Proteomic Study of the Arabidopsis t haliana Chloroplastic Envelope Membrane Utilizing Alternatives...

Proteomic Study of the Arabidopsis thaliana Chloroplastic Envelope

Membrane Utilizing Alternatives to Traditional Two-Dimensional

Electrophoresis

John E. Froehlich,† Curtis G. Wilkerson,† W. Keith Ray,‡ Rosemary S. McAndrew,§

Katherine W. Osteryoung,§ Douglas A. Gage,‡,| and Brett S. Phinney*,‡

Department of Energy Plant Research Laboratory, Michigan Proteome Consortium,Department of Biochemistry and Molecular Biology, and Department of Plant Biology,

Michigan State University, East Lansing, Michigan 48824

Received March 20, 2003

With the completion of the sequencing of the Arabidopsis genome and with the significant increase inthe amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidlybecoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomicsapproach to identify and characterize membrane proteins localized to the Arabidopsis thalianachloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grownArabidopsis plants using a novel isolation procedure, and “mixed” envelopes were subsequentlyisolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimen-sional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresisfollowed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry(Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify392 nonredundant proteins.

Keywords: chloroplastic envelope membrane • proteome • Arabidopsis thaliana • Gel-C-MS/MS

Introduction

Plastids are semiautonomous organelles that are involvedin many essential metabolic processes vital for the properfunctioning of a plant cell. The plastid is surrounded by adouble membrane system, consisting of an outer and an innerenvelope membrane that divides the plastid stroma from thecytosol. The envelope membrane itself is the site of numerousfunctions, such as the biosynthesis of glycerolipids,1 the de-saturation2 and export of fatty acids,3 the transport of numerousmetabolites,4 the breakdown of chlorophyll,5 and the synthesisof lipid-derived signals such as jasmonic acid (for a review, seeref 1). In addition, the plastid envelope membrane containsthe protein import apparatus, which is composed of the Tocand Tic complexes.6-8

The majority of proteins destined for the plastid are nuclear-encoded, synthesized in the cytoplasm as higher molecularweight precursors containing an amino-terminal extensioncalled the transit peptide, which specifically targets these

precursors to the plastid.7,8 Interestingly, the targeting deter-minants that directs some of these proteins to the outer orinner envelope membrane is still poorly defined. The plastidis predicted to contain approximately 2500 to 3500 proteins.9,10

These estimates have been based primarily on computer pre-diction algorithms, such as TargetP.11-13 Unfortunately, theseprograms, while relatively useful, have a high error rate.14,15 Asa result, they are limited in their ability to predict the chloro-plastic subcellular compartment or membrane to which anunknown protein will ultimately be localized. Presently, noprograms can reliably predict whether a protein is targeted tothe outer envelope membrane of the chloroplast.12 Conse-quently, the identification and characterization of proteins tar-geted to the chloroplast envelope remains a significant chal-lenge in plant biology. Complete sequencing of the Arabidopsisthaliana genome9 and the rapid improvements in mass spec-trometry techniques have made it feasible to specifically ana-lyze the proteomes of subcellular compartments of the chlo-roplast, including the envelope membrane. Already, severalproteomic studies focusing on the thylakoids of the chloroplastshave been completed.10,16-18 The methods employed in thesestudies relied heavily on two-dimensional (2D) electrophoresis,followed by excision of protein spots from these gel systemsand subsequent proteolytic digestion and identification ofproteins by either matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) or by elec-trospray liquid chromatography coupled with tandem mass

* To whom correspondence should be addressed. E-mail: [email protected]. Fax: (517) 353-6342.

† Department of Energy Plant Research Laboratory, Michigan StateUniversity.

‡ Michigan Proteome Consortium and Department of Biochemistry andMolecular Biology, Michigan State University.

§ Department of Plant Biology, Michigan State University.| Present address: Discovery Technologies Department, Pfizer Global

Research and Development, Ann Arbor Laboratories, 2800 Plymouth Rd.,Ann Arbor, MI 48105.

10.1021/pr034025j CCC: $25.00 2003 American Chemical Society Journal of Proteome Research 2003, 2, 413-425 413Published on Web 06/20/2003

spectrometry (LC-MS/MS) analysis. However, with regard tothe envelope membranes, only a small fraction of the proteinspresent have been identified and characterized.1,19,20 Morerecently, a limited analysis of the integral membrane proteinsfrom spinach envelopes using one-dimensional (1D) gel elec-trophoresis was completed.20

To date, no systematic analysis of the Arabidopsis chloro-plastic envelope membrane proteome has been achieved. Thisgap in our understanding of the envelope proteome reflectsthe inherent difficulty in studying and identifying hydrophobicenvelope membrane proteins. Because of these inherentdeficiencies, we decided to study the proteome of Arabidopsisthaliana envelope membrane by using two alternatives totraditional 2D electrophoresis, namely off-line multidimen-sional protein identification technology (Off-line MUDPIT)21-23

and traditional 1D electrophoresis followed by LC-MS/MSalternatively referred to as Gel-C-MS/MS. Because both Off-line MUDPIT and Gel-C-MS/MS are complementary yet inde-pendent separation strategies for the identification of proteins,the data sets derived from these two approaches were com-bined to ensure a significant coverage of the envelope mem-brane proteome. In addition, we compared the differences inthe ability of these two techniques to identify both low-abundance and highly hydrophobic envelope membrane pro-teins. Subsequently, a list of envelope membrane proteinsidentified by both methods, while not exhaustive, was com-piled. Additionally, we used both Off-line MUDPIT and Gel-C-MS/MS to examine a known protein complex localized tothe envelope membrane.

Experimental Section

Chemicals. All chemicals were ultrapure grade or better andpurchased through Sigma Chemical. EM science HPLC gradeacetonitrile and Riedel-de Haen 98-100% pure formic acid waspurchased through Fisher Scientific. HPLC-grade water wasproduced by a Barnstead Diamond nano-pure UV system.Sequencing-grade modified Trypsin was purchased from Prome-ga.

Growth of Arabidopsis Plants. Arabidopsis thaliana (ecotypeColumbia 0) seeds were grown on either soil or on agar platessupplemented with 1% w/v sucrose according to the methodof Fitzpatrick and Keegstra.24 Soil-grown plants were incubatedat 20 °C under light conditions of 150 µE m-2 s-1 for 12 h,whereas plate-grown plants were incubated at 22 °C under lightconditions of 100 µE m-2 s-1 for 16 h.

Isolation of Arabidopsis Chloroplasts. Arabidopsis chloro-plasts were isolated from either soil- or plate-grown plants bythe method of Fitzpatrick and Keegstra.24 Intact chloroplastswere collected and washed with Import Buffer (330 mMsorbitol, 50 mM HEPES/KOH, pH 8.0) and recovered again bycentrifugation. Pelleted intact chloroplasts were resuspendedin hypertonic lysis buffer (0.6 M sucrose, 10 mM tricine, pH7.5, 2 mM EDTA), incubated on ice for 20 min, and then storedat -20 °C overnight.

Isolation of Arabidopsis Chloroplastic “Mixed” EnvelopeMembranes. Mixed envelopes from purified Arabidopsis chlo-roplasts were prepared by the method of Keegstra and Yousif25

with the following modifications: chloroplasts in hypertoniclysis buffer were ruptured by several freeze-thaw cycles andby homogenization using a Dounce tissue grinder with a tight-fitting pestle. The suspension of broken chloroplasts wasdiluted with 2 volumes of 10 mM tricine, pH 7.5, 2 mM EDTA(TE) to give a final sucrose concentration of 0.2 M. Ruptured

chloroplasts were centrifuged at 4500g for 15 min to removethylakoids. The supernatant was collected and centrifuged at40000g for 30 min to collect a crude envelope fraction. Thecrude envelope fraction was resuspended in TE buffer supple-mented with 0.2 M sucrose (200 µL) and overlayed onto adiscontinuous sucrose gradient consisting of 1 mL of 0.45 Msucrose/TE and 1 mL of 1.0 M sucrose/TE in a 1.5-mL cen-trifuge tube. The sucrose step gradient was centrifuged in aSorvall RP55S rotor for 2 h at 46 000 rpm. After centrifugation,the “yellowish” band at the 0.45/1.0 M sucrose interface wascollected and diluted with 4 volumes of TE and centrifuged.Pelleted mixed envelopes were resuspended in TE and quan-titated using the method of BioRad. Approximately 300-400µg of mixed envelope membrane protein was recovered from50 mg of chlorophyll. Typically, 100 µg of mixed envelopemembrane protein was used for either Gel-C-MS/MS or Off-line MUDPIT analysis.

Antibodies. Antiserum to allene oxide synthase (AOS) was agift from Gregg Howe et al.26 Antiserum to components of thepea chloroplastic import machinery, translocon at the innerenvelope of chloroplasts (Tic) 110 and translocon at the outerenvelope of chloroplasts (Toc) 75, were prepared as describedin Nielsen et al.27 Antiserum to Light Harvesting Complex(LHCP) was a gift from K. Cline.28

Western Blotting of Mixed Envelope Membranes. Fractionsfrom a typical envelope preparation from soil-grown Arabi-dopsis plants were separated by SDS-PAGE, transferred toImmobilon P, and probed with antibodies prepared againsteither pea Tic110 (inner envelope standard), Toc75 (outerenvelope standard), tomato AOS (peripheral inner envelopeprotein) or LHCP (Thylakoid membrane standard). Primaryantibodies were detected as described in Nielsen et al.27

1D Gel Analysis. Mixed chloroplast membrane protein (100µg) was solubilized in SDS-PAGE sample loading buffer andseparated on a 10-cm 12.5% polyacrylamide gel.29 The gel wassilver stained using the Vorum silver-staining protocol30 andimaged using a Bio-Rad FXPro Plus laser imager. Fifty 2-mmslices were excised from the gel using a Bio-Rad model 190electrophoresis gel slicer and placed in a 96-well plate. In-geldigestion was performed by hand according to the methodpublished in Rowley et al. 31 with the following modifications.After the digest, the digested peptides were extracted in 8 ul of3% TFA.

LC-MS/MS Analysis. Nanoscale liquid chromatography/mass spectrometric analysis was accomplished using LC Pack-ings (San Francisco, CA) Ultimate HPLC system coupled to aThermo Finnigan (San Jose, CA) LCQ DECA XP quadrupole iontrap mass spectrometer through a New Objectives (Woburn,MA) Picoview nanospray source. The peptides from each in-gel digest or strong cation exchange (SCX) fraction wereautomatically loaded and trapped on a Michrom CapTrapcartridge using the LC Packing Famos autosampler and Switchosloading pump. The loading pump operated at 20 µL/min andthe loading solvent consisted of 0.1% TFA, 2% acetonitrile. Thetrapped peptides were then eluted off the trap and separatedby backflushing onto a 75 µm i.d. × 15 cm New Objectivespicofrit column packed with Michrom 5 µm 100 Å MagicC18AQ packing material. Peptides were then eluted from thecolumn using a gradient of 5% B to 40% B in 46 min, 40%-70% B in one min, 70% B for 1 min, and then 5% B until thefinal time of 60 min. The final flow rate was 200 nL/min. Mobilephase A consisted of 0.1% formic acid, whereas mobile phaseB consisted of 0.1% formic acid in a 95/5 acetonitrile/water

research articles Froehlich et al.

414 Journal of Proteome Research • Vol. 2, No. 4, 2003

solution. The picofrit column made electrical contact througha precolumn ZDV titanium union, and terminated in an 8-µmtip id outlet spray needle. Mass spectra were acquired usingthe Top 4 double play mode of operation, where the top 4coeluting peptide ions detected in a MS survey scan triggereddata-dependent MS/MS fragmentation for obtaining production spectra.

Off-line MUDPIT Analysis. Envelope membrane protein (100µg) was digested as previously described Millar et al.32 SCXchromatography was carried out by first adjusting the resultingdigest solution to pH 2.7 and then loading the digest on a 1.0-

mm Michrom Bio Resources (Palo Alto, CA) column packedwith PolySULFOETHYL A. Peptides were separated by meansof a Michrom UMA HPLC system with a 40-min linear gradientof 100 to 350 mM KCL in 5 mM phosphate buffer containing25% acetonitrile at a flow rate of 50 µL/min. Forty 1-minfractions were collected and stored at 4 °C until LC-MS/MSanalysis could be performed on each.

Database Searching and Bio-Informatics. Uninterpretedproduct ion spectra were searched in-house against the Ara-bidopsis thaliana protein database obtained from the Munichinformation services for protein sequences (MIPS) using theMascot search program.33 Mascot data output files for eachin-gel digest or SCX fraction were combined and analyzedusing the DTASelect and contrast programs by David Tabb.DTASelect 1.8 was used which had been modified for use withmascot output files. DTASelect criteria were as follows. Anidentification was considered real if a minimum of 2 peptidesfrom each protein generated a mascot score that was significantwithin a p of 0.05.

Results

Isolation and Characterization of Arabidopsis “Mixed”Envelope Membranes. Our initial attempts to isolate outer andinner envelope membranes from purified Arabidopsis thalianachloroplasts using sucrose step-gradients proved problematic.Consequently, we modified the protocol of Keegstra andYousif25 and used a simple step gradient that allowed for theisolation of a “mixed” envelope fraction containing both outerand inner envelope membranes (see the Materials and Methodssection). Mixed chloroplastic envelopes isolated from eithersoil- or plate-grown Arabidopsis plants were analyzed by SDS-PAGE and silver stained (Figure 1A). The purity of a representa-tive mixed envelope preparation from soil-grown Arabidopsisplants was evaluated by western blotting (Figure 1B). Mixedenvelopes from plate-grown Arabidopsis plants were evaluatedin a similar fashion by western blotting (data not shown). Whilewestern blotting detected the presence of some LHCP (thyla-koid membrane marker) in our envelope preparation (Figure1B), the level of thylakoid contamination of our mixed enve-lopes was further quantitated by measuring chlorophyll. Fromthis analysis, we determined that the envelope fraction wascontaminated with approximately 3.1% thylakoid membranes.This determination was based on the following: 1 mg of chloro-phyll usually corresponds to approximately 10 mg of thylakoidprotein. In Table 1, we determined the total chlorophyll contentof a purified mixed envelope fraction by measuring chlorophyllaccording to the method of Arnon.34 The total chlorophyllcontent for this fraction was 0.0012 mg. Using the chlorophyll/protein ratio above, we estimated that 0.012 mg of totalthylakoid protein was present in our envelope fraction that

Figure 1. Analysis of Arabidopsis purified “mixed” envelopesby SDS-PAGE. Approximately 20 µg of mixed envelope mem-brane proteins isolated from either soil-grown (Lane 1) or plate-grown (Lane 2) Arabidopsis plants were analyzed by 12.5% SDS-PAGE and silver stained according to the method of Mortz et al.30

B, Fractions from a typical envelope preparation from soil-grownArabidopsis plants were separated by SDS-PAGE, transferred toImmobilon P, and probed with antibodies prepared against eitherpea Tic100 (inner envelope standard), Toc75 (outer envelopestandard), tomato AOS (peripheral inner envelope protein) orLHCP (thylakoid membrane standard). The fractions probed areas follows: TM, Total membranes (Lane 1); Str, stroma (Lane 2);Thy, thylakoids (Lane 3); and Env, mixed envelope fraction (Lane4). Primary antibodies were detected as described in the Materialsand Methods section.

Table 1. Chlorophyll Determination of Various Fractionsduring Envelope Isolation from Soil-Grown Arabidopsisthaliana Plantsa

sample

total proteinb

(mg)

total chlorophyllc

(mg)

leaf tissue 950 335thylakoid fraction 87 9.9purified mixed envelopes 0.4 0.0012

a Typical preparation begins with approximately 100 g of tissue from soilgrown plants homogenized in 250 mL of grinding buffer. b Protein deter-mination by the method of BioRad. c Chlorophyll determined by the methodof Arnon.34

Arabidopsis thaliana Chloroplastic Envelope Membrane research articles

Journal of Proteome Research • Vol. 2, No. 4, 2003 415

contained 0.4 mg of total protein. Dividing the total amountof thylakoid protein by the total protein present in our envelopefraction and multiplying by 100, the level of contamination ofour envelope fraction with thylakoid membranes was deter-mined to be 3.1% (see Table 2 for a list of the major thylakoidproteins contaminating our envelope preparations).

Finally, we observed that SDS-PAGE and silver staining ofenvelope membrane proteins isolated from either soil- or plate-grown plants gave essentially the same protein profile (Figure1A). From this observation, we concluded that combiningenvelope preparations originating from either soil- or plate-grown Arabidopsis plants would not compromise the overallprotein composition of the envelope. Therefore, several enve-lope preparations from either soil- or plate-grown Arabidop-sisplants were performed and combined to produce sufficientamounts of purified mixed envelopes for proteome analysis.Routinely, beginning with 100 g of leaf tissue, yields ofapproximately 300-400 µg of purified mixed envelopes were

achieved. Typically, 100 µg of “untreated” purified mixedenvelopes was analyzed by either Gel-C-MS/MS or by Off-lineMUDPIT.

Total Envelope Membrane Proteins Identified by Both Gel-C-MS/MS and Off-line MUDPIT Approaches. The mixedenvelopes isolated in Figure 1 (200 µg) were divided into twoequal portions. For our initial analyses, we decided not toextract our mixed envelope membranes with high salt orsodium carbonate prior to mass spectrometry analysis. Giventhat these extraction procedures would certainly removecontaminating proteins, they would likewise remove authenticperipheral membrane proteins of the envelope membrane.Therefore, to obtain a baseline proteome of the envelopemembrane, we did not pretreat the envelopes. Approximately100 µg of untreated purified mixed envelope membraneproteins were analyzed by Gel-C-MS/MS as described in theMaterials and Methods section. We identified proteins bysearching uninterpreted product ion spectra against the pre-

Table 2. Identified Thylakoid and Stroma Proteins Contaminating Envelope Membranes

no. codea description targetP scoreb TM domainsc MWd (kDa) localizatione

thylakoid1 At5 g42270 cell division protein FtsH 0.829 1 75.2 lumen/env memb?2 At1 g03630 putative protochlorophyllide

reductase0.838 0 43.8 membrane/env memb?

3 At5 g66190 ferredoxin-NADP reductase 0.888 0 40.3 membrane4 At3 g08940 putative chlorophyll

a/b-binding protein0.875 0 31.1 membrane

5 At3 g11630 putative 2-cys peroxiredoxin 0.988 0 29.0 lumen or memb.?6 At2 g34430 putative PSI subunit II 0.708 0 28.1 membrane7 At1 g06680 OEC 23 0.871 0 28.0 lumen8 At1 g11430 DAG 0.808 0 26.2 membrane9 At4 g09650 H-transporting ATP synthase-

like protein0.774 1 25.6 membrane

10 At1 g31330 putative PSI subunit III 0.971 1 24.1 membrane11 At1 g03130 putative PSI reaction center

subunit II0.951 1 22.3 membrane

12 At3 g15360 thioredoxin m4 0.966 0 21.1 lumen or memb.?13 At1 g55670 putative PSI subunit V 0.963 2 17.0 membrane14 At5 g28750 tha4 protein-like 0.838 1 15.7 membrane

stroma1 At4 g24280 Hsp 70-like protein 0.986 0 76.8 stroma and envelope

associated2 At1 g56410 heat shock 70 protein ? 0 68.3 stroma localized3 At1 g26230 chaperonin precursor

(Hsp60 type)0.951 0 66.8 stroma + envelope

association4 At4 g24620 glucose-6-phosphate

isomerase0.344 0 66.8 cytosol/stroma

5 At1 g55490 rubisco subunit binding-protein beta subunit

0.979 0 63.8 stroma localized

6 At5 g38420 ribulose bisphosphatecarboxylase small chain 2bprecursor (RuBisCO smallsubunit 2b)

0.806 0 20.3 cytosol; commoncontaminant of envelopes

7 At3 g55800 sedoheptulose-bisphosphatase precursor

0.937 0 42.4 cytosol

8 At1 g67090 ribulose-bisphosphatecarboxylase small unit

0.772 0 20.2 cytosol localized

9 At5 g38430 ribulose bisphosphate carboxylasesmall chain 1b precursor(RuBisCO small subunit 1b)

0.807 0 20.2 stroma localized;common contaminantof envelopes

10 At2 g42530 cold-regulated proteincor15b precursor.

0.672 0 14.9 stroma localized

11 At4 g25050 acyl carrier-like protein 0.939 0 14.5 stroma localized,some may associatewith envelope

a MIPS accession number. b TargetPv1.01 used to predict subcellular localization (i.e., chloroplasts, mitochondria, etc.). c Transmembrane (TM) domainpredictions were performed using TMHMMv2.0. d Molecular weights (MWs) shown are calculated values. e Localization assignments are derived from variousdatabase searches and published literature annotations.



dicted Arabidopsis proteins derived from the TIGR annotation(ATPEP database) using the Mascot search program.33 TheMascot data output files for each Gel-C-MS/MS run from eachof the digested gel sections were combined and analyzed withthe DTASelect program which resulted in the identification of243 proteins by Gel-C-MS/MS (Figure 2). The standard set fora confident protein identification for this analysis used aminimum identification criterion of 2 polypeptides with aMascot significance level of p < 0.05. (A comprehensive list ofall proteins identified is available in the Supporting Informa-tion.)

Likewise, 100 µg of untreated mixed envelope membraneproteins were analyzed by Off-line MUDPIT. Uninterpretedproduct ion spectra were also searched against the ATPEPdatabase and the Mascot data output files for each analysisset were combined and analyzed by the DTASelect programusing the same protein identification criteria as stated above.Using the Off-line MUDPIT approach, we identified a total of283 proteins (Figure 2). By combining the data sets of Off-lineMUDPIT and Gel-C-MS/MS, we were able to identify 392nonredundant proteins. Further examination revealed that 134proteins (34.2%) were identified by both Gel-C-MS/MS and Off-line MUDPIT approaches, whereas 109 proteins (27.8%) wereexclusively identified by Gel-C-MS/MS and 149 proteins (38%)were exclusively identified by Off-line MUDPIT (Figure 2). Wefurther determined the number of trans-membrane domains(TMDs) present in all the proteins identified in this study (seesupplemental data). Of the 149 proteins exclusively identifiedby Off-line MUDPIT, 68 proteins contained one or more TMDs,whereas of the 109 proteins exclusively identified by Gel-C-MS/MS, only 11 proteins contained one or more TMDs. Finally,of the 134 proteins identified by both mass spectrometry tech-niques, only 34 proteins contained one or more TMDs. Hence,the majority of putative envelope membrane proteins identifiedin this study did not contain a classical predicted TMD.

Evaluation and Classification of Envelope Proteins Identi-fied by Off-line MUDPIT and Gel-C-MS/MS. Informationregarding the subcellular localization of all 392 nonredundantproteins identified in this study was mined from annotationfound in the MIPS database (Figure 3A). From this analysis,26% of the proteins identified were assigned as having authen-tic or tentative envelope membrane localization. Proteins withsignificant database annotation that were designated as au-

thentic envelope membrane proteins (see Tables 3 and 4) werefurther classified according to the following groupings (seeFigure 3B and Table 5): (i) protein import components, (ii)metabolite translocators, (iii) fatty acid metabolism, (iv) oxylipinmetabolism, (v) sugar transporters, (vi) ABC transporters withunknown function, and (vi) envelope proteins with uniquepredicted functions. Furthermore, because we have an ongoinginterest in protein complexes located at the envelope mem-brane, the components of the protein import apparatus (Tocand Tic complexes) were identified and a detailed summaryof the results is given in Table 6.

Interestingly, 63% of the proteins identified could not beassigned any known function (i.e., hypothetical, unknown, orputative). The envelope membrane protein list compiled inthis study was further compared to protein lists derived fromother published chloroplastic or mitochondrial proteome stu-dies.10,16-18,32,35 As expected, no envelope membrane proteinsidentified in this study were similarly detected in the mito-chondrial proteome study.32,35 However, several proteins identi-fied in the thylakoid proteome projects of Peltier et al.10 andSchubert et al.17 were identified in our envelope proteome (seeTable 2).

Figure 2. Comparative distribution of envelope membraneproteins identified by Off-line MUDPIT and/or Gel-C-MS/MS. The392 nonredundant envelope membrane proteins were identifiedby Off-line MUDPIT and Gel-C-MS/MS approaches. The criterionof 2 polypeptides being present per positive identification wasused in this single analysis. The following ven diagram showsthat 134 proteins (34.2%) were identified by both Gel-C-MS/MSand Off-line MUDPIT approaches (dark gray shading), whereas109 proteins (27.8%) were exclusively identified by Gel-C-MS/MS (greenish shading) and 149 proteins (38%) were exclusivelyidentified by Off-line MUDPIT (light gray shading).

Figure 3. Evaluation and classification of envelope membraneproteins identified by Off-line MUDPIT and Gel-C-MS/MS. A. 392nonredundant envelope membrane proteins were analyzed andtheir subcellular localizations are presented. The standard set fora confident protein identification used a minimum identificationcriterion of 2 polypeptides with a Mascot significance level of p> 0.05. A list of all the envelope membrane proteins identifiedis available in the Supporting Information. All protein sequencesobtained from mass spectrometry were subject to BLAST searchesutilizing MATDB (MIPS Arabidopsis Thaliana Database). Likewise,identified proteins were further mined by using the MIPS andTIGR databases to provide additional annotation. B. Identifiedenvelope membrane proteins from Tables 3 and 4 were classifiedaccording to various groups based on known or predictedfunction. Numbers in parentheses represent the number ofmembrane proteins classified in a particular grouping.



Finally, 5% of the proteins identified in this study wereassigned a thylakoid localization, whereas 3% of the proteinsidentified were assigned a stromal localization (see Table 2).The predominant stromal contaminants of the envelope mem-brane were the large and small subunits of RuBisCO (see Figure1A and Table 2). Another 3% of the proteins contaminating ourenvelope membranes were identified as principally ribosomalproteins (see the Supporting Information). Finally, the level ofcontamination of our envelope membranes with either plasmamembrane or mitochondrial proteins was 0.5% and 0.7%,respectively (see the Supporting Information).

Comparing Off-line MUDPIT and Gel-C-MS/MS Datasets.We further compared the dataset generated by both Off-line

MUDPIT and Gel-C-MS/MS derived from a single experimentand examined two general features: (i) the molecular weightdistribution of the proteins (Figure 4) and (ii) the EST frequencydistribution of the proteins (Figure 5). Only proteins with atleast four or more peptides found per protein were used forthis comparison. Requiring this many polypeptides per proteinreduced the likelihood that the proteins found by eitherMUDPIT or Gel-C-MS/MS were due to random chance. Figure4A shows the molecular weight distribution of the proteinsfound by Off-line MUDPIT. The Off-line MUDPIT results reveala broad distribution of molecular weights (from 11 to 110 kD),with the exception of proteins at the extreme ends of the massrange. The majority of proteins identified by Off-line MUDPIT

Table 3. Envelope Membrane Proteins Identified by Off-line MUDPIT and/or Gel-C-MS/MS

no. codea description

targetPb

score

TMc

domains

MWd

(kD)

Off-line

MUDPIT

Gel-C-

MS/MS localizatione

1 At1 g06950 Tic 110 (importapparatus) 3′partial

0.941 2 110 v v inner envelope

2 At3 g48870 Hsp93-III (ClpC) 0.575 0 105 v v inner envelope + stroma3 At5 g50920 HsP93-V (ClpC) 0.849 0 103 v v inner envelope + stroma4 At5 g51070 ERD1 protein

precursor0.969 0 97.0 v envelope

5 At5 g60600 putative protein 0.814 0 82.2 v v envelope?6 At1 g15690 inorganic

pyrophosphatase0.968 14 80.8 v v inner envelope

7 At4 g14070 AMP-binding protein 0.896 0 78.2 v v envelope8 At3 g23790 AMP-binding protein 0.894 0 63.8 v v envelope?9 At5 g35360 acetyl-CoA

carboxylase0.919 0 58.8 v v inner envelope

10 At5 g42650 allene oxide synthase 0.896 0 58.1 v v inner envelope11 At4 g01690 protoporphyrinogen

oxidase0.598 0 57.6 v v both inner env + Thy.

12 At2 g24820 putative Reiskeiron-sulfur (Tic55)

0.897 2 55 v v inner envelope

13 At3 g11170 omega-3 fatty aciddesaturase

0.905 3 51.1 v v envelope

14 At5 g05580 temperature-sensitiveomega-3 fatty aciddesaturase

0.875 3 50.0 v v inner envelope

15 At5 g16620 Tic40-like protein 0.856 1 48.9 v v inner envelope16 At4 g04640 putative protein

(transport ATPase)0.963 0 40.9 v v envelope

17 At3 g63410* putative chloroplastinner envelopeprotein (IEP37)

0.983 1 37.9 v v inner envelope

18 At1 g65260 Inner envelopemembrane protein

30 kDa

0.965 3-4 36.3 v v envelope

19 At5 g09650 inorganicpyrophosphatase

0.564 0 33.3 v inner envelope?

20 At2 g21170 trios phosphateisomerase

0.950 0 33.3 v envelope

21 At1 g24360 putative 3-oxoacyl-ACP reductase

0.545 0 32.2 v inner envelope

22 At5 g19760 oxoglutarate/malatetranslocator-like

? ? 31.9 v inner envelope

23 At4 g33350f Tic22-like protein 0.679 0 30.1 v v inner envelope24 At5 g46110f phosphate/triose-

phosphate translocator(IEP30)

0.607 9 v v inner envelope

25 At4 g12060 putative protein(ClpC related??)

0.936 0 29.9 v v inner envelope + stroma

26 At3 g63160f putative proteinOEP 6.7

1 7.2 v v envelope

27 At3 g04870 putative zeta-carotene desaturase

0.582 0 ? v v envelope + thylakoids

a MIPS accession number. b TargetPv1.01 used to predict subcellular localization (i.e., chloroplasts, mitochondria, etc.). c Transmembrane (TM) domainpredictions were performed using TMHMMv2.0 d Molecular weights (MWs) shown are calculated values. e Localization assignments are derived from variousdatabase searches and published literature annotations. f Indicates proteins likewise identified by the method of Ferro et al.20



were found in the 31-50 kD range. No proteins were foundabove 150 kD by this technique. Figure 4B shows the molecularweight distribution of the proteins found by 1D gel electro-phoresis Gel-C-MS/MS. The distribution of molecular weightsfor these dataset tended to favor smaller proteins (11-50 kDrange) and was more random across the higher molecularweight range when compared to the Off-line MUDPIT results(compare Figure 4A and B). From this analysis, we concludedthat Off-line MUDPIT was in general the better approach fordetecting envelope membrane proteins over a wider range ofmolecular weights.

We likewise compared the sensitivity of Off-line MUDPITand Gel-C-MS/MS to detect and identify low-abundance

proteins of the envelope. For this analysis, we determined theEST frequencies for the proteins identified in this study byusing the BLASTN program from NCBI. Scoring matcheswere considered when the EST sequence matched the genenucleotide sequence with greater than 99% identity (see Fig-ure 5). This analysis is based on the assumption that theEST frequencies reflect the overall abundance of the pro-teins, where a protein with more ESTs is assumed to bemore abundant than a protein with few ESTs. Although thismay not be true for proteins with unusually long or shorthalf-lives, when looking at a large number of proteins thisapproach provides an estimate of the range of protein abun-dance observed in this study. Figure 5, parts A and B, shows

Table 4. Envelope Membrane Proteins Identified Exclusively by Off-line MUDPIT


targetP

scoreb

TM

domainsc

MWd

(kD)

Off-line

MUDPIT

Gel-C-

MS/MS localizatione

1 At4 g02510 chloroplast proteinimport Toc159-like

0 0 94.5 v outer envelope

2 At3 g46740* import-associatedchannel Toc75

0.952 0 89.0 v outer envelope

3 At5 g42480 putative protein similarto AtFN2 (Arc6)


4 At5 g64940 ABC transporter-likeprotein

0.942 6-8 86.0 v inner envelope?

5 At2 g38040 putative alpha-carboxylase 0 0 85.0 v envelope6 At5 g19620 putative protein outer

envelope (IAP75) pea0 0 79.2 v outer envelope

7 At1 g77590 putative acyl-CoAsynthetase

0.260 0 76.1 v envelope

8 At5 g67030 zeaxanthin epoxidaseprecursor

0.735 0 73.8 v envelope

9 At5 g19620 putative protein(similar to Toc75)

0.640 0 73.2 v outer envelope

10 At5 g03910 ABC transporter-likeprotein

0.734 2 69.1 v envelope?

11 At1 g80300 adenine nucleotidetranslocase


12 At3 g53130 cytochrome P450-like 0.936 0 63.5 v inner envelope?13 At4 g14210 phytoene dehydrogenase 0.329 0 62.9 v envelope + thylakoids14 At5 g59250f D-xylose-H+symporter-

like protein0.907 9 62.5 v envelope

15 At5 g64290f 2-oxoglutarate/malatetranslocator


16 At5 g12860f 2-oxoglutarate/malatetranslocator


17 At4 g31780f monogalactosyldiacylglycerolsynthase (MGDG)

0.895 0 58.5 v envelope

18 At5 g16150f sugar transporter-likeprotein (IEP62)

0.674 9-12 56.9 v envelope

19 At4 g15440 hydroperoxide lyase(HPL)-like protein

? 0 53 v outer envelope

20 At4 g30950f chloroplast omega-6 fattyacid desaturase (fad6)

0.853 2 51.2 v envelope

21 At1 g34430 dihydrolipoamideS-acetyltransferase

0.678 0 48.3 v envelope

22 At3 g60620 phosphatidatecytidylytransferase-like


23 At2 g05990 enoyl-ACP reductase 0.888 0 41.2 v envelope24 At2 g38550 putative nongreen plastid

inner envelopemembrane protein


25 At5 g05000f Toc34 0.429 1 34.7 v outer envelope26 At1 g09130 ClpP protease complex 0.863 0 33.9 v envelope + stroma27 At1 g02280 Toc33 0.445 1 32.9 v outer envelope28 At4 g33460 putative protein ABC-type

transporter0.754 2-4 29.8 v inner envelope?

a MIPS accession number. b TargetPv1.01 used to predict subcellular localization (i.e. chloroplasts, mitochondria, etc.). c Transmembrane (TM) domainpredictions were performed using TMHMMv2.0. d Molecular weights (MWs) shown are calculated values. e Localization assignments are derived from variousdatabase searches and published literature annotations. f Indicates proteins likewise identified by the method of Ferro et al.20



the EST frequency distribution of the proteins found by eitherOff-line MUDPIT or Gel-C-MS/MS. We defined low-abundanceproteins as having 0-5 ESTs. Over 80% of the proteinsidentified by Off-line MUDPIT had 0-5 ESTs per protein,whereas only 50% of the proteins from the Gel-C-MS/MSapproach had 0-5 ESTs per protein. Hence, the Off-lineMUDPIT method appeared to identify more low-abundanceproteins than did the Gel-C-MS/MS method. We concludedfrom this result that the SCX chromatography separation oftryptic fragments is definitely beneficial, resulting in morepeptides from low-abundance proteins being identified. Indeed,several studies have recommended that pre-fractionation of thesample by multidimensional chromatography or shallow-gradient RP-HPLC helps to resolve low-abundance proteinsaway from the bulk of the protein mixture prior to massspectrometry, thus increasing our chances of identify them (seeSimpson et al.36 for separation strategies for low-abundanceproteins).

A Case Study: Analyzing the Chloroplastic Import Ap-paratus using Gel-C-MS/MS and Off-line MUDPIT Tech-niques. The majority of cellular processes are performed bymultiprotein complexes. We are interested in applying variousmass spectrometry techniques as a means to identify andanalyze protein complexes associated with the envelope mem-brane. As an initial step toward achieving this goal, we utilizedthe sensitivity of both Off-line MUDPIT and Gel-C-MS/MS toanalyze the known components of the chloroplastic proteinimport machinery from a complex protein mixture. The pro-tein import apparatus is actually composed of two complexescalled Toc and Tic. The Toc complex is composed of thefollowing core components: Toc159, Toc75, and Toc34.37-43

Arabidopsis has two forms of Toc34 designated AtToc34 andAtToc33.6,44 The Tic complex is composed of AtTic110, AtTic20,and AtTic22.45-48 Recently, two additional, though tentative,components of the Tic complex have been identified, AtTic55and AtTic40.49-52 Finally two stromal chaperones, AtHsp93-Vand AtHsp93-III, complete the composition of the importapparatus.

The results of this analysis are presented in Table 6. All thecore components of the Toc complex were identified (i.e.,Toc159, Toc75, Toc34 and Toc33). Most of the components ofthe Tic complex were likewise identified (i.e., Tic110, Tic22,Tic40 and Tic55), only Tic20 was not.

We scrutinized the mass spectrometry data in greater detailto determine why Tic20 was not identified in this analysis. Itshould be noted that Tic20 is an integral protein of the innerenvelope membrane, with four predicted transmembranedomains (TMDs) (see Table 5), and is believed to form at leasta portion of the translocation channel of the inner mem-brane.47,48 In addition, Tic20, like other import components,has a relatively high pI (see Table 6). The simplest explanationfor the inability of either Off-line MUDPIT or Gel-C-MS/MS toidentify Tic20 may be related to how Tic20 behaves whentreated with the protease trypsin. For instance, when weperformed a computational trypsin digest on Tic20, approxi-mately 10 fragments were predicted to be generated (see Table7). Of these 10 fragments, ranging in molecular weight from0.7 to 3.9 kD, seven contain portions of a hydrophobic trans-membrane domain (see Table 7, gray shaded areas indicatehydrophobic domains). Given the large size and hydrophobicityof these hypothetical trypsin fragments, one would predict thatthey would resolve poorly if at all by SCX and RP columnchromatography. Likewise, MS/MS fragmentation of thesetryptic fragments would likewise be problematic due to theirlarge mass. Indeed, failure to identify Tic20 in our actualproteome study may have been a result of our failure to recover,elute or fragment authentically generated Tic20 tryptic frag-ments. By contrast, the component Tic22, a small protein thatis localized in the intermembrane space of the chloroplastenvelope and is peripherally associated with the inner envelopemembrane,47,48 generated five fragments upon trypsin digestionthat were readily identified by either Off-line MUDPIT or Gel-C-MS/MS approaches (see Table 7). Likewise, Tic55, an integralmembrane that has two predicted transmembrane domains,generated nine fragments that were identified by either of themass spectrometry approaches (Table 7). The two predictedTic55 trypsin fragments that included transmembrane domains(amino acid regions 312-341 and 501-523) were not identifiedin this study. The above case study illustrates the profound rolethat the biophysical properties of trypsin-generated peptide

Table 5. Various Assigned Functions of Envelope ProteinsIdentified in This Study


protein import components1 At4 g02510 chloroplast protein import Toc159-like2 At3 g46740 import-associated channel Toc753 At5 g05000 Toc344 At1 g02280.1 Toc335 At1 g06950 Tic 110 (Import Apparatus) 3′partial6 At4 g33350 Tic22-like protein7 At5 g16620 Tic40-like protein8 At2 g24820 putative Reiske iron-sulfur (Tic55)9 At5 g50920 Hsp93-V (ClpC)

10 At3 g48870 Hsp93-III (ClpC)

metabolite translocators1 At5 g19760 oxoglutarate/malate translocator2 At5 g46110 phosphate/triose-phosphate

translocator (IEP303 At1 g80300 adenine nucleotide translocase4 At5 g64290 2-oxoglutarate/malate translocator5 At5 g12860 2-oxoglutarate/malate translocator6 At3 g63410 putative chloroplast inner

envelope protein (IEP37)7 At1 g65260 inner envelope membrane

protein 30 kD

fatty acid metabolism1 At5 g35360 acetyl-CoA carboxylase2 At3 g11170 omega-3 fatty acid desaturase3 At5 g05580 temperature-sensitive omega-3

fatty acid desaturase4 At1 g24360 putative 3-oxoacyl-ACP reductase5 At1 g77590 putative acyl-CoA synthetase6 At4 g31780 monogalactosyldiacylglycerol

synthase (MGDG)7 At4 g30950 chloroplast omega-6 fatty acid

desaturase (fad6)8 At2 g05990 enoyl-ACP reductase

oxylipin metabolism1 At5 g42650 allene oxide synthase2 At4 g15440 hydroperoxide lyase (HPL)-like protein

sugar transporters1 At5 g59250 D-xylose-H+symporter-like protein2 At5 g16150 sugar transporter-like protein (IEP62)

ABC transporters with unknown function1 At5 g64940 ABC transporter-like protein2 At5 g03910 ABC transporter-like protein3 At4 g33460 putative protein ABC-type transporter

a MIPS accession number and MIPS annotation were used to assignproteins to various functional groups.



fragments have on our ability to subsequently identify andanalyze envelope-associated protein complexes by mass spec-trometry.

Discussion

Benefits of Using Multiple Mass Spectrometry TechniquesTo Analyze the Chloroplastic Envelope Membrane. Plantproteomics is still considered to be in its infancy whencompared with the proteomic analyses of prokaryotes, yeasts,and even humans. However, with improvements in massspectrometry and with the availability of large amounts ofsequence data from Arabidopsis, rice, maize, and other plantspecies, the opportunity to identify the complete chloroplastproteome is rapidly becoming a reality.53,54 Already, severalchloroplastic proteome investigations have been completed.10,17,18

More recently, a limited proteomic analysis of the integralmembrane proteins of spinach envelopes has been accom-plished.20 Likewise, the proteome of another plastid type, thewheat amyloplast, has also been studied.55 However, theidentification of the proteins that compose the chloroplasticenvelope proteome remains incomplete. We report here a firstdraft of the Arabidopsis chloroplastic envelope proteome usingmultiple mass spectrometry techniques.

With the completion of the Arabidopsis genome sequence,Arabidopsis has become the obvious choice for various plantproteomic projects.32,35,56 However, for biochemists and cellbiologists, Arabidopsis is a difficult plant with which to work,owing to its small size, which makes organelle purificationparticularly difficult. Even though Arabidopsis leaf tissue canbe harvested in significant amounts, the leaf cells are highlyvacuolated and the majority of the membranes present arethylakoid membranes. As a consequence, it has been estimatedthat envelopes constitute only 2% of the total membranes ofchloroplasts. Given these limitations, isolating Arabidopsischloroplastic envelope membranes in sufficient quantity andpurity proved both difficult and challenging. Certainly, althoughouter and inner envelope membranes can be readily obtained

from both peas and spinach, 25,57 both of these plants have onesignificant disadvantage when using them for proteomicanalysis; namely, they both have a limited amount of availableDNA and protein sequences. This limitation becomes a prob-lem when attempting to correlate tandem mass spectra withprotein and DNA sequences using software such as SEQUEST58

or Mascot.33 Hence, we used a novel chloroplast isolationprocedure24 to obtain sufficient amounts of purified Arabidopsischloroplasts from which mixed envelope membranes wereprepared and from which envelope membrane proteins weresubsequently identified by mass spectrometry.

As previously stated, we used both Off-line MUDPIT and Gel-C-MS/MS to analyze envelope membrane proteins. One benefitderived from using these two complementary approaches isthat they are both very sensitive techniques that do not requirean enormous amount of starting material. For instance, wewere able to identify 392 nonredundant proteins from merely200 µg of mixed envelopes. In a similar fashion, this technologyhas been applied to examine many other systems with thesimilar benefit of identifying a staggering number of proteinsin a very short period of time from a limited amount ofsample.22,23,59,60 For example, Koller et al.60 recently performeda systematic proteome analysis of rice leaf, root, and seed tissueusing two independent technologies, 2D gel electrophoresisfollowed by LC-MS/MS and MUDPIT, which resulted in theidentification of 2528 unique proteins. Likewise, Andon et al.55

performed an initial characterization of the wheat amyloplastproteome using both 1D and 2D electrophoresis followed byLC-MS/MS; they identified 171 novel proteins. In addition toidentifying a large number of envelope proteins, our proteomestrategy exploited the benefits of using a combination of SCXand RP chromatography prior to MS/MS to enhance our abilityto identify a wide range of diverse and novel envelopemembrane proteins (see Tables 3 and 4 and Figures 4 and 5).

Regardless of the rapid advances in mass spectrometrytechniques, one of the major challenges still facing manyproteomic projects in general is the resolution and identifica-

Table 6. Proteins of the Chloroplastic Import Apparatus Identified in This Study


chloroPb

score

TM

domainsc MWd (kD)

Off-line

MUDPIT

Gel-C-

MS/MS pIe localizationf

Toc components1 At4 g02510 chloroplast

protein importToc159-like

0 0 94.5 v (4.17) outer envelope

2 At3 g46740 import-associatedchannel Toc75

0.952 0 89.0 v v 9.10 outer envelope

3 At5 g05000 Toc34 0.429 1 34.7 v 9.42 outer envelope4 At1 g02280.1 Toc33 0.445 1 32.9 v 9.59 outer envelope

Tic components1 At1 g06950 Tic 110 (import

apparatus) 3′partial0.941 2 110.0 v v (5.70) inner envelope

2 At4 g33350 Tic22-like protein 0.679 0 30.1 v v 9.78 inner envelope3 At5 g16620 Tic40-like protein 0.856 1 48.9 v v 5.07 inner envelope4 At2 g24820 putative Reiske

iron-sulfur (Tic55)0.897 2 55.0 v v 8.87 inner envelope

stromal components1 At5 g50920 Hsp93-V (ClpC) 0.849 0 103.0 v v 6.59 inner envelope + stroma2 At3 g48870 Hsp93-III (ClpC) 0.575 0 105.0 v v 6.25 inner envelope + stroma

missing component1 At4 g03320 Tic20 0.611 4 32.5 9.75 inner envelope

a MIPS accession number. b TargetPv1.01 used to predict subcellular localization (i.e., chloroplasts, mitochondria, etc.). c Transmembrane (TM) domainpredictions were performed using TMHMMv2.0. d Molecular weights (MWs) shown are calculated values. e pI calculated value; pI listed in brackets are estimatesfrom the partial sequence. f Localization assignments are derived from various database searches and published literature annotations.



tion of very hydrophobic membrane proteins. Many proteomeprojects have traditionally relied on the use of 2D gel electro-phoresis followed by mass spectrometry to identify proteins.However, traditional 2D electrophoresis has many limitations,particularly when used to analyze hydrophobic membraneproteins.61,62 One benefit of using both Off-line MUDPIT andGel-C-MS/MS is that both of these techniques are compatiblewith hydrophobic membrane proteins and membrane proteinswith extreme pIs. To illustrate this advantage, Tables 3 and 4provide a small list of envelope membrane proteins identifiedin this study, showing the number of known or predictedhydrophobic transmembrane domains present in each protein.Using both Off-line MUDPIT and Gel-C-MS/MS we were ableto identify a variety of proteins having zero to fourteen potentialTMDs (see Tables 3 and 4 and also the Supporting Informa-tion). Interestingly, a similar type of result was observed in theanalysis of the Saccharomyces cerevisiae proteome by MUDPIT,whereby 131 proteins with three or more predicted TMDs weredetected and identified.22 However, the majority of proteinsidentified in this study did not contain a single predicted TMD.

Recently, various strategies have been developed to analyzeand identify “troublesome” integral membrane proteins con-taining multiple TMDs. For instance, Ferro et al.20 performeda limited analysis of the integral membrane proteins fromspinach envelopes using a novel strategy. In their study,purified envelope membranes were initially extracted with

organic solvent and separated by 1D gel electrophoresis,excised proteins were then subjected to tandem mass spec-trometry. Of the 54 proteins identified in their study, 27 weredesignated as new envelope proteins. Furthermore, most of theproteins they identified contained one to four predicted TMDs.Although, we did not extract envelope membranes prior tomass spectrometry analysis, many of the proteins identified inthis study were likewise identified by the novel method of Ferroet al.20 (see Tables 3, 4, and 5). Hence, the method of Ferro etal.20 both complements and enhances the results from ourmultiple mass spectrometry approach by providing additionalinformation on the physical properties of some of the envelopeproteins identified in our study.

Alternatively, Washburn et al.22 pursued a different approachand used a combination of formic acid and CNBr treatment todetect recalcitrant membrane proteins. In this strategy, formicacid was used to partially solubilize the membrane, while CNBrwas used to cleave off soluble portions of the integral mem-brane protein which were subsequently cleaved further bytrypsin prior to mass spectrometry. This multistep approachresulted in the identification of numerous hydrophobic mem-brane proteins containing multiple TMDs.22 Certainly, as wecontinue to further define the Arabidopsis envelope proteomein greater detail, the pretreatment of envelopes by various

Figure 4. Molecular weight distribution of the proteins found byOff-line MUDPIT and Gel-C-MS/MS approaches. The molecularweight in kD of proteins identified by either A, Off-line MUDPITor B, Gel-C-MS/MS from a single envelope preparation. Valueswere determined from the predicted sequences and the numberof proteins having molecular weights within the shown rangeswas plotted (9). In addition, the cumulative number was alsoplotted (-•-).

Figure 5. EST frequency distribution of the proteins found byOff-line MUDPIT and Gel-C-MS/MS approaches. The number ofmatching ESTs for each protein identified by either A, Off-lineMUDPIT or B, Gel-C-MS/MS from a single envelope preparationwere determined using the BLASTN program from NCBI. Thenumber of proteins having an EST number within the shownranges (9) and the cumulative number (-•-) are shown.



extraction techniques (i.e., organic solvent, high salt, sodiumcarbonate) prior to mass spectrometry will become increasinglynecessary if we are to improve our ability to identify recalcitrantmembrane proteins. Likewise, as the next phase of this en-velope proteome analysis develops, it will also be very insightfulto compare the protein composition of the envelope proteomeestablished in this study and the envelope proteome generatedafter membranes have undergone various extraction proce-dures.

Defining the Envelope Proteome and Developing an En-velope Proteome Database. The ultimate goal for any pro-teomic project is to elucidate the localization and function ofall the proteins identified by mass spectrometric analysis. Withregard to the envelope membrane, only a small fraction of theproteins localized to the envelope membrane have beenidentified and characterized.1,20 Consequently, searching theavailable databases to determine the identity or the functionof a newly identified envelope membrane protein has provendifficult. Further complicating the characterization of any newenvelope membrane protein is the lack of prediction programsavailable to determine whether a protein is targeted to the outerenvelope. It should be noted that many of the new envelopeproteins identified in this study had neither a predicted transitpeptide nor any identifiable transmembrane domain regions.Indeed, only 113 proteins of the 392 nonredundant proteinsidentified in this study contained a predicted or known TMD.It should not be too surprising then that the majority of theproteins identified in this study (63%) were assigned anunknown function (i.e., hypothetical, unknown or putativedesignation, see Figure 3A) after database analysis. Certainly,additional experiments will be required to confirm that theunknown proteins identified in this study are authentic enve-lope membrane proteins. Therefore, as a cautionary note theunknown proteins identified in this study should be viewed as

potential candidate envelope membrane proteins until furtherexperiments clarify their final subcellular localization. Interest-ingly, Koo and Ohlrogge14 recently constructed a computationalinner envelope proteome that relied on two selection criteria:the presence of a transit peptide and the presence of predictedmembrane-spanning domains. With this proteome, they wereunable to assign 71% of the candidate proteins to any knownfunction.14 Both our experimental approach and the compu-tational approach of Koo and Ohlrogge14 reflect the inherentdifficulty in studying and identifying proteins of the envelopemembrane. Nevertheless, the computational approach of Kooand Ohlrogge14 did identify 541 proteins as potential integralplastid inner envelope membrane proteins. Many of theenvelope proteins we identified by mass spectrometry (seeTables 3, 4, and 5) were likewise identified by the computationalapproach of Koo and Ohlrogge.14 For example, certain com-ponents of the protein import apparatus such as Tic110, Tic55,and Tic40, were identified by both strategies. Tic20, a compo-nent that was not identified by our mass spectrometry ap-proach (see Tables 6 and 7), was identified by the computa-tional approach of Koo and Ohlrogge.14 In addition, bothapproaches identified proteins involved in fatty acid metabo-lism and metabolite transport (see Figure 3 and Table 5).Finally, because the computational approach of Koo andOhlrogge14 focused primarily on the identification of integralmembrane proteins of the inner envelope, none of the Toccomponents of the import apparatus were identified in theiranalysis. In contrast, this study identified all of the corecomponents of the Toc complex (see Table 6). Regardless ofthese differences, the computational approach of Koo andOhlrogge14 complements our experimental analysis of theArabidopsis chloroplastic envelope membrane and likewiseprovides an additional resource to further define the envelopeproteome.

Table 7. Tryptic-Generated Polypeptides from Protein Import Components Identified by Mass Spectrometry

no. identified polypeptidea

molecular

weight (kD)b

polypeptide

length

residue

positionc

Off-line

MUDPIT

Gel-C-

MS/MS

polypeptide fragments from Tic551 K.NVPEDAPLGLTVYDR.Q 1.7 15 95-109 v v2 K.LSEGQLIDGR.L 1.1 10 135-144 v v3 R.LECLYHGWQFEGEGK.C 1.8 15 144-158 v4 K.IPQLPASAK.I 0.9 9 163-171 v5 K.REDAQPLVFEVTER.S 1.7 14 255-268 v v6 R.FDAPCVLQNNR.E 1.3 11 291-301 v7 K.VPPVVEHAPAGLIAALSASYPAK.G 2.2 23 422-444 v v8 K.GGIGTMHAPNLANR.Y 1.4 14 445-458 v9 R.AINLNTNNFIR.T 1.3 11 533-543 v

polypeptide fragments from Tic221 K.SGTPTTTLSPSLVAK.A 1.5 15 63-77 v v2 R.QEDAEAFLAQAR.L 1.3 12 112-123 v3 K.VVPITLDQVYLLK.V 1.5 13 135-147 v4 R.GDQQIMVGSLEDVL.R 1.7 14 120-133 v v5 R.SYAQHMQDLIK.E 1.5 11 256-266 v

hypothetical trypsin-generated fragments from Tic20d

1 R.EIAPLSATASVDFAAAATSSNQLFANGLPPLAPGLR.R 3.9 36 59-942 R.PIEPAR.V 0.7 6 98-1033 K.IAERPEWWWR.T 1.2 10 118-1274 R.TLACVPYLISLQISDVGFYVQPFLEK.H 2.9 26 127-1545 K.HDAIGDMIYFIPGAINR.W 1.9 17 153-1696 R.WPTWFFMVYCYLGYMWVVK.N 2.1 19 170-1887 K.ELPHYLR.F 0.7 7 191-1988 R.FHMMMGMLLETALQVIWCTSNFFPLIHFK.G 3.2 29 198-2269 R.FGMYYWMAIGFTYICLLLECIR.C 2.4 22 229-250

10 R.CALAGVYAQIPFMTDAASIHTLFNLGGFQR.P 3.3 30 251-280

a Fragments were identified by Off-line MUDPIT and/or 1D-PAGE GEL-C-MS/MS as described in the Material and Methods. b Calculated value from peptidefragment. c Residue position based on published sequence (see Table 5 for MIPS identification number). d Tic20 was subjected to a computational trypsindigest and the fragments predicted are listed. Trypsin-generated fragments containing a transmembrane domain are highlighted by gray shading.



Envelope Proteome Phase II: Strategies To Identify ProteinComplexes Associated with the Envelope Membrane. Onceall proteins of the envelope proteome have been identified, thenext major challenge will be to determine how these proteinsare organized into functional protein complexes. Mass spec-trometry is already being applied to analyze the compositionof large protein complexes from various organisms.21,63-65 Theidentification and characterization of protein complexes as-sociated with the chloroplastic envelope membrane is in itsinfancy. To address this deficiency, both biochemical andgenetic approaches in combination with mass spectrometry willbe needed to identify the components of these novel com-plexes.

To this end, we utilized two mass spectrometry approachesto identify known components of the chloroplastic proteinimport apparatus (see Tables 6 and 7). During this analysis,however, we discovered that one component, Tic20, was notreadily identified by either Off-line MUDPIT or Gel-C-MS/MSapproaches (see Table 7). To discover the reason(s) for thisresult, we performed a computational trypsin-digest on Tic20and examined the hypothetical trypsin-generated polypeptidesderived from digested Tic20. From these hypothetical frag-ments, we were able to develop a strategy that could be appliedin future investigations to facilitate the identification of Tic20in a protein complex. For example, in the case of Tic20, mostof the predicted tryptic peptide fragments conveniently containa methionine residue. Consequently, cyanogen bromide cleav-age of Tic20 could have been performed prior to trypsindigestion, resulting in peptide fragments that are more ame-nable to MS/MS fragmentation (see Table 7). From this smallcase study, it is apparent that creative strategies will be requiredto isolate and identify all of the protein complexes associatedwith the envelope membrane.

For example, novel genetic approaches have been used quitesuccessfully to isolate and characterize protein complexes froma variety of organisms.64,65 In these approaches, a gene-specificcassette containing a specific tag is inserted into either anorganism or a plant by various transformation methodologies.Subsequently, tagged protein complexes are affinity purifiedand analyzed by mass spectrometry. Although many proteintags are available (i.e., FLAG, Myc, His, and so forth), one noveltag, called TAP-tag, has become very useful. For instance,tandem-affinity purification (TAP) has recently been used toisolate protein complexes from whole cells.64,65 Likewise, Gavinet al.64 use TAP and mass spectrometry to characterize multi-protein complexes in Saccharomyces cerevisiae. They defined232 distinct multiprotein complexes and proposed new cellularroles for 344 proteins, including 231 proteins with no previousfunctional annotation.64 Because Arabidopsis plants are easilytransformable, the TAP tag approach may offer researchers aconvenient means of isolating numerous protein complexesfrom various subcellular compartments, including the envelopemembrane. Regardless of the strategy employed, the combina-tion of mass spectrometry and affinity chromatography willcertainly provide researchers a unique opportunity to furtherexpand the envelope proteome by identifying the many novelcomplexes associated with the envelope membrane.

Concluding Remarks

The characterization and identification of hydrophobicmembrane proteins using mass spectrometry requires creativestrategies. In this study, we investigated and attempted todefine the chloroplastic envelope proteome by comparing two

complementary mass spectrometry-based methodologies. Theseapproaches allowed us to identify and characterize a large setof putative membrane proteins localized to Arabidopsis thalianachloroplastic envelope membrane. This high-throughput pro-teomic study of the envelope membrane culminated in theidentification of approximately 392 nonredundant proteins.Regardless of the progress made by various chloroplasticproteome studies, many challenges remain. For instance,strategies will be required to further characterize the majorityof envelope membrane proteins with no known function.Likewise, this particular study provides only an initial draft ofthe total envelope membrane proteome of Arabidopsis; ad-ditional strategies will be needed to further resolve the envelopeproteome into its outer and inner envelope constituents.

Acknowledgment. We wish to thank Dr. KennethKeegstra for allowing portions of this project to be performedin his lab at the MSU-DOE Plant Research Laboratory. Wewould like to thank The Center for Plant Products & Technolo-gies at Michigan State University for its support of this project.We would also like to thank David Tabb at The ScrippsResearch Institute for all of his help with DTASelect. Thisresearch was supported in part by the Cell Biology Program ofthe National Science Foundation (No. MCB-9904524) and theMichigan Life Sciences Corridor Core Technology Alliance,Michigan Proteome Consortium.

Supporting Information Available: The table ofputative envelope membrane proteins identified in this studyby either Off-line MUDPIT or Gel-C-MS/MS. This material isavailable free of charge via the Internet at http://pubs.acs.org.

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