performed for both the chloroform extracted and SPE treated samples in order to provide a more consistent data set for comparison. Since the LC/MS/MS method utilized DDA to identify peptides, the additional LC/MS/MS analysis allowed more unique peptides to be identified and enabled the identification of more proteins with two or more unique peptides. Although each sample was prepared with the same amount of peptides, each sample was processed with either the chloroform extraction or SPE, and thus, some form of normalization was needed to make a comparison. To accomplish this, the SEQUEST threshold values pertaining to each charge state were independently determined for each sample at a 5% FDR and appear in Table 1. While the search parameters and DTA generation were the same for both data sets, the quality of the precursor and product ion spectra 2758

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Figure 5. Functional analysis of proteins identified using the chloroform extraction and SPE methods. The AGI number for each identified protein was submitted to SUBA to determine the protein localization using the WoLF PSORT algorithm. On the basis of the number of identified proteins, the percentage of each class was calculated and plotted for the (A) chloroform extraction and (B) SPE method.

depends on the sample preparation, and thus at a given Xcorr threshold, the FDR could be different for each sample. Consequently, a 5% FDR was applied to each data set and the SEQUEST score threshold values at each charge state were determined. In this manner, a more meaningful qualitative comparison between the chloroform extraction and SPE methods can be made to reveal the advantages and disadvantages

Multiple Organic Solvent Method for Membrane Proteomic Analysis

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Figure 6. Hydropathy comparison of the identified proteins and peptides unique to the chloroform extraction and SPE methods. The hydropathy plot for (A) proteins and (B) peptides was based on SUBA analysis and the ProtParam algorithm, respectively. Each histogram was generated by plotting the number of proteins or peptides per 0.1 GRAVY value increment (within a range of (0.05 of each value). The distributions for the chloroform extraction and SPE approaches are indicated in each histogram by the white and black bars, respectively. For hydrophilic peptides possessing GRAVY values in excess of -1.85, the chloroform extraction produced 29 peptides while the SPE method generated 10 peptides.

between the two methods to facilitate the identification of plasma membrane proteins. Protein and Peptide Identification. On the basis of the 5% FDR and the SEQUEST protein designations, a total of 5356 peptides corresponding to 2636 unique peptides for an estimated 1362 proteins were identified with the chloroform extraction method compared to a total of 4413 peptides corresponding to 2339 unique peptides for an estimated 1306 proteins identified with the SPE method (Tables 1, and S1 and S2 in Supporting Information). In terms of overlap, 645 proteins and 600 peptides were common to both methods. In comparison to the SPE method which used the same amount of material (250 µg of peptides), the chloroform extraction procedure provided an increase of 21.4%, 12.7%, and 4.3% in the number of peptides, unique peptides, and proteins identified, respectively. This increase at both the peptide and protein level for the chloroform extracted sample is not due to lower Xcorr

thresholds since they are very similar to those of SPE (all within 0.09 Xcorr units); in fact, the average Xcorr value for the chloroform extraction was higher for each charge state than that for SPE (Table 1) suggesting higher quality product ion spectra. Although these Xcorr increases are not statistically significant, it is important to note that the coefficient of variation (CV) is less than 2% across the same charge state for each method, indicating that the data sets can be compared and that the measured differences are not simply attributable to unusual Xcorr values but are the direct result of the nature of the peptides contained within each sample. To better understand the increases for peptide and protein identifications by LC/MS/MS using the chloroform extraction procedure, the data were analyzed using several bioinformatic approaches. The first comparison was based on functional classification as determined by SUBA as shown in Figure 5. The overall composition between the two samples is very similar, Journal of Proteome Research • Vol. 8, No. 6, 2009 2759

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Table 2. The Comparison of the Peptide and Protein Identifications for Various Membrane Protein Categories for the Chloroform Extraction and SPE Methods chloroform extraction category

a

Plasma Membrane Proteins Cellular Transporters Membrane-Specific Transporters ATPases PIPs LRR RLKs Ribosomal Proteins Rubisco

peptides/proteins

b

SPE ratio

c

peptides/proteins ratio

511/272

1.9

302/169

1.8

271/123 194/77

2.2 2.5

196/82 93/40

2.4 2.3

97/46 23/18 30/20 329/178 14/4

2.1 1.3 1.5 1.8 3.5

78/40 13/13 16/10 317/176 24/4

1.9 1.0 1.6 1.8 6.0

a These categories were chosen because two-phase partitioning was used to enrich for plasma membranes from microsomal fractions. Because of their high abundance, Rubisco and ribosomal proteins serve as a measure of protein contamination (i.e., nonspecific enrichment). b The number of unique peptides/the number of assigned proteins. c The ratio is the value for the number of unique peptides divided by the number of assigned proteins.

but when considering differences of at least 2%, there is an increase in plasma membrane protein identifications (7%) with a concomitant decrease in plastid (5%) and cytosol (2%) protein identification in the chloroform extracted sample. To better understand these findings, we examined the nature of protein and peptide hydrophobicity specific to each approach using GRAVY analysis as shown in Figure 6. At the protein level (Figure 6A), the distribution between the chloroform and SPE methods was very similar and produced an average protein GRAVY value of -0.236 and -0.261, respectively. When considering just the hydrophilic proteins, 566 proteins (79%) were unique to the chloroform extraction method and 557 proteins (84%) were unique to the SPE method, each eliciting an average GRAVY value of -0.362 and -0.352, respectively. For the hydrophobic proteins, the number of identifications is lower, but a difference emerges between the two samples: 144 proteins (20%) possess an average GRAVY value of +0.258 for the chloroform extraction and 96 proteins (15%) possess an average GRAVY value of +0.271 for SPE. The 50% increase in the detection of hydrophobic proteins by the chloroform extraction over SPE supports the increase in membrane protein detection (Figure 5 and Table 3). At the peptide level (Figure 6B), the GRAVY distribution for the peptides obtained only by chloroform extraction has a similar profile as the peptides obtained only by SPE, but it is dramatically shifted toward the hydrophilic side. The average GRAVY value for peptides unique to the chloroform extraction and SPE methods is -0.446 and -0.139, respectively. Further analysis revealed that for the chloroform extraction method 1500 peptides (74%) are hydrophilic and possess an average GRAVY value of -0.746, whereas with SPE, 1024 peptides (59%) are hydrophilic and have an average GRAVY value of -0.553. In terms of the hydrophobic peptides, SPE has the advantage with 706 peptides (41%) which elicit an average GRAVY value of +0.460 compared to the chloroform extraction which contained 528 (26%) hydrophobic peptides with an average GRAVY value of +0.399. Overall, the chloroform extraction method resulted in more peptides being identified with a greater percentage being more hydrophilic than those of the SPE method. Since each sample originated from the same tissue and preparation procedure using methanol-assisted solubilization 2760

Journal of Proteome Research • Vol. 8, No. 6, 2009

and tryptic digestion (Figure 4), the GRAVY analysis is consistent with peptide losses based on each extraction method. When considering the SPE method, very hydrophilic peptides may not be retained on column during the loading and/or washing steps prior to elution; thus in effect, SPE depletes hydrophilic peptides (i.e., enriches for hydrophobic peptides) relative to the chloroform approach. However, an alternative explanation for the enhanced hydrophilicity using the chloroform extraction method is that some of the hydrophobic peptides partition into the chloroform layer during the extraction with the 10% methanol buffer solution after the tryptic digestion. Although the presence of methanol could provide a needed polar protic component to facilitate hydrogen bonding to peptides and thus promote their solubility in the chloroform layer, the lack of detection for this phenomenon using our test peptide (Figure 3) with a -0.923 GRAVY value (which is probably lower due to N-acetylation) or its digestion products (-1.175 for M1 and -0.520 for M2 of Figure 3) does not preclude the possibility of extracting very hydrophobic peptides. Even if some hydrophobic peptides partition into the chloroform layer, this type of extraction is not immediately useful in this current study since a significant amount of phytochemicals (and probably some lipid-based components) would be present in the chloroform layer (Figure 1) and thus require removal prior to LC/MS/MS analysis, thus, promoting further peptide losses that would affect detection of these highly hydrophobic peptides. Because this study utilized a bottom-up proteomics approach, protein identification was based on peptide detection, but since more hydrophilic peptides were detected with the chloroform extraction method than with SPE, how does that translate into an increase in membrane protein detection and a decrease in cytosolic proteins? Part of this can be attributed to the nature of integral membrane proteins which contain various hydrophilic extra- and intracellular domains that often comprise the majority of the protein’s sequence. These regions provide a higher number of tryptic peptides that can be generated over the number of hydrophobic peptides that span the transmembrane region, which, in effect, increases their probability of being detected by DDA-based LC/MS/MS analysis. When considering all plasma membrane proteins and some important functional classes, each method produces an average peptide/protein ratio of around 2.0 as presented in Table 2. When considering the plasma membrane proteins, there was a 69% increase in the number of peptides identified with the chloroform extraction approach compared to SPE which corresponds to a 61% increase in protein identification, a trend that was observed for all the plasma membrane protein classes and was consistent with the overall detection of more hydrophobic proteins as shown in Figure 6A. When considering the highly abundant proteins from plant tissue that are present due to nonspecific binding, such as Rubisco and ribosomal proteins which represent contaminants present in the enriched plasma membranes, each method produces different results. For Rubisco, chloroform reduces the number of identified peptides by 42%, whereas both methods perform similarly for ribosomal protein identification (Tables S1 and S2 in Supporting Information). On the basis of these data, chloroform extraction as a cleanup step is a better method for membrane protein identification using a bottom-up LC/ MS/MS proteomics approach than SPE since it minimizes identification of nonspecific proteins while promoting peptide identification of membrane proteins, both important aspects

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Multiple Organic Solvent Method for Membrane Proteomic Analysis

Table 3. Membrane Transporters Identified by LC/MS/MS Using the Chloroform Extraction and SPE Methods AGI number

protein

TMDsa GRAVYb

[M + H]+ [M + H]+ no. calc.d meas.d chg.e Xcorrf ∆Cng peptidesh

peptidec Chloroform Extraction R.EIGVFEADEDISSR.Si K.EASDM#VLADDNFSTIVAAVGEGR.S

1567.6 2384.6

1567.5 2384.3

2 2

4.06 4.41

0.44 0.48

5

0.48

R.FVEDGEYGNALEM#GK.N R.GVDDVSQEFDDLVAASK.E

1675.8 1795.9

1674.3 1796.3

2 2

2.59 3.27

0.18 0.36

2

11

0.36

R.KLELPADPSYLYDVDDIIAAEGSM#K.G K.ASVSGFGSDQFDETEPK.E

2771.1 1801.8

2771.1 1801.6

2 1

3.39 2.94

0.40 0.22

2

PDR7 (ATPase, coupled to transmembrane movement of substances)

14

0.04

K.TGGYIEGDVR.V K.KAQLTILK.Dj

1067.1 915.2

1066.7 914.7

2 2

3.38 3.07

0.39 0.24

16

AT1G15520m PDR12 (ATPase, coupled to transmembrane movement of substances)

13

0.02

R.PGVLTALM#GVSGAGK.Tk K.TTLM#DVLAGR.Kl

1374.6 1093.3

1375.0 1093.2

2 2

2.79 3.22

0.19 0.36

2

6

0.14

K.NGTQNTTVAPDGLTQSPS*LR.N K.APTEFVGDVSAR.L

2138.2 1249.4

2137.9 1248.8

2 2

4.74 3.27

0.13 0.45

4

10

0.53

R.GTVM#ELGITPIVTSGLVM#QLLAGSK.In R.PFLAFLPEVQSADR.Ko

2549.0 1590.8

2548.2 1590.4

2 2

4.41 3.92

0.35 0.46

3

AT1G07810

ACA3, ECA1 (CALCIUMTRANSPORTING ATPASE 1)

8

0.06

AT1G11260

STP1 (SUGAR TRANSPORTER 1) carbohydrate transmembrane transporter

12

AT1G12110

NRT1.1 (NITRATE TRANSPORTER 1.1)

AT1G15210

AT1G17840

ABCG11/COF1/DSO/ WBC11 (DESPERADO); ATPase, coupled to transmembrane movement of substances

AT1G29310m Protein transport protein sec61, putative AT1G52190

Proton-dependent oligopeptide transport (POT) family protein

9

–0.14

R.VNGVREEEELIDIVGK.G R.LCTTDKVEELK.A

1800.0 1336.5

1800.2 1335.3

2 2

3.49 3.21

0.35 0.35

2

AT1G59870

PDR8 (ATPase, coupled to transmembrane movement of substances)

14

0.05

R.RTQSVNDDEEALK.W R.GGQVIYAGPLGQNSHK.V

1505.6 1626.8

1505.5 1626.2

2 2

4.12 4.60

0.26 0.48

18

AT1G71880

SUC1 (SUCROSE-PROTON SYMPORTER 1) sugar:hydrogen ion symporter

12

0.47

K.TSSVPLFGEIFGAFK.V K.DAAALETQSPEDFDQPSPLR.K

1600.8 2188.3

1600.3 2187.3

2 2

2.81 4.07

0.45 0.46

2

AT2G39480

PGP6 (ATPase, coupled to transmembrane movement of substances)

12

–0.16

R.VPTIEPDDTSALSPPNVYGSIELK.N R.FYDPTLGEVLLDGENIK.Np

2543.8 1924.1

2544.6 1924.4

2 2

2.74 4.29

0.22 0.46

2

AT2G47000

PGP4 (P-GLYCOPROTEIN4) putative ABC type auxin efflux transporter

12

0.11

R.FYDPDSGEITLDGVEIK.S K.VLLLDEATSALDAESER.Vq

1899.0 1833.0

1898.3 1833.3

2 2

5.57 3.71

0.35 0.38

6

AT3G21180

ACA9 (autoinhibited Ca2+-ATPase 9); calcium-transporting ATPase

9

0.07

K.DGGEVEISGSPTEK.A R.LSACETM#GSATTICSDK.Tr

1405.4 1849.0

1404.9 1848.9

2 2

2.91 3.31

0.32 0.35

2

AT3G57330

ACA11 (autoinhibited Ca2+-ATPase 11) calcium-transporting ATPase

9

0.14

K.IASISDVIEGFASEALR.T K.AAFQFIDAGARPEYK.L

1779.0 1684.9

1779.1 1685.4

2 2

4.93 3.15

0.47 0.29

2

AT4G29900

ACA10 (autoinhibited Ca2+-ATPase 10); calcium-transporting ATPase

9

0.04

R.TFEADKIPTDEEQLSR.W R.DQNIGALQELGGVR.G

1880.0 1470.6

1879.6 1469.6

2 2

3.14 4.94

0.28 0.45

3

AT4G39850

PXA1 (PEROXISOMAL ABC TRANSPORTER 1)

4

–0.80

R.DDSALLTDAEIDSVK.S K.AAFVRLIGLSVLQSGASSIIAPSLR.H

1592.7 2528.0

1591.4 2526.9

2 2

3.74 2.70

0.48 0.17

2

AT5G43350m PHT1 (PHOSPHATE TRANSPORTER 1) inorganic phosphate transmembrane transporter

12

0.35

R.ALSTPPQVDYIWR.Is K.AATM#NATHEVFR.Is

1546.8 1364.5

1546.2 1364.0

2 2

3.14 2.55

0.46 0.27

2

9

0.03

R.TYEAEKVPTGEELSK.W R.SQSSILHAFPFNSEK.K

1681.8 1692.9

1681.2 1692.2

2 2

3.49 3.42

0.54 0.56

5

AT5G57110

ACA8 (autoinhibited Ca2+-ATPase isoform 8)

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Table 3. Continued AGI number

protein

[M + H]+ calc.d

[M + H]+ meas.d

chg.e

Xcorrf

∆Cng

SPE 0.06 K.LADIGVAM#GISGTEVAK.Et K.ALEALKEIQSQQATVM#R.Dt

1648.9 1933.2

1648.0 1931.9

2 2

4.43 2.96

0.56 0.34

3

0.26 R.LYGTHENQSYLAR.P R.VLQQLCGR.E

1552.7 974.1

1552.5 973.7

2 2

3.07 2.52

0.39 0.41

2

8

0.20 R.MVT*GDNLT*TAKAIAR.E K.M#LVTTVGM#R.Tu

1722.7 1040.3

1721.5 1040.2

2 2

2.60 2.63

0.14 0.27

2

TMDsa

GRAVYb

peptidec

no. peptidesh

AT1G07810m

ACA3, ECA1 (CALCIUMTRANSPORTING ATPASE 1)

AT1G20840

TMT1 (TONOPLAST MONOSACCHARIDE TRANSPORTER1)

AT1G27770

ACA1 (autoinhibited Ca2+-ATPase 1)

AT1G59870

PDR8 (ATPase, coupled to transmembrane movement of substances)

14

0.05 R.GGQVIYAGPLGQNSHK.V K.VVEYFESFPGVSK.I

1626.8 1488.7

1626.2 1489.8

2 2

4.04 3.76

0.43 0.50

9

AT2G36380

PDR6 (ATPase, coupled to transmembrane movement of substances)

13

0.05 R.GGQVIYAGTLGHHSQK.L K.DISGIIKPSR.M

1653.8 1086.3

1653.7 1086.0

2 2

3.71 2.65

0.48 0.24

2

AT5G43350m

PHT1 (PHOSPHATE TRANSPORTER 1) phosphate transmembrane transporter

12

0.35 R.IYYFNPESAK.Ps R.ALSTPPQVDYIWR.Is

1232.4 1546.8

1232.0 1546.4

2 2

2.58 2.51

0.32 0.13

2

AT5G57110

ACA8 (autoinhibited Ca2+-ATPase isoform 8)

0.03 K.TTGPATPAGDFGITPEQLVIM#SK.D R.SQSSILHAFPFNSEK.K

2348.7 1692.9

2348.2 1692.4

2 2

2.45 3.66

0.16 0.44

2

8

12

9

a The number of putative transmembrane domains (TMDs) was determined for each protein by ARAMEMNON Arabidopsis integral membrane protein database (http://aramemnon.botanik.uni-koeln.de/). b The grand average of hydropathicity (GRAVY) values for each protein were obtained using the Arabidopsis Subcellular Proteomic Database (SUBA) (http://www.plantenergy.uwa.edu.au/applications/suba/index.php). c Amino acid residues before and after the period are not part of the identified peptide, but are those occurring within the protein sequence and illustrate trypsin substrate cleavage specificity. Peptide mass modifications are indicated after the corresponding amino acid: # for oxidation (+16.0 u) and * for phosphorylation (+80.0 u). d Average mass calculated based on the peptide sequence (Calc. [M + H]+) and the deconvoluted mass-to-charge (m/z) ratio based on the measured centroid mass (Meas. [M + H]+). e Charge state of the precursor ion selected for collision-induced dissociation. f SEQUEST cross-correlation score (Xcorr) of the peptide is based on the match of the obtained product ion spectrum to the theoretical ion distribution for the corresponding peptide contained in database. g SEQUEST difference of cross correlation scores (∆Cn) is the “difference” between the top two Xcorr values for a given product ion spectrum. h The number of distinct peptides detected for each protein which may not be unique to a single protein isoform. The two highest scoring representative peptides are listed for each protein. i This peptide also matches AT1G07670 exactly. j This peptide also matches AT1G59870 exactly. k This peptide also matches AT1G15210, AT1G59870, AT3G16340, and AT3G30842 exactly. l This peptide also matches AT2G26910, AT1G66950, AT2G36380, AT2G29940, AT3G30842, AT1G59870, AT3G16340, and AT1G15210 exactly. m Ambiguous assignment due to multiple matches to closely related family members. n This peptide also matches AT1G29310 exactly. o This peptide also matches AT2G34250 exactly. p This peptide also matches AT3G55320 exactly. q This peptide also matches AT3G62150 exactly. r This peptide also matches AT4G29900 and AT5G57110 exactly. s This peptide also matches AT5G43370 exactly. t This peptide also matches AT1G07670 exactly. u This peptide also matches AT2G22950, AT2G41560, and AT3G57330 exactly.

for meaningful qualitative and quantitative analysis for membrane protein abundance measurements. To understand these ramifications further in a biological context, we examined in detail two of the plasma membrane protein classes: transporters and LRR RLKs. Membrane Transporters. Transporters are highly hydrophobic membrane bound proteins involved in the movement of ions, small molecules or macromolecules across biological membranes. Numerous membrane transporters were identified by both chloroform extraction (Table S3 in Supporting Information) and SPE (Table S4 in Supporting Information) methods. On the basis of SUBA analysis, a total of 77 membranespecific transporters were detected with 194 unique peptides using our chloroform extraction method as compared to 40 membrane-specific transporters identified with 93 unique peptides using the SPE method. Of the 194 and 93 unique peptides matching to chloroform extraction and SPE methods, respectively, 13 peptides were found to match to multiple proteins, but all belonging to the same family of proteins. The implementation of the chloroform extraction basically doubled the coverage of this important, highly hydrophobic class of proteins which included some of the major classes of transporters: ABC transporters, ATPases, aquaporins, plasma membrane integral proteins (PIPs), nitrate transporters, phosphate transporters and sugar transporters. Since both methods 2762

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produced an average of two unique peptides per protein identification (Table 2), this constraint was used to provide a basis for comparing the transporters between the two methods and the results are presented in Table 3. We identified five sucrose transporters with our chloroform extraction method and only one could be identified using the SPE method (Table S1 in Supporting Information). Sugar transporter 1 (STP1) is an important member of this family with two unique peptides identified for this protein in our chloroform extraction method. In Arabidopsis, the disaccharide sucrose is transported by members of the sucrose carrier family protein (SUC), whereas the monosaccharide hexose is transported by members of the sugar transport family protein (STP).16 Out of the 14 members in the Arabidopsis STP family, only STP1 has been characterized electrophysiologically and determined to be a proton/monosaccharide symporter.17 Phosphoproteomic studies by Nuhse et al.18 have also revealed that the protein is serine phosphorylated. Using our chloroform extraction method, we also identified SUC1 (At1g71880) by two unique peptides which are highly hydrophobic membrane carriers with 12 TMDs. Arabidopsis SUC1 is highly expressed in pollen, trichomes and roots and has been recently shown to be important for sugar signaling in vegetative tissue as well as for normal male gametophyte function19 and SUC-dependent signal transduction leads to anthocyanin accumulation.

Multiple Organic Solvent Method for Membrane Proteomic Analysis

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Figure 7. Sequence coverage of membrane transporter PDR8 using the chloroform extraction and SPE methods. PDR8 (At1g59870) is an integral membrane protein containing 14 putative TMDs and has 1469 amino acid residues. The sequence coverage obtained by the identification of tryptic peptides with chloroform extraction (red), SPE (blue), and both methods (purple) is shown. The TMD regions are in bold capital letters. The hydrophilic loop and regions connecting the TMDs are in lower case. The peptide identified by the chloroform extraction method containing TMD8 is hydrophobic and has a GRAVY value of +0.714.

The nitrate transporter is a highly hydrophobic membrane transporter with 11 TMDs and was only identified using our chloroform extraction method by two unique peptides. Although phosphate, hexose sugar, and zinc transporters have been identified in other proteomic studies by Nuhse et al.18 and Marmagne et al.,20 identification of NRT1 was unique to our study. Two gene families NRT1 (with 53 members) and NRT2 (with 7 members) have been identified in Arabidopsis that potentially encode transporters responsible for nitrate uptake by roots or distribution within the plant.21 The NRT1 family of proteins transport many nitrogen containing substrates including a dicarboxylate.22 Another class of highly hydrophobic membrane proteins PHT1/PHT2 (phosphate transporter 1/2) was identified in our study and was common to both the extraction methods. Phosphate transporters play an active role in phosphate uptake from the soil and involve 6 members in the family. PHT1/2 identified in our study contains 12 TMDs interrupted by a large hydrophilic loop between TM8 and TM9. Functional analysis of PHT1/2 indicated that it is a proton/phosphate symporter dependent on the electrochemical gradient across the plasma membrane.23 Identification of this protein by the chloroform extraction method promoted the detection of peptides ALSTPPQVDYIWR and AATM#NATHEVFR in which each C-terminal portion is part of TMD6 and TMD8, respectively. This illustrates the ability of the methanol-bicarbonate buffer to extract integral membrane proteins from the lipid bilayer to facilitate solubilization and denaturation so that trypsin can cleave within a TMD region. In our previous study using the methanol approach to solubilize membrane proteins in microsomal fractions,13 we identified PDR5 and PDR8, two integral membrane proteins from the ATP binding cassette (ABC) family of transporters. In the present study using the effective methanol solubilization

and digestion technique of plasma membrane enriched samples combined with chloroform extraction, we were able to identify 10 ABC transporters belonging to three different families (Table 3, and Tables S1 and S2 in Supporting Information): multidrug resistance proteins (MDR), multidrug associated proteins (MRP), and pleiotropic drug resistance protein (PDR). The PDR family is unusual in that it is only found in fungi and plants.24 In our study of plasma membranes, we found PDR6, PDR7, PDR8, and PDR12 from the PDR family and MDR1, MDR4, MDR6, MDR8, MDR11, and MDR16 from the MDR family with both single peptide matches (Tables S1 and S2 in Supporting Information) and multiple peptide matches (Table 3), suggesting that members of both PDR and MDR families of ABC transporters are plasma membrane bound which is consistent with other experimental studies employing both experimental and bioinformatic approaches.24 These findings exemplify the advantage of our chloroform extraction method by noting that in previous Arabidopsis proteomic studies by Nuhse et al.18 and Alexandersson et al.25 only one family member, PDR8, was identified. We also identified multiple unique peptides of a peroxisomal ABC transporter (PXA1) and another ABC family protein At5g06530 identical to white-brown complex homologue protein 23 (WBC23) both of which were not reported in previous membrane proteomic studies. PXA1 protein promotes germination and represses embryo dormancy, whereas WBC23 has been shown to play a major role in the transportation of monolignols as glycosides into the extracellular space.26 In animals, fungi, and plants, Ca2+-ATPases are involved in the regulation of cytosolic calcium concentration.27 Our membrane proteomic analysis identified 6 proteins belonging to the calcium transporting ATPase family of proteins with multiple peptide identifications. Of the 6 Ca2+-ATPases identified in our study, only two of them, ACA8 and ACA10, have been previously reported by proteomic analysis.18 ACA3 (At1g07810) and ACA8 Journal of Proteome Research • Vol. 8, No. 6, 2009 2763

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Table 4. LRR RLKs Identified by LC/MS/MS Using the Chloroform Extraction and SPE Methods AGI number AT4G20450 AT2G37050

AT4G33430

AT2G26730 AT3G02880

[M + H]+ calc.b

peptidea

protein

Chloroform Extraction 1303.4

[M + H]+ meas.b

chg.c

Xcorrd

∆Cne

LRR subfamily

1302.2

2

3.10

0.57

LRR I

Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase

R.DDTYTTTTGSLK.L

K.RLEIAEDAAR.G K.KPNYLVDVAAGTVR.V

1144.3 1503.7

1144.1 1503.4

2 2

3.41 3.74

0.38 0.39

LRR I LRR I

Leucine-rich repeat transmembrane protein kinase (BAK1) Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase

R.ERPESQPPLDWPK.R

1579.7

1580.4

2

2.74

0.25

LRR II

K.KEFETQM#EVVGK.I

1441.6

1440.7

2

2.75

0.34

LRR III

K.SFGEFDLDGLLK.Af R.LRDVVVPEK.Ef R.AISYLHSR.Df R.FNSLSGPIPSDFSNLVLLR.Y R.NVEAPVAAATSSAAIPK.E

1341.5 1055.3 947.1 2077.4 1597.8

1341.7 1055.1 946.8 2076.3 1597.2

1 2 2 2 2

2.48 2.56 2.78 3.73 4.99

0.26 0.20 0.36 0.40 0.42

LRR LRR LRR LRR LRR

III III III III III

AT5G16590

Leucine-rich repeat transmembrane protein kinase

K.ESNGPPAVVANGASENGVSK.N R.LATLYLQDNQLTGPIPEIK.I

1885.0 2128.5

1886.9 2129.7

2 2

3.37 4.89

0.41 0.49

LRR III LRR III

AT1G66150

Leucine-rich repeat transmembrane protein kinase (TMK1) Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase (SRF7)

R.DNSFTGPVPASLLSLESLK.V

1976.2

1976.5

2

3.13

0.41

LRR IX

R.DTVTLVTEGNANM#GK.N

1566.7

1567.2

2

3.65

0.51

LRR IX

R.DNDLTGIVPPTLLTLASLK.N

1982.3

1982.1

2

2.79

0.29

LRR IX

R.DSPYNALGTGLVYTSDVGLVSSGK.T

2401.6

2400.3

2

3.41

0.44

LRR RLK

K.SVQNPPLVETK.K K.IDSSALPTDTADDFTEIVSK.I

1212.4 2126.3

1213.9 2126.2

2 2

2.57 2.81

0.36 0.35

LRR V LRR V

Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase

R.GLDLQTGSFTLK.Q

1280.5

1279.9

2

2.71

0.37

LRR VIII-2

K.DTYTDDLNSR.G

1200.2

1199.8

2

2.60

0.35

LRR VIII-2

K.DFNIVDEAK.G K.LLEASVNNEKDEESVR.A K.VATDNFDPANK.I K.YDADTWDTPGYYDSK.N

1051.1 1832.9 1192.3 1797.8

1050.3 1832.8 1192.4 1797.8

1 2 2 2

2.60 2.55 2.99 3.83

0.23 0.24 0.44 0.46

LRR LRR LRR LRR

Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase (PEPR1) Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase

K.LGDLM#AATNNFSSGNIDVSSR.T

2186.3

2186.1

2

2.65

0.32

LRR X

K.DAYVFTQEEGPSLLLNK.V

1925.1

1924.7

2

2.69

0.32

LRR X

R.IPVFFSSPSYSSDDFSGNK.G

2082.2

2082.8

2

2.95

0.43

LRR X

K.KPVGEFGDGVDIVQWVR.S

1902.1

1901.6

2

4.32

0.47

LRR XI

R.DISSGNILLGEDYEAK.I

1724.8

1723.9

2

4.07

0.45

LRR XI

K.SNNILLDENLTAK.V

1445.6

1445.5

1

3.18

0.40

LRRVIII-1

AT1G34210i

somatic embryogenesis receptor-like kinase 2 (SERK 2)

R.LADGTLVAVKR.Lg R.GTIGHIAPEYLSTGK.Sh

1143.4 1544.7

1143.1 1543.9

2 2

2.58 2.85

0.33 0.35

LRR II LRR II

AT5G16590

Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase

R.INLAQNNFLGR.I

1260.4

1260.4

2

3.88

0.48

LRR III

R.AISYLHSR.Df R.NVEAPVAAATSSAAIPK.E R.INLGENKFSGR.I K.VSDYGLAPIISSTSAPNR.I K.ATGSESGAVNKDLTFFVK.S

947.1 1597.8 1235.4 1849.0 1872.1

947.2 1597.2 1235.1 1849.4 1871.5

2 2 2 2 2

2.45 2.48 2.67 3.15 3.35

0.27 0.13 0.16 0.44 0.35

LRR LRR LRR LRR LRR

AT2G01820 AT3G23750 AT1G51805 AT3G14350

AT1G53440 AT1G53430 AT3G14840

AT1G27190 AT1G73080

AT3G28450 AT3G49670 AT4G08850 AT5G49760

VIII-2 VIII-2 VIII-2 VIII-2

SPE

AT3G02880

2764

Journal of Proteome Research • Vol. 8, No. 6, 2009

III III III III III

research articles

Multiple Organic Solvent Method for Membrane Proteomic Analysis Table 4. Continued AGI number AT3G23750 AT2G29000

AT1G53730

AT3G14350

AT3G14840 AT1G27190 AT3G02130

protein

peptidea

[M + H]+ calc.b

[M + H]+ meas.b

chg.c

Xcorrd

∆Cne

LRR subfamily

Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase

K.VTIDHNVFCTTK.A

1435.6

1435.2

2

2.73

0.34

LRR IX

R.LPIT*KSEILT*K.K R.HCY*DLS*VKQGTNYLIR.A

1403.5 2128.1

1404.7 2127.0

2 2

2.74 2.57

0.12 0.27

LRR RLK LRR RLK

Leucine-rich repeat transmembrane protein kinase (SRF 6) Leucine-rich repeat transmembrane protein kinase (SRF 7) Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase Leucine-rich repeat transmembrane protein kinase (TOAD2)

K.YLNLGHNQFK.G

1234.4

1234.4

2

2.55

0.35

LRR V

K.YLNLAHNQLK.Q

1214.4

1214.4

2

3.04

0.29

LRR V

R.VAGTYGYM#APEYAM#R.G

1712.9

1712.3

2

2.97

0.36

LRR VIII-2

R.LSLAGNDLSGTIPSELAR.F

1815.0

1815.9

2

2.55

0.34

LRR X

R.KWHPKS*KIMAT*T*K.R

1796.8

1795.5

2

2.52

0.15

LRR X

a Amino acid residues before and after the period are not part of the identified peptide, but are those occurring within the protein sequence and illustrate trypsin substrate cleavage specificity. Peptide mass modifications are indicated after the corresponding amino acid: # for oxidation (+16.0 u) and * for phosphorylation (+80.0 u). For identifications based on only one peptide, their corresponding product ion spectra were manually inspected to validate the SEQUEST assignment. b Average mass calculated based on the peptide sequence (calc. [M + H]+) and the deconvoluted mass-to-charge (m/z) ratio based on the measured centroid mass (meas. [M + H]+). c Charge state of the precursor ion selected for collision-induced dissociation. d SEQUEST cross-correlation score (Xcorr) of the peptide is based on the match of the obtained product ion spectrum to the theoretical ion distribution for the corresponding peptide contained in database. e SEQUEST difference of cross correlation scores (∆Cn) is the “difference” between the top two Xcorr values for a given product ion spectrum. f This peptide also matches AT5G16590 exactly. g This peptide also matches AT1G71830 and AT4G33430 exactly. h This peptide also matches AT2G13790, AT4G33430, AT2G13800 and AT5G10290 exactly. i Ambiguous assignment. The peptide listed could also match to BAK1 or other closely related SERK subfamily members.

(At5g57110) were identified by both methods (Table 3), whereas ACA1 (At1g27770) was specific to the SPE method and ACA9 (At3g21180), ACA10 (At4g29900) and ACA11 (At3g57330) were identified by the chloroform extraction method, clearly demonstrating once again that the chloroform extraction approach has distinct advantages over the SPE method. While the localization of ACA328 and ACA129 has been reported to be in the chloroplast inner envelope and endoplasmic reticulum, respectively, the localization of other ACAs obtained in the present study suggest that a number of these proteins reside in the plasma membrane. The efficacy of using chloroform extraction to promote identification of membrane proteins over SPE is illustrated for one of the ABC transporters PDR8 (At1g59870). The extent of sequence coverage obtained by the identification of 9 and 18 distinct peptides using the SPE and chloroform extraction methods, respectively, is shown in Figure 7. The significant increase in sequence coverage afforded by the chloroform extraction approach is due to the enhanced detection of hydrophilic peptides and is consistent with the trend observed when considering the peptide GRAVY distribution presented in Figure 6. Moreover, 7 out of the 9 peptides (3 are hydrophobic and 4 are hydrophilic based on GRAVY values) identified by SPE were also detected with the chloroform extraction approach. Although an increase in hydrophilic peptide identification occurs, detection of hydrophobic peptides is not precluded by the chloroform extraction since the peptide GVTGAFRPGVLTALM#GVSGAGK which is essentially TMD8 of PDR8 was identified using the chloroform extraction method but not by SPE. By enhancing detection of hydrophilic peptides, but not at the expense of hydrophobic peptides, the use of chloroform extraction provides a means to better characterize integral membrane proteins.

Leucine-Rich Repeat Receptor-Like Kinases. Our interest in plant membrane proteomics centers around the functional analysis of components involved in various developmental signaling cascades, with a particular focus on LRR RLKs. LRR RLKs are found widely in plants as multigene families that includes over 220 members in Arabidopsis and nearly 400 in rice.5 LRR RLKs have an organization of functional domains similar to mammalian receptor tyrosine kinases (RTKs) and transforming growth factor beta (TGF-β) serine/threonine receptor kinases, including an extracellular domain potentially involved in ligand binding and receptor oligomerization, a single pass transmembrane sequence, and a cytoplasmic kinase domain that propagates the signal downstream in a phosphorylation-dependent manner.30 In our present study (Table 4), we identified 20 LRR RLKs by the chloroform extraction method and at least 10 by the SPE method, of which 6 or 7 were found to be common to both methods (depending on the origin of peptides with an exact sequence match to multiple LRR RLKs). One of the wellcharacterized members of the LRR RLK family identified by the chloroform extraction method is Somatice Embryogenesis Receptor Kinase 3/BRI1 Associated Kinase (SERK3/BAK1) which plays an important role in steroid hormone-mediated signal transduction31 as well as having independent roles in programmed cell death32 and various defense responses.33,34 With the SPE method, peptides were identified matching to BAK1 and other members of the SERK family, such as SERK2 along with the functionally redundant SERK1, which have been recently reported to play an essential role in tapetum specification and pollen development during male sporogenesis.35 In addition, two proteins were detected from the StrubelligReceptor Family (SRF): SRF6 that has been proposed to play a role in defense responses against fungi and SRF7 which may Journal of Proteome Research • Vol. 8, No. 6, 2009 2765

research articles

Mitra et al. 36

act in primary cell wall biosynthesis. We also identified Toadstool2 (TOAD2) by our SPE method which has been implicated in radial pattern formation in early embryos.37 The identified peptide has phosphorylated serine and threonine residues that map to the juxtamembrane region of the kinase domain. Transmembrane Kinase1 (TMK1) identified in our study has been cloned and characterized in tobacco38 and has been found to play a role in plant defense. Using the chloroform extraction method, we detected Barely Any Meristem2 (BAM2), which along with other members of the BAM receptor family, has been determined to regulate peripheral vascular development, leaf shape, size and symmetry, as well as male and female gametophyte development.39 In our previous work,13 14 LRR RLKs were identified using a combination of methanol-assisted solubilization, two-phase partitioning, and SPE, which is similar to the 10 LRR RLKs we identified in this study using the same SPE method. However, substituting chloroform extraction for SPE increased the number of LRR RLKs identified to a total of 20 (Table 4) which compares well to other proteomic studies in which purified plasma membranes were isolated from intact Arabidopsis plants. For example, Alexandersson et al.25 identified 7 LRR RLKs in plasma membranes isolated from leaves of Arabidopsis plants, while Nelson et al.4 used purified plasma membranes from whole Arabidopsis seedlings grown in shaking liquid culture to identify 21 LRR RLKs. Interestingly, 8 of the 14 LRR RLKs identified by our previous method13 were also identified by the current chloroform protocol. One of the these LRR RLKs, At3g02880, has been identified by all of our extraction approaches and was also detected in four other proteomic analyses of the Arabidopsis plasma membrane,2,4,18,25 suggesting it is either an abundant LRR RLK or one that is easily extracted from the membrane. Two other LRR RLKs, At3g28450 and At4g08850, identified in our previous study13 and our current chloroform protocol (Table 4), were also identified in three other proteomic studies.4,18,25

Conclusions The presence of chlorophylls, phytochemicals, and lipidbased components in both microsomal and, to a lesser extent, plasma membrane fractions, adversely affect sample preparation and down stream analysis. Although these contaminants do not impede the use of methanol-assisted solubilization to promote the extraction of membrane proteins from the lipid bilayer and their subsequent tryptic digestion, they do require removal for reproducible LC/MS/MS analysis. Our developed chloroform extraction was determined to be exceptionally suited for extracting chlorophyll a and b, two of the most highly abundant phytochemicals present in the sample while facilitating a 70% increase in unique peptides for plasma membrane protein identification when compared to SPE. Although used in conjunction with our methanol-bicarbonate buffer, it can potentially be adapted to other organic-based buffers containing miscible chloroform/methanol ratios used for membrane protein solubilization by increasing the volume of chloroform added after protease digestion. In addition, our chloroform extraction approach allows for effective precipitation of trypsin with nondetectable peptide losses. In this aspect, it could be used to remove the protease or undigested proteins after any digestion procedure or for other situations where protein removal is required. This would produce an aqueous peptide mixture more suitable for the addition of a second protease or isotope-coded labeling reagent such as ICAT or iTRAQ to 2766

Journal of Proteome Research • Vol. 8, No. 6, 2009

improve protein sequence coverage by bottom-up quantitative measurements using mass spectrometry analysis.

Acknowledgment. This work was supported by a grant from the National Science Foundation (MCB-0419819) as part of the Arabidopsis 2010 Project. The authors thank Uma Kota, Andrew Debrecht, and John A. Gantt for providing assistance in initial sample preparation and data analysis. The authors also thank the research agencies of North Carolina State University and the North Carolina Agricultural Research Service for continued support of our biological mass spectrometry research. Supporting Information Available: Comprehensive lists of all identified peptides and proteins obtained from the plasma membrane preparations of Arabidopsis seedlings using the chloroform extraction (Table S1) and SPE (Table S2) approaches. Comprehensive lists of all identified membrane transporters using the chloroform extraction (Table S3) and SPE (Table S4) methods. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Schwacke, R.; Flugge, U. I.; Kunze, R. Plant membrane proteome databases. Plant Physiol. Biochem. 2004, 42 (12), 1023–1034. (2) Marmagne, A.; Ferro, M.; Meinnel, T.; Bruley, C.; Kuhn, L.; Garin, J.; Barbier-Brygoo, H.; Ephritikhine, G. A high content in lipidmodified peripheral proteins and integral receptor kinases features in the Arabidopsis plasma membrane proteome. Mol. Cell. Proteomics 2007, 6 (11), 1980–1996. (3) Benschop, J. J.; Mohammed, S.; O’Flaherty, M.; Heck, A. J.; Slijper, M.; Menke, F. L. Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis. Mol. Cell. Proteomics 2007, 6 (7), 1198– 1214. (4) Nelson, C. J.; Hegeman, A. D.; Harms, A. C.; Sussman, M. R. A quantitative analysis of Arabidopsis plasma membrane using trypsin-catalyzed 18O labeling. Mol. Cell. Proteomics 2006, 5 (8), 1382–1395. (5) Shiu, S. H.; Karlowski, W. M.; Pan, R.; Tzeng, Y. H.; Mayer, K. F.; Li, W. H. Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 2004, 16 (5), 1220–1234. (6) Becraft, P. W. Receptor kinase signaling in plant development. Annu. Rev. Cell Dev. Biol. 2002, 18, 163–192. (7) Whitelegge, J. P.; Laganowsky, A.; Nishio, J.; Souda, P.; Zhang, H.; Cramer, W. A. Sequencing covalent modifications of membrane proteins. J. Exp. Bot. 2006, 57 (7), 1515. (8) Loo, R. R.; Dales, N.; Andrews, P. C. The effect of detergents on proteins analyzed by electrospray ionization. Methods Mol. Biol. 1996, 61, 141–160. (9) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 1999, 17 (10), 994–999. (10) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3 (12), 1154–1169. (11) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R., III. A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 2003, 21 (5), 532–538. (12) Whitelegge, J. P. Plant proteomics: BLASTing out of a MudPIT. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (18), 11564–11566. (13) Mitra, S. K.; Gantt, J. A.; Ruby, J. F.; Clouse, S. D.; Goshe, M. B. Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques. J. Proteome Res. 2007, 6 (5), 1933–1950. (14) Larsson, C.; Sommarin, M.; Widell, S. Isolation of highly purified plant plasma membranes and separation of inside-out and rightside-out vesicles. Methods Enzymol. 1994, 228, 451–469. (15) Schwert, G. W.; Takenaka, Y. A spectrophotometric determination of trypsin and chymotrypsin. Biochim. Biophys. Acta 1955, 16 (4), 570–575. (16) Norholm, M. H.; Nour-Eldin, H. H.; Brodersen, P.; Mundy, J.; Halkier, B. A. Expression of the Arabidopsis high-affinity hexose

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PR801044Y

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