Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques Srijeet K. Mitra,† John A. Gantt,‡ James F. Ruby,‡ 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 October 5, 2006
This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58. Keywords: Arabidopsis • proteomics • liquid chromatography • mass spectrometry • membrane proteins • phosphorylation • leucine-rich repeat receptor-like kinases
Introduction The promise of mass spectrometry (MS) analysis to identify large numbers of proteins, quantify their abundances, and characterize post-translational modifications has attracted many plant biologists into the field of proteomics, which has expanded dramatically since the completion of the Arabidopsis genome sequence.1,2 Numerous proteomic analyses have been undertaken in Arabidopsis whole plants and cell suspension cultures subjected to a variety of stimuli and physiological conditions, as well as extensive profiling of the protein composition of purified subcellular organelles.3-8 To increase proteomic coverage, all of these studies invested significant efforts to enrich and fractionate plant proteins obtained from whole cells or purified plastids, mitochondria, and membrane components prior to MS-based proteomic analysis. Obtaining * To whom correspondence should be addressed. 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. ‡ Department of Molecular and Structural Biochemistry. 10.1021/pr060525b CCC: $37.00
2007 American Chemical Society
complete proteome coverage of membrane fractions offers unique challenges due to the wide variation in physicochemical properties of these proteins, including the hydrophobic nature of many integral membrane proteins and the hydrophilic nature of porins and a range of peripheral membrane proteins. Moreover, many important membrane-associated proteins involved in signaling pathways are of low abundance and must be enriched to the level of detection by MS-based approaches. Therefore, successful membrane proteomics requires effective methods for solubilization of both hydrophobic and hydrophilic proteins from purified membranes that are efficient and compatible with downstream MS applications. Our interest in plant membrane proteins centers on plant cell signaling involving the family of leucine-rich repeat receptor-like kinases (LRR RLKs), which consist of an extracellular domain putatively involved in ligand binding, a single-pass transmembrane domain (TMD), and an intracellular kinase that is activated by phosphorylation.9,10 Several of these plant RLKs have proven functional roles in the regulation of plant growth, morphogenesis, disease resistance, and responses to the environment,9,11 but the functions of most LRR RLKs remain Journal of Proteome Research 2007, 6, 1933-1950
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research articles unknown. To eventually generate a large database of LRR RLK in vivo phosphorylation sites, we chose whole Arabidopsis seedlings subjected to a variety of physiological and environmental light-grown conditions as our model for phosphoproteomic analysis of this important family of membrane-localized signaling molecules. As a foundation for these future studies, we needed to establish an effective and reliable enrichment protocol to isolate Arabidopsis membrane proteins for analysis by strong cation exchange chromatography (SCXC) and reversedphase liquid chromatography-tandem mass spectrometry (LCMS/MS). Such a procedure could be used to screen a large number of plant samples to identify growth conditions where LRR RLKs are highly expressed and phosphorylated, prior to a more in-depth analysis of specific phosphorylation sites using highly purified plasma membranes to enrich for LRR RLKs. The use of nonionic detergents such as Triton X-100 and Brij58 for membrane protein solubilization was reported nearly 30 years ago and has been widely employed in numerous recent studies involving membrane proteomics.12-15 Although effective in solubilizing many types of membrane proteins, such detergents often interfere with downstream LC-MS/MS analysis by affecting chromatographic separations and suppressing peptide ionization and MS detection, unless removed by time-consuming purification procedures with potential sample losses.16-18 The use of MS-compatible organic solvents such as methanol for membrane protein solubilization, followed by direct insolution tryptic digestion and LC-MS/MS, proved effective in proteomic studies of bacterial and human membranes.17,19-23 The application of this protocol to plant membrane proteomics has not been reported, although differential solubilization of membrane proteins in chloroform/methanol mixtures has been widely used in the extraction of highly hydrophobic proteins of the chloroplast envelope and mitochondrial membrane.3,7,24 This procedure is biased toward hydrophobic membrane proteins and is often combined with extraction by basic and saline solutions to increase the diversity of membrane proteins obtained. Multiple extractions of purified plasma membranes from Arabidopsis cell suspension cultures with chloroform/ methanol, sodium hydroxide, and Triton X-100 followed by 1-D gel electrophoresis and in-gel tryptic digestion, permitted the identification of 100 plasma membrane proteins by multiple MS analyses.25 However, only two LRR RLKs were found in that study. Subsequent studies in Arabidopsis using purified plasma membranes from cell suspension cultures incorporated additional enrichment and labeling techniques, which substantially increased the number of LRR RLKs identified.26 Although plant cell suspension cultures provide an abundant and uniform population of cells responsive to a variety of external stimuli, the undifferentiated state of cells in these cultures makes them less desirable for studying signaling proteins, such as LRR RLKs, involved in complex regulatory pathways associated with growth and development of tissues and organs. Thus, we chose light-grown whole Arabidopsis seedlings for functional proteomic studies of plant LRR RLKs. Based on the current literature, relatively few studies of the Arabidopsis plasma membrane proteome have used tissues derived from whole plants. Alexandersson et al.27 used phase partitioning followed by vesicle inversion with Brij-58 treatment to purify plasma membranes from leaves of Arabidopsis plants. The purified membrane fractions were subjected to gradient SDS-PAGE, in-gel tryptic digestion, and LC-MS/MS analysis to identify 238 putative plasma membrane proteins, 7 of which were LRR RLKs. Nelson et al.28 used whole Arabidopsis seed1934
Journal of Proteome Research • Vol. 6, No. 5, 2007
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lings grown in shaking liquid culture to isolate 31 RLKs from purified plasma membranes, 21 of which were annotated as LRR RLKs. Here we report two complementary approaches to effectively extract and solubilize membrane proteins from total microsomal fractions isolated from whole plant tissue. Samples were treated with either 60% methanol or 2% Brij-58 in a bicarbonate-based buffer in an effort to increase the diversity of the identified membrane proteins with particular attention to the LRR RLKs. Treatment of total membranes with methanol rather than Brij-58 resulted in a much higher number of LRR RLKs identified. When applied to highly purified plasma membranes, the methanol approach was even more effective in LRR RLK identification. Additionally, our study revealed several important distinctions that can be applied to other organelle fractionation techniques to enhance membrane protein solubilization and identification using 2-D LC-based proteomic analysis.
Materials and Methods Materials. Arabidopsis thaliana (ecotype Columbia-0) seedlings were grown in the light for 11 days in shaking liquid culture as previously described29 and used for all proteomic analyses. Sequencing grade-modified trypsin was from Promega (Madison, WI). Acetonitrile (HPLC grade) and formic acid (ACS reagent grade) were from Aldrich (Milwaukee, WI). Ammonium bicarbonate and ammonium formate were from Fluka (Milwaukee, WI), and sodium carbonate was from Fisher Scientific (Suwanee, GA). Brij-58 (polyoxyethylene monocetyl ether) was from Sigma (St. Louis, MO), and methanol (HPLC grade) was from Fisher Scientific. Cytochrome c and bacteriorhodopsin were from Sigma and Fluka, respectively. Water was distilled and purified using a High-Q 103S water purification system (Wilmette, IL). Preparation of Cytochrome c and Bacteriorhodopsin using Methanol. A stock solution of 1.0 mg/mL of each protein was prepared by dissolving the lyophilized protein in 50 mM ammonium bicarbonate, pH 8.0. An aliquot of 0.1 mL was removed from each stock solution, and then methanol was added to produce a final concentration of 60% (v/v). Each sample was sonicated for 5 min in an ice-cold sonicating bath. Once the samples reached room temperature, trypsin was added in a 1/20 (w/w) trypsin-to-protein ratio followed by incubation for 6 h at 37 °C. After digestion, the samples were reduced in volume by evaporating the methanol to approximately 10% using a stream of nitrogen. The samples were frozen in liquid N2 and stored at -80 °C until reversed-phase LC-MS/MS analysis. For LC-MS/MS analysis an aliquot of each sample was diluted with 5% mobile phase B/95% mobile phase A (v/v) to produce a concentration of 25 ng/µl and analyzed by reversed-phase LC-MS/MS as described below. Preparation of Microsomal and Purified Plasma Membrane Fractions. Microsomal fractions were isolated by grinding 20 g of plant tissue in liquid nitrogen followed by further grinding in 50 mL of cold 20 mM Tris-HCl (pH 8.8), 150 mM NaCl, 1 mM EDTA, 20% glycerol, 1 mM phenylmethylsulfonylfluoride, 20 mM NaF, 50 nM microcystin, and protease inhibitor cocktail tablets (product no. 11873580001, Roche Diagnostics, Indianapolis, IN). The extract was centrifuged at 6000× g for 15 min at 4 °C. The supernatant was decanted and was further subjected to ultracentrifugation at 100 000× g for 2 h at 4 °C. The pellet was washed with 100 mM sodium carbonate, pH 11, followed by a wash with 100 mM ammonium bicarbonate.30
Membrane Proteomic Analysis of Arabidopsis thaliana
Each wash step consisted of intermittent vortexing and sonication using a sonicating bath (Bronson Model 1510, Danbury, CT). The pellet was resuspended in 50 mM ammonium bicarbonate, pH 8.0, via intermittent vortexing and sonication. The membrane protein concentration was determined at this stage using the BCA assay with BSA standards diluted with bicarbonate buffer as previously described.21,22 The sample was divided into two 250 µg aliquots, and each aliquot was treated by the addition of Brij-58 to produce a final concentration of 2% (w/ v) or treated by the addition of methanol to produce a final concentration of 60% (v/v). Using intermittent vortexing and sonication, the proteins were solubilized. A 30 M excess of TCEP (tris(2-carboxyethyl) phosphine, Pierce Chemical Co., Rockville, IL) to protein was added to the sample, which was incubated at 37 °C for 60 min to reduce disulfide bonds. After reduction, the cysteinyl residues were alkylated by adding a 30 M excess of iodoacetamide (Sigma) to protein. The solution was slowly agitated using a shaker/rotisserie (Labquake, Conroe, TX) for 90 min at room temperature in the dark. Proteolysis was performed overnight at 37 °C using a 1:20 (w/w) trypsin/protein ratio, then quenched by rapid freezing in liquid nitrogen. Samples were stored at -80 °C until solid-phase extraction was performed. Plasma membranes were isolated by phase partitioning as described in Larsson et al.31 and then solubilized in 50 mM ammonium bicarbonate, pH 8.0, in a final concentration of 60% methanol. The proteins were digested with trypsin as described above for the microsomal membrane preparations. Solid-Phase Extraction. Following digestion, peptides were purified via solid-phase extraction (SPE) using a Prevail C18 Extract-Clean column (Alltech Associates, Inc., Deerfield, IL) connected to a vacuum manifold (Fisher Scientific) with a flow rate of about 1 mL/min. The column was equilibrated with 1 mL of water and 1 mL of 10% methanol/90% 50 mM ammonium bicarbonate, pH 8.0, followed by sample loading. The column was then washed with 2 mL of 1:9 (v/v) of acetonitrile/ 50 mM ammonium bicarbonate, pH 8.0. Peptides were eluted with 1 mL of 19:1 (v/v) of acetonitrile/water with 0.1% formic acid, lyophilized, and then stored at -80 °C until SCXC could be performed. Strong Cation Exchange Chromatography of Peptides. SCXC was performed as previously described.32 Briefly, a 4.6 mm × 200 mm, 5 µm polySULFOETHYL aspartamide SCX column (PolyLC Inc., Columbia, MD) was connected to an Agilent 1100 Analytical HPLC system equipped with a UV-vis diode array detector (Agilent Technologies, Inc., Palo Alto CA). The mobile phases consisted of (A) 75% 10 mM ammonium formate, pH 3.0/25% acetonitrile (v/v) and (B) 75% 200 mM ammonium formate, pH 8.0/25% acetonitrile (v/v). Using a flow rate of 1.0 mL/min, approximately 250 µg of peptides was loaded onto the column and separated using a linear gradient from 0 to 100% mobile phase B over 30 min, which continued isocratically at 100% mobile phase B for 25 min. Peptide elution was monitored at 214, 254, 260, and 280 nm with spectra from 190 to 400 nm acquired every second using the diode array detector, whereas fractions were collected every 0.5 min. Collected fractions were lyophilized and stored at -80 °C until further analysis. Microcapillary Reversed-Phase LC-MS/MS Analysis of SCXC Fractionated Peptides. Peptides collected during SCXC were analyzed by microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (µrpLC-MS/MS) using
research articles an Agilent 1100 Series high-performance capillary LC system (Agilent Technologies, Inc., Palo Alto, CA) coupled to a LCQ Deca ion trap mass spectrometer (Thermo Electron, San Jose, CA) using an in-house manufactured electrospray interface operating in the positive ion mode at 2.25 kV. A reversed-phase capillary column was manufactured in-house by slurry packing 5 µm 300 Å Jupiter C18 stationary phase (Phenomenex, Torrance, CA) into a 55 cm × 360 µm o.d. × 150 µm i.d., capillary (Polymicro Technologies Inc., Phoenix, AZ) incorporating a 2 µm retaining mesh in a HPLC stainless steel union (Valco Instruments Co., Houston, TX) containing a flame-pulled capillary tip. The mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. After a volume of 8 µL was loaded onto the reversed-phase column, a 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. After washing the column with 95% B for 20 min, the column was equilibrated with 5% B for 60 min prior to the next injection. The LCQ Deca was operated in the data-dependent MS/MS mode in which the four most intense ions detected in the precursor MS scan were selected for collision-induced 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 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 Arabidopsis thaliana database using TurboSEQUEST (BioWorks 3.1, Thermo Electron, San Jose, CA). Only tryptic peptides displaying a charge dependent cross-correlation score (Xcorr) of 2.1, 2.2, 2.5 and 2.9, for +1, +2 of
