Identification of Novel Centrosomal Proteins in Dictyostelium d

Identification of Novel Centrosomal Proteins in Dictyostelium discoideum by Comparative Proteomic Approaches. Yvonne Reinders, Irene Schulz,† Ralph ...
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Identification of Novel Centrosomal Proteins in Dictyostelium discoideum by Comparative Proteomic Approaches Yvonne Reinders, Irene Schulz,† Ralph Gra1 f,† and Albert Sickmann* Protein Mass Spectrometry and Functional Proteomics Group, Rudolf-Virchow-Center for Experimental Biomedicine, Julius-Maximilians-University Wuerzburg, Versbacher Strasse 9, 97078 Wuerzburg, Germany Received October 18, 2005

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidatess currently subject of further investigationssfor the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome. Keywords: centrosome • dictyostelium discoideum • relative quantification • mass spectrometry

Introduction During interphase, the nucleus of an animal or fungal cell is associated with a single centrosome, a nonmembranous organelle that represents the main microtubule-organizing center in the cell.1 Additionally, it plays an important role in cell cycle control during mitosis. Since perturbation of core centrosomal and centrosome-associated proteins is linked to cell-cycle misregulation and potentially to cancer,2 there is increasing interest in determining the protein composition of this organelle. The latter is a prerequisite for the understanding of its function, which seems to be very similar in different eukaryotic organisms although centrosomal morphologies in animals, fungi and Dictyostelium are strikingly diverse.3 Centrosomes are the largest known protein complexes with a size of several hundred nanometers and are composed of more than a hundred different proteins.4,5 Comparison of the protein inventory of ultrastructurally very different centrosome types, such as centriole-containing centrosomes of animals, acentriolar centrosomes of Dictyostelium and spindle pole bodies in yeast, now offers the chance to identify the key-centrosomal proteins required for the general and conserved centrosome functions. Despite its size the analysis of centrosomal proteins is complicated by several problems. Since a normal eukaryotic * To whom correspondence should be addressed. Protein Mass Spectrometry and Functional Proteomics Group, Rudolf-Virchow-Center for Experimental Biomedicine, Julius-Maximilians-University Wuerzburg, Versbacher Strasse 9, 97078 Wuerzburg, Germany, Tel.: +49 931 201 48730, Fax: +49 931 201 48123. E-mail: [email protected]. † Irene Schulz and Ralph Gra¨f’s address is Adolf-Butenandt-Institute for Cell Biology, Ludwig-Maximilians-University Munich, Schillerstrasse 42, 80336 Munich, Germany. 10.1021/pr050350q CCC: $33.50

 2006 American Chemical Society

cell contains a single centrosome during interphase the copy number of most centrosomal proteins per cell and their corresponding mRNA-levels are usually very low.3 Therefore, high amounts of starting material are required to purify centrosomes for proteomic studies. Sequences encoding centrosomal proteins are usually underrepresented in cDNA libraries3 and may, therefore, not be included in databases used for MS/MS spectra interpretation. Moreover, the dynamic behavior of the centrosome during cell cycle6 hampers the identification of protein components. Since many proteins are merely needed for centrosome duplication or spindle organization they are recruited to the centrosome for a limited period of time. Therefore, absolute purification of this huge, low-abundant and also highly dynamic protein complex is rather difficult. Additionally, some of the already known constituents are very large as for instance DdCP224 or DdDhcA thereby interfering with proteomic analysis. Distinction between contaminants and genuine complex constituents is usually not possible in later analysis stages using current separation and detection methods. Therefore, differential proteomic approaches have to be applied using samples of specifically enriched and depleted fractions of the respective protein complex. In contrast to the protein profiling data of human centrosomes generated by Andersen et al.,7 this work aims at the application of techniques allowing the relative quantification of centrosome-enriched and centrosome-depleted proteins in order to circumvent false positives caused by contaminations. Therefore, the proteins of two centrosomedepleted fractions (NC1 and NC2) are compared to those of a centrosome-enriched fraction (C). Two alternative strategies such as 16-BAC-PAGE (16-benzyldimethyl-n-hexadecylammoJournal of Proteome Research 2006, 5, 589-598

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research articles niumchloride-polyacrylamid gel electrophoresis) and 2D-PAGE (two-dimensional polyacrylamid gel electrophoresis) are both based on differential image analysis of expression patterns. Classical 2D-PAGE8 followed by mass spectrometric identification of the proteins represents a standard approach for differential proteome studies. Although 2D-PAGE features both high resolution and the capability to separate protein isoforms, this technique is known to be less suitable for hydrophobic, highly acidic and alkaline as well as very small and large proteins.9 These limitations can be overcome by parallel application of further two-dimensional gel electrophoresis methods such as 16-BAC/SDS-PAGE.10 In contrast to classical 2D-PAGE this established technique uses ionic detergent based systems both in first and in second dimension. Thereby, protein solubilization especially for hydrophobic proteins is increased and precipitation effects are diminished. Since the resolution of 16-BAC/SDS-PAGE is usually inferior to classical 2D-PAGE, MALDI mass fingerprinting may not be sufficient for protein identification and nano-LC-MS/MS is recommended. Additionally, the DIGE (differential gel electrophoresis) technology was applied for both gel systems. To detect proteins that cannot be separated using gel-based techniques LC-based methods may be applied as third strategy. Since LC-MS/MS approaches are not suitable for direct quantification,11 stable isotope labeling techniques such as iTRAQ (isobaric Tagging for Relative and Absolute Quantitation)12 are necessary to enable MS-based quantification of peptides. The label binds specifically to all accessible amino groups of peptides. Since each label reagent has the same total mass, a single distinct signal is obtained for the comparative peptides in mass spectrometry. Upon fragmentation, different reporter ions are obtained due to the stable isotopes within the label so their individual peak areas can be used for quantification. Thus, a correlation between the peak areas of the reporter ions and the ratio of the respective protein’s concentrations within the samples can be concluded. In this work, a combination of these alternative techniques is used to provide an unbiased overview of centrosomal protein components providing a future basis for understanding its biogenesis and function.

Experimental Procedures Chemicals. All chemicals, the CyDyes and materials for 2DPAGE were purchased from GE Healthcare, Freiburg, Germany. The iTRAQ labeling reagents were supplied by Applied Biosystems, Darmstadt, Germany. Sequencing grade modified trypsin was obtained from Promega, Mannheim, Germany. All other chemicals and HPLC-solvents were acquired from Merck KGaA, Darmstadt, Germany. Isolation of Dictyostelium discoideum Centrosomes. Centrosomes were purified according to a modified protocol,13 which was based on the original isolation method of Gra¨f et al.14,15 Briefly, ∼8 × 109 sedimented cells were lyzed by Triton X-100 treatment, and nuclei with attached centrosomes were pelleted by ultra-centrifugation at 80 000 × g for 40 min in a sucrose solution. Centrosomes were separated from the nuclei by treatment with pyrophosphate buffer and shear forces being the key in the isolation procedure. After two ultra-centrifugation steps using sucrose density gradients and DNase treatment, highly enriched centrosomes were sedimented by centrifugation at 17 500 × g for 20 min. After each sucrose density gradient centrifugation, a noncentrosomal fraction was collected (NC1 and NC2, respectively), which consisted of 1 mL 590

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of the separated proteins that were fractionated directly after the centrosomal fraction, i.e., at lower sucrose densities. Immunoblot Analysis. Protein concentrations were determined using a BCA-assay (Pierce, Bonn, Germany). Equal protein amounts from the centrosome-enriched and -depleted fractions were separated by 1D-PAGE and transferred onto nitrocellulose membrane (Hybond ECL, GE Healthcare) by semi-dry blotting. Detection of the proteins was performed using the SuperSignal West Pico Chemiluminescent Kit (Pierce, Bonn, Germany) with an 1:8000-fold dilution of the polyclonal anti-gamma-tubublin antibody, no dilution for anti-DdCP224 and the anti-filamin described in ref 16, and a dilution of 1:8000 of the secondary anti-rabbit HRP-conjugated anti-IgG antibody (Sigma-Aldrich, Munich, Germany). X-ray films were exposed to the immunoblot for one minute and developed with the X-OMAT 1000 X-ray developer (Kodak, Stuttgart, Germany). 2D-PAGE. The protein content of each sample was determined using a BCA-assay (Pierce). Equal amounts of both purified centrosomes and proteins of the centrosome-depleted fractions were separated by 2D-PAGE. Therefore, isoelectric focusing was performed using the Ettan IPGphor II IEF System (GE Healthcare) according to the manufacturer’s instructions. IPG strips (24 cm, pH 3-10, nonlinear, GE Healthcare) were incubated in 450 µL reswelling buffer overnight (7 M urea, 2 M thiourea, 2% CHAPS, 2% carrier ampholytes pH 3-10, 30 mg/mL DeStreak (GE Healthcare)). The proteins were solubilized in 60 µL reswelling buffer and loaded on IPG strips using sample cups at the acidic end of the IPG strip. Before transferring IPG strips onto 12.5% SDS polyacrylamide gels, proteins were reduced using 65 mM DTT and alkylated by 280 mM iodoacetamide. Electrophoresis was performed using the EttanDalt system (GE Healthcare). Finally, gels were subjected to silver staining according to Blum et al.17 For image analysis, gels were scanned with standardized parameters and gel images were processed by the Proteomweaver Software (Definiens AG, Munich, Germany). Spots solely present in the centrosomeenriched 2D-gel and spots with an increased volume (intensity integrated by area) of a factor g 1.5 were assigned and excised for subsequent MS analysis. This rather low factor was chosen because not all centrosomal components are expected to be enriched as high as the major constituents such as gammatubulin or DdCP224. All analyzed spots were verified manually to avoid false positives derived from the imaging software. 16-BAC/SDS-PAGE. 16-BAC/SDS-PAGE separation was performed as described previously.18 Briefly, the first dimension was accomplished using a 10% 16-BAC gel, pH 2.1 for approximately 60 min at 200 V. Afterward, the excised gel lanes were rebuffered for 20 min in 100 mM Tris, pH 6.8, and incubated for 15 min in reducing SDS-sample buffer (65 mM DTT in 2× NuPAGE sample buffer). Proteins were separated in the second dimension using 12% SDS-Tris-Glycine gels (Invitrogen, Karlsruhe, Germany). Gels were silver stained using the protocol of Blum et al.17 Image analysis was done analogue to the analysis of the 2D-PAGE. DIGE of Centrosome-Enriched and -Depleted Proteins. For 2D-PAGE and 16-BAC/SDS-PAGE equal protein amounts of centrosome-enriched and -depleted proteins were individually tagged using one of the three fluorescent CyDyes according to the manufacturer’s instructions. Both separations were performed in the dark to minimize photobleaching of the dyes. Gels were documented using a Typhoon 9400 scanner (GE Healthcare). Images were processed by the ImageQuant soft-

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ware (GE Healthcare). Up-regulated spots of the centrosomeenriched fraction were excised and peptides resulting from tryptic in-gel digestion were subjected to nano-LC-ESI-MS/ MS. iTRAQ Labeling. Peptide labeling with iTRAQ reagent was performed according to the manufacturer’s instructions and propositions (addition of CHAPS and urea enhancing protein denaturation). Briefly, equal protein amounts of the corresponding samples (50 µg each) were dissolved in 20 µL of 0.5 M triethylammonium bicarbonate, pH 8.5, 0.1% CHAPS, 0.2 M urea and 0.1% SDS; iTRAQ reagents were resolved in 70 µL ethanol. The samples were reduced for 60 min at 37 °C using DTT, alkylated for 10 min at room temperature using iodoacetamide and digested with trypsin overnight. Afterward, each sample was incubated with a different iTRAQ reagent for 1 h. After pooling the samples, the mixture was separated directly by cation exchange chromatography according to Wagner et al.19 Before nano-LC-MS/MS each fraction was concentrated in a vacuum centrifuge to diminish the acetonitrile concentration. In-Gel Digestion and Peptide Extraction for MS. Sample preparation of all spots was performed according to the protocol of Shevchenko et al.20 Briefly, gel spots were washed alternately twice for 10 min with 50 mM NH4HCO3 and 25mM NH4HCO3 in 50% acetonitrile. In case of 16-BAC/SDS-gels samples were reducted using 10 mM dithiothreitole and alkylated with 5 mM iodoacetamide and then a final washing step as described above was applied. The protein spots were vacuum-dried prior to tryptic digestion. Gel pieces were rehydrated with 12.5 ng/µL trypsin in 50 mM NH4HCO3. Incubation was performed overnight at 37 °C. The resulting peptides were extracted by addition of 15 µL 5% formic acid for 10 min. Additionally, for distinct proteins tryptic digests were performed in 50% H218O according to Schnolzer et al.21 in order to distinguish between b- and y-ions in fragment spectra facilitating de-novo-sequencing using MALDI-MS as additional ionization method. These results were used for confirmation of the proteins identified by LC-MS/MS-measurements of the 2D-gel spots. LC-MS/MS-Analysis. The extracted peptides were separated using reversed phase liquid chromatography coupled to ESIMS. The sample was preconcentrated on a C18 precolumn (C18 PepMap RP, 300 µm ID × 1 mm, particle size 5 µm, Dionex, Idstein, Germany) for 5 min at a flow rate of 25 µL/min using an HPLC system consisting of a Famos autosampler, a Switchos microcolumn switching module and an Ultimate micropump (all Dionex). Afterward, peptides were eluted and separated on a C18 main column (C18 PepMap RP, 75 µm ID × 150 mm, particle size 3 µm, Dionex) by a binary gradient composed of solvent A (0.1% formic acid) and solvent B (0.1% formic acid in 84% acetonitrile). Solvent B was increased linearly from 5% to 50% within 30 min, then to 95% for 10 min, and afterward the column was reequilibrated to 5% solvent B. The flow rate was set to 250 nL/min. Peptides were directly eluted into an ESI-mass spectrometer. For mass spectrometric analysis an ion trap LCQ Deca XPplus (Thermo Electron, Dreieich, Germany), a Quad-TOF QStarXL or a linear-iontrap QTrap 4000 (both Applied Biosystems, Darmstadt, Germany) was used. MSacquisition duty cycle was set up with 1 s survey scan and three dependent scans (each approximately 1 s) when using an ESIion-trap mass spectrometer or 1 s survey scans and two independent scans, each 3 s, for the ESI-Quad-TOF-MS.

Figure 1. Western blot analysis of centrosome-enriched and centrosome-depleted fractions (∼10 µg each). The presence of two known centrosomal proteins, DdCP224 and gamma-tubulin, and one cytosolic protein were proved by immonublot analysis. The centrosome-enriched fraction C contains DdCP224 in contrast to the depleted fractions NC1 and NC2; also gamma-tubulin is perceptibly enriched. As presumed, the cytosolic protein filamin is not a constituent of fraction C but present in the centrosomedepleted fraction NC2.

Database Search. Mass spectra were transformed into peak lists as dta- or mgf-format using two in-house software solutions wiff2dta22 and raw2dta, respectively. The default parameters for generating mgf- or dta-files were applied (minimum of 35 signals per spectrum, mass range 400-2,000 m/z). Generated data was processed using the search algorithms SEQUEST (version 27, Thermo Electron)23,24 and MASCOT (version 2.05, Matrix Science, London, UK).25 Database searches were accomplished using the dictyBase database (version October 2004, http://www.dictybase.org).26 Fixed modification carbamidomethylation of cysteines was selected and oxidation of methionine as an optional modification. As further parameters tryptic digest and up to one missed cleavage was considered for the search algorithms. For the iTRAQ experiments, side reactions with tyrosine were also taken into account. Doubly and triply charged ions were considered, respective mass tolerances were set individually for each instrument (0.4 Da for linear ion trap; 1.5 Da for ion trap; 0.2 Da for ESI-TOF-MS). As filtering criteria for SEQUEST only peptide hits with a minimum cross-correlation factor of 2.5, a ∆CN of 0.25 and a preliminary ranking of 1 were accepted. For MASCOT the minimum score was set to 35 for each peptide. All spectra were verified manually in order to avoid false positive hits derived from the search algorithms. Furthermore, each protein identification was based on at least 2 validated MS/MS-spectra.

Results Enrichment of Centrosomes. After the isolation of nucleus/ centrosome complexes in the first step of centrosome preparation, our approach revealed a further enrichment of centrosomes compared to the centrosome-depleted fractions. The enrichment of the two known centrosomal proteins DdCP22427-29 and gamma-tubulin30 is shown by immunoblotting (Figure 1). In the centrosome-depleted fraction either a weak signal or no signal of anti-gamma-tubulin antibody is detectable. The weak signal in NC1 most likely resulted from co-purified, noncentrosomal gamma-tubulin that makes up the majority of the cellular gamma-tubulin.31 Furthermore, the cytoskeletal, noncentrosomal protein filamin could only be detected in centrosome-depleted fraction NC 2. Centrosomal enrichment was monitored by electron microscopy and indirect immunofluorescence microscopy in addition to the reported western blots.14The analysis of centrosomal proteins separated by one-dimensional SDS-PAGE followed by nano-LC-MS/MS displayed the co-purification of several ribosomal proteins. In Journal of Proteome Research • Vol. 5, No. 3, 2006 591

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Figure 2. (A) ∼80 µg proteins from a centrosome-enriched fraction from Dictyostelium discoideum were separated by 2D-gel electrophoresis, silver stained according to Blum et al.15 and protein expression levels were compared to respective levels on gels from centrosome-depleted fractions. A representative gel image is depicted, with spots enriched in the centrosomal fraction numbered; respective mass spectrometric identifications are listed in Table 1. (B) Enrichment of CABP in the centrosome-enriched fraction (spot 3). Several centrosome candidate proteins such as CAPB were identified to be enriched with a factor g 2. C: area in a 2D-gel of a centrosome-enriched fraction C. NC1: area in a 2D-gel of centrosome-depleted fraction NC1. NC2: area in a 2D-gel of centrosomedepleted fraction NC2. (C) Enrichment of gamma-tubulin in the centrosome-enriched fraction (spot 4). The known centrosomal protein, gamma-tubulin, was identified which was enriched by a factor of nearly 5 in the centrosome-enriched fraction. C: area in a 2D-gel of a centrosome-enriched fraction C. NC1: area in a 2D-gel of centrosome-depleted fraction NC1. NC2: area in a 2D-gel of centrosomedepleted fraction NC2.

addition, a complete removal of actin-associated proteins could not be achieved although their amount was significantly reduced. Since full depletion of contaminants in purified centrosomes was not possible, the centrosome-enriched fraction had to be quantified in comparison to the centrosomedepleted fractions. Therefore, orthogonal quantification strategies were applied such as iTRAQ, differential 2D-PAGE and differential 16-BAC/SDS-PAGE. Separation of Centrosomal Components by 2D-PAGE. The separation capacities provided by one-dimensional systems are usually not sufficient for differential analysis because proteins have to be completely separated for comparative image analysis. Therefore, the application of multidimensional separation strategies using orthogonal systems is mandatory. Furthermore, the use of additional separation strategies enables access to various protein species.32 592

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The differential annotated centrosomal spots of the classical 2D-PAGE are shown in Figure 2 by a representative gel. Accordingly, 17 spots were analyzed and identified (Table 1). The proteins varied between 150 and 24 kDa in size with pI values ranging from 4.8 to 7.9. The known centrosomal protein gamma-tubulin as well as several previously unknown centrosomal candidate proteins such as CABP33 were identified. The protein identities were initially obtained by nano-LC-MS/ MS and confirmed by MALDI-MS/MS using de-novo-sequencing (Figure 3). Sequencing was facilitated by introduction of one or two 18O-atoms upon tryptic digest in 50% H218O at the C-terminus of peptides, resulting in a characteristic doublet isotope pattern of y-ions. Thereby, differentiation between yand b-ion patterns is easily possible. In total, 10 proteins were identified using IEF/SDS-PAGE, thereof five unknown proteins and the known centrosomal protein gamma-tubulin.

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Figure 3. Example of MALDI-MS (A) and MALDI-MS/MS (B) spectra using de-novo-sequencing. Panel A shows the fingerprint spectrum of the CABP1 protein of spot 3 from a classical 2D-PAGE. In panel B the fragment ion spectrum of m/z of 1522.763 is depicted (LVFAVTIHQPELR). The protein spot was digested in trypsin-solution containing 50% H218O. Due to the incorporation of one or two 18O-atoms at the C-terminus of the peptide, a differentiation between y- and b-ion patterns is facilitated. In panel C a zoom-in of the MS/MS spectrum is depicted showing typical y- and b-ion patterns. Table 1. All Identified Differential Protein Spots of the 2D-PAGE spot no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

protein name

mass in Da

pI

sequence coverage (%)

accession no.

CABP1-related protein p31/p34 CABP1-related protein p31/p34 CABP1-related protein p31/p34 gamma-tubulin ubiquitin-conjugating enzyme E2 CABP1-related protein p31/p34 CABP1-related protein p31/p34 CABP1-related protein p31/p34 GTP-binding nuclear protein RAN/TC4 DDB0204561 GTP-binding nuclear protein RAN/TC4 DDB0184253 heat shock protein DDB0206211 DDB0206211 DDB0206211 DDB0190611 DDB0206211 DDB0206211 DDB0190611

30922 30922 30922 52234 66063 30922 30922 30922 24047 31439 24047 39926 45669 149065 149065 149065 113274 149065 149065 113274

5.89 5.89 5.89 5.44 5.06 5.89 5.89 5.89 6.96 5.56 6.96 7.87 6.92 4.78 4.78 4.78 6.44 4.78 4.78 6.44

8.7 8.7 39.7 22.1 73.0 15.0 13.2 11.8 58.0 40.6 34.4 13.9 17.5 1.5 7.3 8.9 4.2 4.8 9.8 4.8

DDB0185023 DDB0185023 DDB0185023 DDB0185068 DDB0218828 DDB0185023 DDB0185023 DDB0185023 DDB0215409 DDB0204561 DDB0215409 DDB0184253 DDB0215016 DDB0206211 DDB0206211 DDB0206211 DDB0190611 DDB0206211 DDB0206211 DDB0190611

Separation of Centrosomal Components by 16-BAC/SDSPAGE. In addition to 2D-PAGE, 16-BAC/SDS-PAGE was used as further separation method to increase the number of identified genuine centrosomal proteins. In contrast to 2DPAGE, 16-BAC/SDS-PAGE is less biased against separation of

hydrophobic or basic proteins. Five spots were analyzed and identified, thereof the known centrosomal protein spindle pole body component 98 (Table 2). In addition to these classical image analyses, proteins were labeled with different CyDyes and separated together on a Journal of Proteome Research • Vol. 5, No. 3, 2006 593

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Table 2. All Identified Differential Protein Spots of the 16-BAC/SDS-PAGE spot no.

1 2

3 4 5

protein name

mass in sequence Da coverage (%)

heat shock protein 90 80094 DDB0191699 65450 DDB0189463 145021 spindle pole body 94947 component 98 DDB0206211 149065 DDB0186471 35611 DDB0188601 21984 DDB0186471 35611 GTP-binding nuclear 24218 protein RAN/TC4 CABP1-related protein p24 31036

accession no.

3.00 18.00 9.00 1.00

DDB0191163 DDB0191699 DDB0189463 DDB0191482

15.00 57.00 32.00 12.00 17.00

DDB0206211 DDB0186471 DDB0188601 DDB0186471 DDB0215409

21.00

DDB0185023

To explain the shift in the molecular weight it has to be noticed that for the DDB0189463 protein only peptides of C-terminal part of the protein could be identified.

Figure 5. Display of a 16-BAC/SDS-PAGE labeled using CyDyes. Cy2 (blue) labels the centrosome-enriched fraction, Cy3 (green) and Cy5 (red) the centrosome-depleted fractions 1 and 2, respectively.

Figure 4. (A) Display of a 2D-PAGE labeled using CyDyes. Cy3 (green) labels the centrosome-enriched fraction, Cy2 (blue) and Cy5 (red) the centrosome-depleted fractions 1 and 2, respectively. (B) Silver stain from the DIGE-labeled gel in panel A. Only centrosome-enriched spots as judged by manual inspection of the DIGE-gel were annotated and excised for further analysis.

single 2D-gel or 16-BAC/SDS-gel, respectively. Proteins that were only displayed in the centrosome-enriched fraction but not in the depleted fractions were considered as centrosomal. 21 differential protein spots were identified using DIGE tech594

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nology, thereof 18 with 2D-PAGE (Figure 4) and three with 16BAC-SDS/PAGE (Figure 5). There is an overlap of eight proteins identified with and without DIGE labeling. Nevertheless, 14 proteins were only displayed using the DIGE technology leading to the assumption that image analysis using DIGE may be slightly more sensitive than classical image analysis. Since the variances of conditions during gel electrophoresis are minimized separating all samples in a single gel, fluorescence labeling of the different samples simplifies differential image analysis, especially for 16-BAC/SDS-PAGE. In total, 28 proteins could be identified by all gel based separation approaches, including three of 10 known centrosomal and 14 hypothetical proteins. Analysis of the Centrosomal Proteome Using the iTRAQ Methodology. Introduced by Ross et al.12 iTRAQ represents a relative quantification technology using stable isotope labeling. For this work, centrosome-enriched and depleted fractions were differently labeled and relatively quantified (Figure 6). Thereby, 59 proteins could be identified as centrosomeenriched proteins which varied between 8 and 245 kDa in size. Furthermore, only significantly enriched proteins, that mean proteins enriched by a factor of at least 1.5, were considered to be genuine centrosomal proteins. Thereby, the maximal error of the quantification was generally below 25%. Proteins identified with a Pro QUANT confidence factor (also see manufacturer’s instruction Pro QUANT) of less than 98 or less than two manually verified spectra were discarded. In comparison to gelbased analysis, a larger number of proteins with a size below 15 kDa were displayed (15% of all LC-analyzed proteins). Moreover, 49 proteins were only identified using the iTRAQ technology. Furthermore, two proteins, DDB0206211 and DDB0191988, could only be identified by the gel-based approaches while no significant changes were obtained using the iTRAQ approach. Thus, only one isoform of each of the proteins is enriched in the centrosomal fraction whereas the total amount of all isoforms of these proteins (as measured by the iTRAQ approach) remains constant. Hence, merely one isoform each may be a centrosomal component. Altogether, we identified 76 different centrosome-enriched proteins, thereof 39 unknown proteins. To increase the reliability of the identifications all experiments were repeated three times with independently prepared samples. Table 3 gives an

Identification of Novel Centrosomal Proteins

Figure 6. Example of an MS/MS spectrum using iTRAQ. The fragment ion spectrum of a peptide (VCENIPIVLCGNK) of the GTP-binding nuclear protein Ran is depicted. The enlarged picture shows the 8-fold enrichment of the protein in the centrosome-enriched fraction labeled with iTRAQ116. Thereby, the centrosome-depleted fractions NC1 and NC2 were labeled with iTRAQ114 and iTRAQ117. The peak at m/z 115 is resulted by slight contaminationy of the heavy iTRAQreagents with lighter isotopes.

overview of all identified proteins including the respective identification technique.

Discussion In this study, we present the first comprehensive proteome study on centrosomes of Dictyostelium discoideum. Thereby, an easily transferable strategy to distinguish genuine protein complex constituents from contaminations is established. Recently, several different approaches for identification of the complete centrosomal, mitotic spindle, or basal body inventory by mass spectrometry-based proteomic analysis were demonstrated.34-39 The first differential proteome approach using relative quantification on centrosomes isolated from a human cell line was published by Andersen et al.7 A protein correlation profiling of the human centrosome was performed to exclude contaminating proteins. Therefore, different fractions of an ultra-centrifugation gradient were measured by LC-MS/MS and XICs (extracted ion chromatograms) of peptides of known centrosomal proteins were used for the generation of protein elution profiles. The elution profiles of the other identified proteins were compared to the profiles of the known centrosomal components and similar profiles were used as an indicator for centrosomal localization. This approach resulted in a rather large dataset of novel centrosomal proteins giving the opportunity screening for homologue proteins in other organisms. However, definition of criteria which have to be met by centrosomal candidate proteins to be categorized as genuine centrosomal components is still a major problem. Unfortu-

research articles nately, there is no clear structural border of this organelle. Therefore, the localization of a part of the proteins identified by the protein correlation profiling approach has been shown by fluorescence microscopy of tagged proteins. To circumvent the initial need for localization studies a strategy using relative quantification was applied in this work analogue to Andersen et al.7 However, in contrast to the protein profiling technique image analysis of 16-BAC/SDS-PAGE and conventional 2D-PAGE as well as an iTRAQ approach were performed. Due to the application of three different approaches using relative quantification 76 centrosomal proteins, thereof 73 novel centrosomal proteins, could be detected. Analysis of Centrosomal Components Using Three Orthogonal Approaches. Altogether, 22 centrosomal proteins in Dictyostelium discoideum, 20 of them previously unknown in centrosomes, were identified with both 2D-PAGE techniques. Thereby, 10 proteins were identified using the classical 2DPAGE and 18 proteins by the DIGE approach. Additionally, two known centrosomal proteins were analyzed such as DdCP224 and gamma-tubulin. Orthologues of the newly identified proteins include GTPase Ran3 and HSP90.40 The small GTPase Ran is known to be related to the centrosome in several organisms and is required for nuclear import but also fulfills functions at the centrosome.41 The heat shock protein HSP90 is an integral component of the centrosome of Drosophila melanogaster,40 so a similar function of the heat shock protein DDB0215016 may be assumed. In Drosophila melanogaster, the chaperone HSP90 is required for the stability of the centrosomal protein Polo kinase and it is possible that several other centrosomal proteins also require this chaperone for their stability.42 Using 16-BAC/SDS-PAGE 10 centrosomal proteins in Dictyostelium discoideum, thereof eight unknown as centrosomal proteins so far, were analyzed. With the complementary DIGE approach one additional protein was elucidated. 59 centrosomal proteins in Dictyostelium discoideum could be detected using stable isotope labeling, 25 of them unknown in centrosomes so far. In total, 76 proteins were identified using all three techniques. Comparison of the Different Methods. Regarding the different data sets obtained from all three quantification techniques, we found 22 proteins from 2D-PAGE compared to 10 hits from 16-BAC/SDS-PAGE and 59 proteins from the stable isotope labeling approach. It has to be noticed that the protein ratios of the centrosome enriched compared to the depleted fractions varies in range for all used techniques. In addition to the unknown centrosomal proteins, three known proteins were identified such as DdCP224, spindle-polebody 98 and gamma-tubulin. Further seven protein constituents of the centrosome are known which have not been identified in this study. This may partially be due to their low abundance or only temporary recruitment to the centrosome but also limitations of the applied methods (proteins of high molecular weight in 2D-PAGE, fragmentation behavior in tandem mass spectrometry, etc.) cannot be excluded. 12 Proteins (16%) were identified by more than one analysis strategy. In particular, a large number of small or basic proteins could be only displayed by the iTRAQ method presumably due to the application of multidimensional liquid chromatography rather than gel-based technologies. This emphasizes the fact that each technique is preferably suited for different protein species and produces a different protein subset. Several homologous proteins such as tubulinJournal of Proteome Research • Vol. 5, No. 3, 2006 595

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Reinders et al.

Table 3. All Identified Protein Spots Which Were Enriched in the Centrosome-Enriched Fraction expression factors of identification methods

protein name

21D7 2-oxoglutarate dehydrogenase E2 component alpha tubulin beta tubulin CABP1-related protein p31/p34 Calreticulin precursor CCT chaperonin gamma subunit. centromere/microtubule binding protein CBF5 CG11796 protein clathrin heavy chain coactosin, cyclic AMP-regulated protein conserved hypothetical protein DD-1 protein DEAD-box protein abstrakt dipeptidyl-peptidase II precursor fttB, 14-3-3 gamma tubulin GTP-binding nuclear protein RAN/TC4 GTP-binding protein Sar1A guanine nucleotide Exchange Factor guanine nucleotide-binding protein beta subunit-like protein heat shock cognate protein Hsc70-2 heat shock factor heat shock protein heat shock protein 90 interaptin kinesin-related motor protein Eg5 1 M04G12.2 protein major vault protein-alpha microtubule-associated protein CP224 microtubule-associated protein-like (AT5g57210/MJB) orfSGP pkiA polyubiquitin PSMD1 protein (Fragment) putative nuclear transport factor similar to nuclear transport factor 2 RE62270p spindle pole body component 98 ubiquitin ubiquitin ligase subunit SKP1 ubiquitin/actin fusion protein 2 ubiquitin-conjugating enzyme E2 DDB0167169 DDB0168319 DDB0169112 DDB0184253 DDB0186471 DDB0187447 DDB0188190 DDB0188601 DDB0188657 DDB0188920 DDB0188978 DDB0189463 DDB0189496 DDB0189501 DDB0189754 DDB0189869 DDB0190611 DDB0190801 DDB0191699 596

mass in Da

2D-PAGE

56201 47634

6.59 9.19

DDB0219362 DDB0230198

1.6 2.8

50961 51336 31036 47163 58601 60804

5.34 5.21 5.89 4.61 6.41 9.45

DDB0191380 DDB0191169 DDB0185023 DDB0191384 DDB0204641 DDB0204297

5.8 3.2 3.4

41607 193601 16004

5.85 5.45 5.25

DDB0231604 DDB0185029 DDB0215369

34966 14683 76532 57262 27757 49427 24218 21215 166712 36573

6.24 5.14 6.38 4.60 4.78 5.44 6.96 7.78 8.95 7.64

DDB0168077 DDB0219436 DDB0187443 DDB0188558 DDB0190707 DDB0185068 DDB0215409 DDB0229965 DDB0217753 DDB0185122

69791 40090 45668 80094 161809 143433 32662 94131 225581 70846

5.47 4.57 6.92 5.02 6.26 9.03 5.15 6.05 8.53 5.62

DDB0185047 DDB0186469 DDB0215016 DDB0191163 DDB0191136 DDB0201557 DDB0185484 DDB0191259 DDB0189914 DDB0190669

32470 14055 34212 107939 14318

6.36 9.43 7.76 6.01 5.56

DDB0214941 DDB0216234 DDB0184145 DDB0187705 DDB0167060

101510 94947 42822 18718 15018 66063 91901 33533 37387 39926 35611 205051 172400 21984 115507 55905 16012 145021 19472 33269 24547 50815 113274 73672 65450

5.83 5.81 7.00 4.74 6.09 5.06 6.97 7.72 6.02 7.87 10.68 4.99 5.44 8.91 8.00 5.46 5.16 5.40 6.90 5.76 6.71 5.87 6.44 4.64 8.84

DDB0188394 DDB0191482 DDB0214921 DDB0191107 DDB0188457 DDB0218828 DDB0167169 DDB0168319 DDB0169112 DDB0184253 DDB0186471 DDB0187447 DDB0188190 DDB0188601 DDB0188657 DDB0188920 DDB0188978 DDB0189463 DDB0189496 DDB0189501 DDB0189754 DDB0189869 DDB0190611 DDB0190801 DDB0191699

3.2 3.2

conv.

5.0

DIGE

iTRAQ

accession no.

Journal of Proteome Research • Vol. 5, No. 3, 2006

DIGE

16-BAC/SDS-PAGE

pI

9.0

conv.

4.8

4.3 2.1 1.8 9.1 7.7 8.5 3.0 6.9 1.7 2.5 1.6 1.5

4.9 5.3

4.9

7.1 1.9

2.7 1.8

4.5 4.5

1.7 1.9 2.2 12.1

5.5 2.8 7.1 1.8 4.3

3.0 2.3 5.3 6.3 3.2 2.4 4.7

2.0

3.0 6.2

1.6 2.1 3.1 2.8 3.2

3.0 3.3 4.3 10.2 1.5

1.6 2.7 1.7 2.0 1.6

1.5 11.1 2.1 18.0 1.7 2.8 10.5 36.4

13.8 2.2 1.8

1.5

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Identification of Novel Centrosomal Proteins Table 3. (Continued)

expression factors of identification methods 2D-PAGE

protein name

mass in Da

pI

accession no.

DIGE

DDB0191988 DDB0192051 DDB0192069 DDB0202301 DDB0203397 DDB0204075 DDB0204076 DDB0204561 DDB0205913 DDB0206211 DDB0206256 DDB0206497 DDB0216598 DDB0217404 DDB0218175 DDB0219564

248529 68636 33448 33802 56745 31247 31216 31439 104482 149065 107773 8531 17971 30869 71118 80231

4.60 5.16 9.56 8.28 5.56 5.89 5.70 5.56 4.97 4.78 6.56 7.97 7.65 11.2 9.00 5.22

DDB0191988 DDB0192051 DDB0192069 DDB0202301 DDB0203397 DDB0204075 DDB0204076 DDB0204561 DDB0205913 DDB0206211 DDB0206256 DDB0206497 DDB0216598 DDB0217404 DDB0218175 DDB0219564

7.2 2.6

alpha and tubulin-beta were also detected by the dataset of the human centrosome confirming the results of both studies. Future studies will investigate in the interaction partners of several selected proteins. Obviously, not all known centrosomal proteins can be identified by a 2D-PAGE based approach due to the known limitations of this method such as precipitation of very large or hydrophobic proteins as well as gel-to-gel variances. These problems can partly be overcome by application of the DIGE technology due to parallel separation of all samples within a single gel. On the other hand only lysine-containing proteins are susceptible for CyDye-labeling and introduction of the label may cause shifting of the proteins, particularly in the lower molecular weight range, hence hampering protein identification.43 LC-based approaches are known not to suffer from biases against distinct classes of proteins, since they are not prone to typical electrophoretic limitations such as protein precipitation.11 Transferring the separation task from proteinto peptide-level comes along with an increase in sample complexity countervailing the superior resolution of LC-based peptide- against gel-based protein-separations. Furthermore, the iTRAQ-label, required for relative quantification by mass spectrometry, is introduced at a rather late stage of the analysis.11 Thereby, specific protein loss prior to pooling of the respective samples cannot be fully excluded and the identification of an utmost complete protein inventory of an entire cell organelle by a single proteomics approach is rather difficult. Moreover, there is probably no point of time when all proteins were expressed simultaneously in a detectable concentration. An analysis of centrosomes in an arrested cell cycle may yield additional information. Furthermore, some proteins will still escape detection by the available separation and identification methods. Nevertheless, the combination of orthogonal methods in this work results in an increase of the number of known centrosomal proteins from 10 to 76 in Dictyostelium discoideum. Therefore, the newly identified candidate proteins may provide novel targets for functional research granting deeper insight into composition and function of centrosomes.

Conclusion For this work, different separation and relative quantification strategies including techniques such as iTRAQ and DIGE were

16-BAC/SDS-PAGE

conv.

DIGE

iTRAQ

conv.

1.3 2.3 13.3 2.3 11.2 24.0 3.5

3.1

3.8

3.2

2.1 1.1 3.0 5.2 2.4 1.5 1.8 2.6

established to distinguish between centrosomal and contaminating proteins. More than 70 new candidates for the protein inventory of the Dicytostelium centrosome were identified. However, the combination of orthogonal methods in this work results in an increase of the number of known centrosomal proteins from 10 to 76 in Dictyostelium discoideum. Thereby, a deeper insight into composition and function of centrosomes may be facilitated soon.

Acknowledgment. We thank Dr. Kai Stu¨hler and Prof. Dr. Helmut E. Meyer, Medical Proteom-Center, Ruhr-University of Bochum, for providing the fluorescence scanner Typhoon (GE Healthcare). This work was supported by the DFG (Deutsche Forschungsgemeinschaft; FZT 82; SI 835 1-1, GR 1642/11). References (1) Doxsey, S.; McCollum, D.; Theurkauf, W. Centrosomes in Cellular Regulation. Annu. Rev. Cell Dev. Biol. 2005. (2) Badano, J. L.; Teslovich, T. M.; Katsanis, N. The centrosome in human genetic disease. Nat. Rev. Genet. 2005, 6, 194-205. (3) Gra¨f, R.; Daunderer, C.; Schulz, I. Molecular and functional analysis of the dictyostelium centrosome. Int. Rev. Cytol. 2004, 241, 155-202. (4) Kalt, A.; Schliwa, M. Molecular components of the centrosome. Trends Cell Biol. 1993, 3, 118-128. (5) Keller, L. C.; Romijn, E. P.; Zamora, I.; Yates, J. R., 3rd; Marshall, W. F. Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr. Biol. 2005, 15, 1090-1098. (6) Gra¨f, R.; Brusis, N.; Daunderer, C.; Euteneuer, U.; et al. Comparative structural, molecular, and functional aspects of the Dictyostelium discoideum centrosome. Curr. Top Dev. Biol. 2000, 49, 161-185. (7) Andersen, J. S.; Wilkinson, C. J.; Mayor, T.; Mortensen, P.; et al. Proteomic characterization of the human centrosome by protein correlation profiling. Nature 2003, 426, 570-574. (8) Westermeier, R.; Postel, W.; Weser, J.; Gorg, A. High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients. J. Biochem. Biophys. Methods. 1983, 8, 321-330. (9) Santoni, V.; Molloy, M.; Rabilloud, T. Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-1070. (10) Hartinger, J.; Stenius, K.; Hogemann, D.; Jahn, R. 16-BAC/SDSPAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal. Biochem. 1996, 240, 126-133. (11) Putz, S.; Reinders, J.; Reinders, Y.; Sickmann, A. Mass spectrometrybased peptide quantification: applications and limitations. Expert Rev. Proteomics 2005, 2, 381-392.

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