Caenorhabditis elegans Proteome during ... - ACS Publications

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Dynamic Changes of the Caenorhabditis elegans Proteome during Ontogenesis Assessed by Quantitative Analysis with 15N Metabolic Labeling Kerstin E. Geillinger,*,†,# Katja Kuhlmann,‡,# Martin Eisenacher,‡ Helmut E. Meyer,‡ Hannelore Daniel,† and Britta Spanier† †

ZIEL Research Center of Nutrition and Food Sciences, Abteilung Biochemie, Technische Universität München, Gregor-Mendel-Strasse 2, 85350 Freising, Germany ‡ Medizinisches Proteom-Center, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany S Supporting Information *

ABSTRACT: The development of the nematode Caenorhabditis elegans is a highly dynamic process. Although various studies have assessed global transcriptome changes, information on the dynamics of the proteome during ontogenesis is not available. We metabolically labeled C. elegans by using 15N ammonium chloride as a precursor in Escherichia coli feeding bacteria grown in minimal media as a new cost-effective technique. Quantitative proteome analysis was performed by LC−MS/MS in animals harvested at different times during ontogenesis. We identified and quantified 245 proteins at all larval stages in two independent replicates. Between larval stages (20 and 40 h after hatching) 61 were found to change significantly in level. Among those ribosomal proteins, aminoacyl tRNA synthetases and enzymes of energy metabolism increased in abundance, while extracellular matrix proteins and muscle proteins dominated groups displaying reduced levels. Moreover, changes observed for selected proteins such as VIT-6 and SOD-1 matched with previously published findings confirming the validity of our approach. The metabolic labeling technique applied seems well suited to assess changes in the proteome changes of C. elegans in a quantitative manner during larval development. The data set generated provides the basis for further exploitation of the role of individual proteins or protein clusters during ontogenesis. KEYWORDS: Caenorhabditis elegans, larval development, 15N metabolic labeling, nanoHPLC−ESI−MS/MS, quantitative proteomics

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INTRODUCTION Embryogenesis, growth, development, and aging are dynamic processes that are tightly regulated by various signaling cascades that change gene expression, protein biosynthesis, and degradation in order to coordinate the development of the biological system. The pattering in Caenorhabditis elegans embryonic and postembryonic development for example is mainly regulated via the LIN-12/Notch pathway by mediating cell−cell interactions (for review see refs 1 and 2). In adulthood and in the aging trajectory, the insulin/IGF-1 signaling cascade (IIS) is crucial for controlling metabolism and lifespan (for review, see ref 3). Its downstream target, the FOXO class transcription factor DAF-16, critically controls lifespan via changes in gene expression of a multitude of proteins.4 Functional genomics studies in C. elegans have assessed changes in the transcriptome during embryogenesis, larval development, and adulthood over eight defined developmental stages5 and depending on sex.6 In addition, large-scale RNA interference (RNAi) approaches were employed to define gene functions by observed phenotypic changes.7,8 Although mRNA levels may partially reflect the programming of the processes that mediate growth and development in an organism, they cannot a priori © 2012 American Chemical Society

be taken as a measure of protein abundance and function in the biological system. Comprehensive analysis of the protein complement of the transcriptome is more difficult to achieve. One of the few examples in C. elegans research is the work by Tabuse et al. (2005), which explores changes in the proteome during the larval development using 2D-DIGE with a special focus on the transition from embryo to L1 larvae.9 This approach delivered 165 proteins that were present in all six developmental stages (egg, L1-L4 larvae and adult). Also using 2D gel analysis but in combination with MALDI-TOF analysis, stage-specific proteome markers were identified by Madi et al (2003).10 Of 55 protein entities defined by mass spectrometry only 9 proteins were shown to be differentially expressed resulting in at least one specific marker protein per larval stage (except for L2 larvae). LC−MS/MS based analysis platforms in proteomics in contrast to gel-based technologies have generally higher resolution and also allow protein categories such as membrane associated, hydrophobic, and/or transmembrane proteins to be Received: April 23, 2012 Published: August 17, 2012 4594

dx.doi.org/10.1021/pr300385v | J. Proteome Res. 2012, 11, 4594−4604

Journal of Proteome Research

Article

Protein Extraction

resolved. Further improvements are achieved by in vivo labeling with stable isotopes by incorporation into proteins allowing easy quantification. The first metabolic labeling approach in C. elegans was reported by Krijgsveld et al. in 2003,11 and to date there are five studies that employed 15N-labeling techniques in C. elegans. Venable et al. (2004) introduced a novel approach for protein analysis via tandem mass spectra reflecting complex peptide mixtures which were validated using 15N-labeled C. elegans.12 Drosophila melanogaster and C. elegans were used by Gouw et al. (2008) as model organisms to improve identification and quantitation for metabolic labeling approaches with varying degrees of labeling.13 Dong et al. (2007) reported on a list of target proteins of the IIS pathway in worms with quantitative analysis using metabolic labeling.14 The most recent study by Tops et al. (2010) compared the proteome of hermaphrodites and male worms.15 These studies have used commercially available 15N-enriched media as their basis for the complete labeling of the feeding bacteria Escherichia coli OP50. Growing sufficient quantities of worms under these conditions is expensive and even more so when stable-isotope labeled amino acids are used (SILAC methods). Very recently Fredens et al. (2011) and Larance et al. (2011) published such approaches with one study using isotope-labeled heavy lysine in nuclear receptor 49 knockout animals16 while the other study used lysine and arginine to assess heat-shock responses.17 However, a quantitative profiling of the proteome during the ontogenesis of C. elegans as asked for by Tabuse et al. (2005) has not yet been delivered.9 To better understand the molecular processes underlying the developmental changes, we employed a metabolic labeling method with minimal media containing 15N ammonia chloride for growing the feeding bacteria and labeling of C. elegans. Quantitative proteome analysis was performed via LC−MS/MS for selected time points during ontogenesis, providing a novel data set for a higher eukaryotic organism which may serve as reference material in developmental research.

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Nematodes were harvested and extensively washed with M9 buffer and the resulting pellet was disrupted using glass beats in combination with mechanical force (3 × 30 s, level 5, FastPrep, MP Biomedicals, Germany). To avoid proteolysis, lysis buffer (100 mM Tris/HCl pH7.4, 200 nM NaCl, 2 μM EDTA, 8% glycerol, 1.25 mM DTT) containing 1 mM PMSF was used and all steps were performed on ice or 4 °C. The whole worm lysate was centrifuged in order to remove cell debris (1 min, 10000g at 4 °C) and the supernatant was used for further protein analysis. Western Blot Analysis

Ten micrograms of protein extract was loaded onto a 10% SDS−PAGE. Western blot analysis was conducted with minor changes as previously described by Benner et al. (2011).19 Membrane was blocked for 1 h at room temperature using 1% BSA solution in PBST. Primary antibodies were incubated overnight at 4 °C. Anti-SOD-1 (P34697) used at a concentration of 1:5000 was kindly provided by Valeria Cizewski Culotta. Anti-ATPase5a (Q9XXK1) used at a concentration of 1:500 was purchased from Abcam. As loading controls actin (anti-Actin; 1:5000) was detected after stripping the membrane using low pH (25 mM glycine-HCl pH 2, 1% SDS) and SDS−PAGE was stained with Coomassie Brilliant Blue. 2D Gel and MALDI-TOF Analysis

Protein Separation. Proteins were separated by twodimensional electrophoresis with minor modifications as described in ref 20. Briefly, before precipitation, using acetone, the protein solution was diluted with 1 vol. bidest water. Pelleted proteins were resuspended in a minimal volume of lysis buffer. To increase protein recovery, the solution was sonified. Remaining protein aggregates were removed by centrifugation. 250 μg of recovered protein was incubated with 24 cm IPG-strip (pH 3−10) overnight to allow reswelling. IEF was carried out at 4000 V gradually increasing for 1.5 h to 8000 V until 25000 V h were reached. After equilibration in buffer (6 M urea, 50 mM TrisHCl pH 8.8, 30% glycerol, 2% SDS) containing 1% DTT for 15 min, strips were incubated another 15 min in the same buffer then containing 4% IAA instead of DTT and subsequently placed onto a 12.5% SDS− PAGE. Separation was performed for the duration of 21 h at 144 mA. Proteins were fixed using acetic acid and ethanol before staining in Coomassie G250 for 4 days and destaining in water. Spots were randomly picked, and Coomassie fully removed and subjected to in-gel tryptic digest overnight. Extraction of resulting peptides was done by incubation with TA30 (30% ACN in 0.1% TFA) in combination with sonification as recommended by Bruker Daltonics. One microliter of sample or peptide calibration standard was placed on an 384 anchor chip steel target and allowed to cocrystallize with HCCA in TA30, before remaining salts were washed off using 1 μL of 10 mM ammonium hydrogen phosphate in 0.1% TFA. Mass spectrometry was conducted using Bruker Ultraflex3 in the positive ion reflection mode. Obtained mass spectra were processed using FlexAnalysis 3.3.65.0 software and protein identification was conducted using Biotools 3.2 in combination with MASCOT search engine (Database: Swissprot release 51.6 with species constraint on C. elegans (2998 sequences), peptide mass tolerance: ±150 ppm, max. missed cleavage of 1, fixed

MATERIAL AND METHODS

Bacterial Culture

Minimal medium was freshly prepared each time, containing 0.042 M Na2HPO4, 0.02 M KH2PO4, 0.009 M NaCl, 0.01 mM CaCl2, 0.002 mM MgSO4, 0.11 mM glucose, 0.18 mM uracil, and 0.02 M 15N- or 14N-containing NH4Cl. Bacterial culture of the feeding bacteria started from a glycerol stock of either 15Nor 14N-labeled E. coli OP50, and subsequently fresh overnight culture was used to inoculate larger volumes. Culture of C. elegans

Wild-type N2 (var. Bristol) were grown at 20 °C on agarose plates as described by Krijgsveld et al. (2003).11 Before bacteria were seeded onto plates, cells were pelleted and resuspended in about a third of the original volume. Synchronization of worms was conducted using hypochloride treatment as previously described.18 Briefly worms were washed off the plates and treated with hypochloride in combination with mechanical force to release eggs from adult worms. L1 larvae hatched overnight in M9 buffer and were placed on fresh agarose plates seeded with either 15N- or 14N-labeled E. coli OP50 the next day. Worms were harvested at 20, 40, and 60 h after L1 larvae were placed on corresponding plates. 4595



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engine (www.matrixscience.com).22 A decoy version of the database was used that was complemented with a duplicate of itself in which the amino acid sequence of each protein entry was randomly shuffled in order to enable the calculation of a false discovery rate. Search parameters were as follows: Tryptic specificity, maximum one missed cleavage site, oxidation of methionine as variable modification, precursor mass tolerance: 4 ppm, fragment mass tolerance: 0.5 Da. Protein identifications from Mascot were ranked by Mascot score and the lists were truncated at a false discovery rate of 1%. A minimum of 1 peptide per protein was required. For peptide and protein quantitation, the 15N metabolic method in the Mascot Distiller Quantitation toolbox was used. Impurity of labeling was set to 92%. Protein ratio was calculated based on the average of quantified peptides with at least two peptides meeting the selection criteria of a correlation threshold above 0.9 and a standard deviation below 0.2 whereby at least one peptide had to be unique to the protein.23 Lists of identified proteins were first merged for the two biological replicates and three time points, to give one combined list of identified proteins. Proteins that were not quantified in all the samples and in each replicate were eliminated from this list, as progression of protein abundances during the time course was the major focus (lists of proteins specific for time points can be extracted from the supplementary). Statistical analysis of the obtained data set was conducted using the free available R software package applying a one-way ANOVA following a Tukey Posthoc test with p < 0.05 set as significance threshold. Analysis of enriched GO terms was done using the Cytoscape (Smoote et al 2010) plugin BiNGO (Maere et al. 2005). The significance level was set to p < 0.01 as determined by hypergeometric test and Benjamini & Hochberg false discovery rate correction. For mapping of proteins to KEGG pathways, the “search & color pathway” tool on the KEGG Web site was used.24

modifications: carbamidomethyl (C), variable modifications: oxidation (M)). LC−MS/MS Analysis

Gel Electrophoresis and Tryptic Digestion of Proteins. For sample cleanup, 10 μg of total protein per sample were shortly subjected to one-dimensional SDS-polyacrylamide gel electrophoresis (1-D SDS-PAGE) using 4−12% NuPageTM Bis-Tris gradient gels (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions and visualized by colloidal Coomassie Brilliant Blue G-250. One slice per lane was cut out. Destaining, trypsin digestion, peptide extraction, and preparation for LC−MS/MS analysis were performed as described in ref 21. Peptide concentration after extraction was determined by amino acid analysis, and 300 ng total peptide per sample was used for LC−MS/MS analysis. NanoHPLC−ESI−MS/MS Analysis. Peptide mixtures were analyzed by nanoHPLC−ESI−MS/MS using the UltiMateTM 3000 RSLCnano system (Dionex, now Thermo Fisher Scientific, Bremen, Germany) online coupled to an LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany). Peptide mixtures were loaded onto a C18 RP precolumn (75 μm inner diameter × 20 mm; PepMap, Dionex) equilibrated with 0.1% (v/v) TFA, washed and preconcentrated for 7 min at a flow rate of 7 μL/min. The precolumn was then switched in line with a C18 RP nano LC column (75 μm inner diameter × 500 mm, 2 μm particle size; PepMap, Dionex). Peptides were separated with a binary solvent system consisting of 0.1% (v/v) FA (solvent A) and 0.1% (v/v) FA in 84% (v/v) ACN (solvent B) using the following gradient: 7−25% solvent B in 128 min, 25−40% B in 20 min, and 40−95% solvent B in 2 min. The flow rate was 400 nL/min and the column was heated to 40 °C. The LTQ-Orbitrap velos was equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) and distal coated SilicaTips (New Objective, Woburn, USA). To provide high mass accuracy, lock masses (derived from a set of distinctive air contaminants) were routinely used for internal calibration of each MS spectrum acquired. The general mass spectrometric parameters were as follows: spray voltage, 1.3− 1.5 kV, capillary temperature, 300 °C. For data-dependent MS/ MS analyses, the software XCalibur 2.1 (Thermo Fisher Scientific) was used. Full scan MS spectra ranging from m/z 300 to 2000 were acquired in the Orbitrap with a resolution of 30 000 at m/z 400. Automatic gain control (AGC) was set to 1 × 106 ions and a maximum fill time of 500 ms. The 20 most intense multiply charged ions were selected for fragmentation by low energy collision-induced dissociation (CID) in the linear ion trap. The AGC of the LTQ was set to 5000 ions and a maximum fill time of 120 ms. Fragmentation was carried out at a normalized collision energy of 35% with an activation q = 0.25 and an activation time of 10 ms. The ion selection threshold was set to 500. Fragmentation of previously selected precursor ions was dynamically excluded for the following 30 s.

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RESULTS AND DISCUSSION

Determination of 15N Labeling Efficiency Using QuantiSpec

To enable a quantitative analysis of the C. elegans proteome for different stages during larval development, we employed a costeffective variation of 15N metabolic labeling by using feeding bacteria E. coli OP50 grown in minimal medium with 15NNH4Cl as the nitrogen source.25 In a first set of experiments, the labeling rate of wild-type (WT) N2 was determined. Culture started with 10 L4 larvae placed on agarose plates seeded with a lawn of either 14N- or 15N-enriched bacteria. To ensure a high rate of labeling, C. elegans culture was conducted over three generations before nematodes were extensively washed and subsequently lysed as described in Material and Methods. Protein samples were combined 1:1 and 250 μg of the resulting protein mixture was separated on a 2D-gel. Eight spots were randomly picked covering a pI range from 4 to 8 and a mass range from 30 kDa to 100 kDa. After a tryptic digest of the protein spots peptides were extracted and analyzed using MALDI TOF/TOF following Mascot database search. Labeling rates of the respective peptides were determined using QuantiSpec,26 which was specifically developed for analysis of metabolically labeled protein data generated by a MALDI-TOF instrument. Protein identification was accomplished based on the 14N peptides, which were then used to calculate theoretical isotope distributions. Between 4 and 23 peptides per protein were subjected to quantification and resulted in an average

Data Analysis, Protein Identification, Quantitation and Statistics

Generation of peak lists from mass spectrometric data and the peptide/protein quantitation were done using Mascot Distiller version 2.4.0.0. Parameters for peak picking were set according to recommendations of the software supplier; briefly, the correlation threshold was set to 0.7 with a S/N of 2. Peak lists were correlated to the Uniprot/Swissprot database (Uniprot/ Swissprot release 2011_06) with taxonomy restricted to C. elegans (3332 sequences) using the Mascot (version 2.3) search 4596



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Table 1. Proteins Identified from 2D-PAGE and MALDI-TOF Analysis with the Corresponding Mascot Score and the Number of Peptides Used to Determine 15N Labeling Rate Using QuantiSpec protein heat shock protein 90 (abnormal dauer formation protein 21) heat shock 70 kDa protein C precursor vacuolar ATP synthase catalytic subunit A (EC 3.6.3.14) (V ATPase subunit A) (vacuolar proton pump subunit alpha) calreticulin precursor tropomyosin isoforms a/b/d/f (levamisole resistant protein 11) kinesin light chain (KLC) elongation factor 2 (EF 2) enolase (EC 4.2.1.11) (2 phosphoglycerate dehydratase) (2 phospho D-glycerate hydrolyase)

Mascot score

MW [Da]

pI

labeling rate [%]

# peptides

93 81 168

80689.00 73093.00 66874.00

4.96 4.1 5.08

96.1 88 91.9

15 4 23

87 169 60 118 59

45816.00 32984.00 60529.00 95477.00 46759.00

4.59 4.66 5.59 10.6 5.56

93.5 90.5 87.8 93.5 93.2

10 15 6 13 6

Figure 1. (A) Venn diagrams giving an overview of identified versus identified and quantified proteins. Only proteins identified in both independent biological samples were included. (B) Randomly picked L/H ratios of experiment 1 divided by L/H ratios of experiment 2 display reproducibility of experimental setup. (C) L/H ratios of experiment 1 plotted against L/H ratios of experiment 2 for each time point show a better reproducibility at later developmental stages. (D) Scatterplot of mean L/H ratios of 20 h vs 40 and 20 h vs 60 h illustrates large changes in the proteome while scatterplot of 40 h vs 60 h indicates rather stable protein abundances during this period of development.

resulting in 325 (20 h), 400 (40 h), and 370 (60 h) quantified proteins, respectively (Figure 1A). Plotting the L/H ratios obtained from experiment 1 against the values of the biological replicate (experiment 2) showed a high reproducibility of the chosen experimental setup (Figure 1B,C). Interestingly, variability between experiments was larger in the early stage of development, while in the later stages only protein abundances with high values displayed a wider distribution. This phenomenon might be explained by differences in the developmental processes, which seem to have a stronger impact in the early stages of development. Although the worm population was synchronized, the fertilization of eggs did not occur isochronously, adding further variation which probably aligns during development. The visualization of protein abundances of samples collected at 20 h against 40 and 40 h against 60 h shows that major changes take place during the early stages of development (Figure 1D), while the variation between 40 and 60 h is low.

labeling rate of 92% (Table 1). Therefore, a slightly diminished labeling rate (∼4%) and two times prolonged culturing time were accepted taking into account that costs for labeling are considerable lower (∼25%). Analysis of the Proteome during Larval Development

For the large-scale analysis of larval development wild-type C. elegans L1 larvae were placed on fresh plates seeded with 14Nbacteria, and after 20, 40, and 60 h, respectively, the nematodes were harvested, extensively washed, and snap frozen in liquid nitrogen. In order to compare all samples with each other we decided to use indirect quantification. For this purpose an internal standard was generated containing equal amounts of protein extract of every 15N-labeled sample to ensure integrity. After combining each sample with an equal amount of 15N standard, sample cleanup by a short SDS-PAGE and tryptic digestion was performed and extracted peptides were analyzed by LC−MS/MS. To minimize false-positive identifications, only proteins found in both replicates were considered. In total, 650 proteins were identified in both replicates, with 464 for the 20 h-time point, 556 proteins for the 40 h-samples and 501 proteins for the 60 h-samples (Figure 1A and Supplementary Table S1, Supporting Information). Quantification was done using the Mascot Distiller quantitation toolbox, since this software allows adjustment for incomplete labeling rate,

Protein Quantitation and Statistical Analysis of Differentially Expressed Proteins

Significance of regulation was tested employing a one-way analysis of variance with a subsequent Tukey posthoc test to distinguish between groups. In total, 61 proteins were found to be differentially expressed with p-values below 0.05 (Supple4597



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Figure 2. Validation of MS data using Western blot analysis. (A) Protein extracts of worm cultures either 20 or 40 h after hatching were immunoblotted and probed with antibodies against P34697 (SOD-1) and Q9XXK1 (ATP5a). Detection of actin and (B) Coomassie staining were used as loading control. (C) Corresponding fold changes obtained by MS are presented for each experiment for comparison.

Identification of Pathways Possibly Involved in Ontogenesis

mentary Table S2, Supporting Information). The posthoc test revealed 46 proteins to be differentially expressed between 20 and 40 h, between 20 and 60 h 52 proteins showed significant changes in abundance, while only 11 proteins were classified significantly changed between 40 and 60 h. These results confirmed evidence obtained from scatter plots (see Figure 1D) with major changes taking place during the early stages of development. This is in agreement with Madi et al. (2003) reporting the largest differences for the transition from the L1/ L2 to the L3 stage, which probably best accounts for the 20 and 40 h sample in our experimental set up.10 For selected proteins, our data confirm findings from earlier studies. Vitellogenin-6 (VIT-6, P18948), a yolk protein with tissue, sex, and stage specific expression patterns,27 had significantly elevated abundances between 20 and 60 h and 40 and 60 h, respectively (Supplemetary Table S1, Supporting Information). The monitored progression of VIT-6 abundance is in line with observations reported by Tabuse et al. (2005).9 They first describe a sharp decrease in yolk protein abundance after hatching followed by a gradual increase as the worm reaches adulthood. Interestingly, a second protein of the vitellogenin class, VIT-4, was found as a unique entity in the 20 h sample. Its abundance in both replicates was very low compared to the mean of the L/H ratios, giving rise to the speculation of a decrease in protein abundance after hatching comparable with the profile of VIT-6, but with no increase toward adulthood. Detected vitellogenin proteins in the 20 h sample are probably residues of maternally provided yolk proteins, as vitellogenin genes are not expressed during larval development.27 A second protein quantified and identified as significantly changed is the superoxide dismutase 1 (SOD-1, P34697). Here the protein level dropped by more than 50% between 20 and 40 h and remained unchanged between 40 and 60 h. SOD-1 was already classified as a stage specific marker protein during the first larval stage using 2D-gel analysis.10 It was speculated that the high abundance of SOD-1 in young larvae might result from increased expression induced by hypochlorite treatment of worms during synchronization. These results are in agreement with our results, as the protein levels show the same trends of change and the higher sensitivity of our LC−MS/MS based study allowed the detection of the lower levels in the 40 and 60 h samples. Further experimental validation was conducted applying Western blot analysis for SOD-1 and Q9XXK1 (ATP5a). Results of Western blot analysis confirmed differential expression for both proteins, with SOD-1 showing decreased levels at 40 h while levels of Q9XXK1 increased compared to 20 h after hatching (Figure 2).

First indications on biological relevance of differentially expressed proteins were obtained by grouping based on overrepresented gene ontology (GO) categories within the set of differentially expressed proteins using the Cytoscape plugin BiNGO. For the biological process ontology, the enriched terms fall into the main categories “growth”, “reproduction”, and “development” as well as “metabolic processes” including translation. The high number and significant enrichment of the proteins in the first category shows that our data set depicts indeed mainly proteins relevant for larval development. Growth and development require increased protein biosynthesis rates and increased energy production and this is reflected in the data set as well. Molecular function annotations show mainly enrichment in ribosomal proteins and general protein binding. Subcellular localization as determined by the cellular component part of GO highlighted again an enrichment of ribosomal components as well as cytoplasmic proteins (Figure 3 and Supplementary Table S3, Supporting Information). To graphically depict the involvement of ribosomal components, all identified ribosomal proteins were mapped to the KEGG ribosome reference pathway and the direction of regulation over time is indicated by color (Figure 4). In total 54 ribosomal proteins were identified and quantified, but interestingly only 13 of them showed a significant regulation over time. The abundance of 12 proteins increased between 20 and 40 h remaining at a constant level at later stages (marked in red in Figure 4), while only one of them (RPS-28) was downregulated as marked in blue. Regulation of Selected Ribosomal Proteins Suggest Functions beyond Protein Synthesis

Although the overrepresentation of ribosomal proteins among the up-regulated fraction is not surprising, it is noteworthy that some of the identified ribosomal proteins did not show alterations in level and stayed rather constant. Kamath et al. (2003) conducted a large scale RNAi screen on gene functions by observing reproducible phenotypes including “slow postembryonic growth”.28 Several of the investigated genes encode for ribosomal proteins. Eight of them where significantly upregulated during larval development (RPA-0, RPL-4, RPL-7, RPS-14, RPL-21, RPL-20, RPL-33, and RPL-36) but only two of them showed a slower postembryonic growth compared to control when silenced by RNAi (rpl-7 and rpl-33). As one would expect the same phenotype upon silencing the various ribosomal proteins if only the ribosome genesis and function would be affected, the phenotype based findings suggest extra4598



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phenotypes of knock-down of ribosomal genes were compared with the phenotype caused by knock-down of the gene of interest. Animals treated with RNAi for rpl-4 exhibited sterility and a small and sickly morphology classified as abnormal development. Although knock-down of five other ribosomal proteins resulted in the same phenotype (rpl-3, rpl-5, rps-2, rps7, and rps-9), we found no evidence that those proteins change their levels during the passage from larvae to adulthood. This may as well serve as an indication for extra-ribosomal functions and therefore different regulations during ontogenesis. Cluster Analysis Depicts Possible Coregulations

To get insights into potential coregulation of proteins, protein abundances were depicted in a heat map after being sorted according to the value at 20 h of life. As obtained from Figure 1D, major changes in protein levels occur during the progression from 20 to 40 h, while protein abundances of the 40 h sample match largely those determined in the 60 h sample. In addition, a hierarchical clustering was done to further visualize possible grouping of proteins depending on their progression over time (Figure 5). The analysis revealed four main clusters. The first two clusters show increases up to 40 h. While cluster 1 increases further with time, cluster 2 represents proteins with unchanged levels between 40 and 60 h. Proteins with decreasing levels during development are grouped in cluster 4. Cluster 3 includes six proteins that show a rather unique pattern. Abundance of VIT-6, FAR-2 (P34383) and CGH-1 (Q95YF3) is similar between 20 and 40 h followed by an increase in later larval developmental stages. In contrast, abundance of RPL-7 (O01802), RPS-14 (P48150), and ACO-1 (Q23500) culminates at 40 h. Especially FAR-2 and CGH-1 show a high correlation with only a slight increase in protein level from 20 to 40 h and a stronger increase from 40 to 60 h. As there are no other proteomics studies published that can be compared to our data set, we inspected transcriptomics data sets. The conserved germline RNA helicase 1 (CGH-1) is specifically expressed in the gonad with increasing mRNA

Figure 3. Over-represented gene ontology categories for significantly changed proteins. 61 differentially expressed proteins were used as input for the Cytoscape-Plugin BiNGO 2.44. Nodes show GO categories, with node size representing the number of protein hits and node color representing the p-value for enrichment. Redundant terms within one path and terms with less than three protein hits were removed for clarity.

ribosomal role or variable importance for ribosome function/ assembly for some of these proteins. The study by Kerins et al. (2010) describes the effects of pre-mRNA splicing on germline sex determination and the decision between mitotic and meiotic development.29 To ensure that the observed effects are not only mediated by decreased overall protein biosynthesis,

Figure 4. Detected and differentially expressed ribosomal proteins. All identified ribosomal proteins were mapped to the KEGG ribosome reference pathway. Boxes with green filling represent ribosomal C. elegans proteins. Red script: up-regulated, purple script: not regulated, blue script: downregulated proteins from 20 to 40 h after hatching. 4599



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Figure 5. Heat-map of mean L/H ratios. 61 proteins showing significant regulation after 1faq ANOVA are depicted in rows, time points 20, 40, and 60 h after hatching in columns. Hierarchical cluster analysis classifies four groups depending on their expression pattern. Group one stays stable after an increase at 40 h, while group two proteins rise only at 60 h. Proteins of category three show peak abundances at 40 h and class four proteins steadily decrease in abundance over time.

abundance toward young adulthood as shown by in situ hybridization.30 Also mRNA levels of far-2 at various larval stages31,32 correspond well to the protein pattern found in our study. FAR-2, a member of the fatty acid/retinol binding protein family that consists of eight proteins, was cloned and studied by Garofalo et al. and shown to bind retinol (Vitamin A1).31 Vitamin A1 is already known to be involved in spermatogenesis of C. elegans.33 Use of reporter constructs located expression of FAR-2 to body wall and vulva muscle cells and the increase in abundance of this protein during development might be explained with the differentiation of the vulva. Interestingly, a second member of the FAR-family, FAR-1 (P34382), showed differential expression as well, but was inversely related to FAR-2. This only partially correlates with mRNA expression data found in the literature,31 but here the exact sampling time is not described making a comparison unfeasible. Little is known about function, expression, and localization of FAR-1, and therefore the reciprocal changes in protein levels of FAR-1 and FAR-2 deserve further studies. For the second subgroup of proteins containing ribosomal proteins RPS-14 and RPL-7 and aconitase-1, literature searches did not retrieve any potential connection between the ribosomal proteins and ACO-1. However, the changes in protein levels of aconitase-1 are in accordance with earlier published data of Tabuse et al. (2005).9 We next classified changes in protein levels between the different time points for differential expressed entities by foldchanges with threshold values of ≤0.67 or ≥1.5. This resulted

in a list of 47 proteins (Table 2). In this way, gradually increasing protein levels which are only significantly changed between the end points of the analysis (20 and 60 h) and proteins with very small changes are excluded. Of those proteins, 25 were up-regulated between 20 and 40 h, with 14 proteins thereof involved in protein biosynthesis as either ribosomal proteins or aminoacyl tRNA synthetases. From 20 to 40 h 19 proteins were found to be down-regulated, and the corresponding functions are very different from those of the upregulated proteins with extracellular proteins (six) and muscle proteins (five) as the main categories. The extracellular aspartic protease ASP-6 was also identified at various larval stages in the 2D-gel approach of Tabuse et al. (2005), but contradictory to our data they did not detect any change over time.9 mRNA levels of asp-6 retrieved from data sets of McCarroll et al. (2004) and Jiang et al. (2000) were in accordance with the changes in protein levels we observed.6,34 The mRNA levels of all the identified members of the categories of extracellular and muscle proteins did not reveal a high correlation. While for two candidates (Y54G2A.23 and lev-11) no mRNA data are available, only three (far-1, mlc-3 and pcbd-1) of the remaining eight candidate proteins depicted mRNA expression patterns that appear to correlate to those observed here for the proteins. MIF-2 has previously been shown to increase about 100-fold during dauer formation,35 and therefore the strong decrease seen at 40 h might indicate favorable culture conditions preventing entry into dauer state. Tropomyosin (LEV-11) was as well detected by Tabuse et al. (2005) and Madi et al. 4600



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Table 2. Significantly Changed Proteins during C. elegans Larval Developmenta UniProt ID

Cosmid Number; Gene name

O01802 O02056 O18650 O44480 P34334 P48150 P49180 P49181 Q93572 Q94300 Q9N3 × 2 Q21276 Q9U1Q4 Q03577 P34575 Q23500 Q10663 P55954 Q9XXK1 Q19749 Q22633 P45971

F53G12.10;rpl-7 B0041.4;rpl-4 T05F1.3 rps-19 E04A4.8;rpl-20 C14B9.7;rpl-21 F37C12.9;rps-14 F10E7.7;rpl-33 F37C12.4;rpl-36 F25H2.10;rpa-0 T22F3.4;rpl-11.1 Y43B11AR.4;rps-4 K07C5.4 Y87G2A.5;vrs-2 B0464.1;drs-1 T20G5.2;cts-1 ZK455.1;aco-1;gei-22 C05E4.9;gei-7 Y37D8A.14;cco-2 H28O16.1 F23B12.5 T21C12.2;hpd-1 T09A5.11

Q10454

F46H5.3

Q18787 Q21215

C52E4.4;rpt-1 K04D7.1;rack-1

O01530 O76840 P34382 P34500 P55955 Q9N3B0 P53014 Q09665 Q18785 Q22866 Q9TZH6 O01805 P34697 P52013 Q09610 Q10121 Q22037 Q23680 Q95Y04

F21F8.7;asp-6 C37C3.6;mig-6;ppn-1 F02A9.2;far-1 K03H1.4;ttr-2 Y5F2A.1;ttr-16 Y54G2A.23 F09F7.2;mlc-3 ZK673.7;tnc-2 C52E4.2;mif-2 Y105E8B.1;lev11;tmy-1 T10B11.1;pcbd-1 C44E4.6;acbp-1 C15F1.7;sod-1 F31C3.1;cyn-5;cyp-5 R07B1.10;lec-8 C23G10.2 F42A6.7;hrp-1;rbp-1 ZK970.4;vha-9 Y41D4B.5;rps-28

P18948

K07H8.6;vit-6

protein name Up-regulated 40 versus 20 h after Hatching 60S ribosomal protein L7 60S ribosomal protein L4 40S ribosomal protein S19b 60S ribosomal protein L18a 60S ribosomal protein L21b 40S ribosomal protein S14b 60S ribosomal protein L35ab 60S ribosomal protein L36b 60S acidic ribosomal protein P0b 60S ribosomal protein L11 40S ribosomal protein S4 uncharacterized NOP5 family protein K07C5.4 valyl-tRNA synthetase aspartyl-tRNA synthetase, cytoplasmic probable citrate synthase, mitochondrial probable cytoplasmic aconitate hydratase bifunctional glyoxylate cycle proteinc cytochrome c oxidase subunit 5A, mitochondrial ATP synthase subunit alpha, mitochondrial pyruvate dehydrogenase complex component E2 4-hydroxyphenylpyruvate dioxygenase probable dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit probable arginine kinase F46H5.3 26S protease regulatory subunit 7 guanine nucleotide-binding protein subunit beta-2-like 1 Down-regulated 40 versus 20 h after Hatching aspartic protease 6b abnormal cell migration protein 6;papilin fatty-acid and retinol-binding protein 1b transthyretin-like protein 2 transthyretin-like protein 16 ARMET-like protein myosin, essential light chainb troponin C, isoform 2 MIF-like protein mif-2 tropomyosin isoforms a/b/d/f 4-alpha-hydroxy-tetrahydropterin dehydrataseb acyl-CoA-binding protein homologue 1 superoxide dismutase [Cu−Zn]c peptidyl-prolyl cis−trans isomerase 5 probable galaptin lec-8 UPF0076 protein C23G10.2 heterogeneous nuclear ribonucleoprotein A1 probable V-type proton ATPase subunit F 40S ribosomal protein S28 Up-regulated 60 versus 40 h after Hatching vitellogenin-6c b

FC 40−20 FC 60−40

category

3.38 2.77 2.08 3.39 3.98 3.22 3.07 5.17 2.24 2.70 2.85 7.28 4.57 3.76 3.56 3.17 2.97 1.51 3.26 2.24 3.24 4.95

0.78 0.93 1.13 0.95 0.88 0.53 0.92 1.11 1.10 1.01 0.89 0.91 1.27 0.97 1.00 0.73 1.11 0.97 1.03 1.31 1.04 1.52

ribosome ribosome ribosome ribosome ribosome ribosome ribosome ribosome ribosome ribosome ribosome ribosome biogenesis tRNA synthetase tRNA synthetase TCA cycle TCA cycle glyoxylate cycle/TCA cycle oxidative phosphorylation oxidative phosphorylation pyruvate dehydrogenase amino acid degradation glycosyl-transferase

3.78

1.10

5.73 3.01

1.46 0.95

arginine and prolin metabolism proteasome small G-protein

0.43 0.38 0.46 0.25 0.37 0.53 0.57 0.28 0.28 0.39

1.28 1.26 0.84 3.09 1.39 1.13 1.02 0.82 1.15 1.17

extracellular extracellular extracellular extracellular extracellular extracellular muscle protein muscle protein muscle protein muscle protein

0.34 0.56 0.36 0.42 0.37 0.51 0.39 0.49 0.51

1.31 1.18 1.05 1.15 1.44 0.97 1.08 1.07 0.92

muscle protein lipid transport and storage oxidative stress response protein folding carbohydrate binding translation inhibitor telomere component ion transport ribosome

3.54

306

P34383

F02A9.3;far-2

fatty-acid and retinol-binding protein 2

3.14

3.56

P34500

K03H1.4;ttr-2

transthyretin-like protein 2

0.25

3.09

secreted egg-yolk precursor protein secreted, lipid transport and storage secreted, lipid transport and storage

a Only proteins that showed statistically significant changes either between 20 and 40 or 40 and 60 h and fold changes ≥1.5 in either direction were taken into account. Proteins are grouped into up-regulation from 20 to 40 h, down-regulation from 20 to 40 h, and up-regulation from 40 to 60 h, with only one protein (TTR-2) appearing in more than one group. Proteins were manually classified into functional categories using information from publicly available databases and literature. FC, fold changes. bmRNA levels correspond to protein expression (McCarroll et al. 2004, Jiang et al. 2001). cProtein levels correspond to literature (Tabuse et al. 2005, Madie et al. 2003).

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(2003).9,10 Tropomyosin isoform III was seen to decrease during development, while isoform I was described as a L3 specific marker.10 Tabuse et al. (2005) could not distinguish the isoforms and only reported an up-regulation in L1 larvae compared to embryos.9 It therefore remains unclear whether these isoforms display stage specific regulation. This holds true also for MIG-6, which is found expressed either as MIG-6 L or MIG-6 S with tissue and stage specific expression profiles and defined as essential for embryogenesis and proper cell migration.36 Another protein of interest is RPS-28, the only ribosomal protein that shows a decrease in abundance as development progresses. This suggests the protein to have a distinct function only during early phases of ontogenesis. Only mRNA expression data are available from transcriptomic studies on RPS-28. McCarroll et al. used the fer-15(b26) II; fem-1 (hc17) IV mutant strains for mRNA profiling during the development of C. elegans as these nematodes are sterile which diminishes transcript contaminants of embryos. Sampling was done at various stages of development, whereby 16, 28, and 52 h samples probably best match with our times of collection.34 Although for the 16 h time point no data were presented, between 28 and 52 h rps-28 mRNA levels dropped by more than 90%, but increased again strongly at 96 h. Abundance of RPS-28 mRNA at 40 h accounted for only 50% of the level at 20 h which resembles grossly our findings on protein levels. Comparison of transcript data sets reported by McCarroll et al. (2004) and Jiang et al. (2001) with our group of upregulated ribosomal proteins (Table 2) displayed some similarities but also discrepancies.6,34 Although transcriptome data were collected from mutant C. elegans strains, a good correlation between those and our data was observed for most of the relevant genes (except for rpl-7). Six of the mRNA species showed the same pattern of change during development as our protein profiles (RPA-0, RPS-14, RPL-21, RPL-33. RPS19, and RPL-36) but for four other proteins (RPL-4, RPL11.1, RPL-20 and RPL-7A) no such relationship existed. Discrepancies found between the regulation of mRNA and protein levels were also seen in several previously published studies, reporting only good correlation for a subset of investigated genes/proteins.37,38 Several mechanisms are involved in post-transcriptional regulation of mRNAs (for review39) as well as post-translational regulation of protein stability. Translation or stability of the mRNA is affected by for example differential loading of mRNAs onto polysomes40,41 and binding of specific repressors to the target mRNA,42 while posttranslational modifications, like sumoylation, alter protein stability.43 Interestingly, post-translational modifications were also found to regulate ribosomal protein abundance in various organisms ranging from E. coli and Drosophila to humans.42,40,41,43 Thus, it might be possible that similar mechanisms are involved in the specific differential expression of certain ribosomal proteins.

dehydrogenase and isocitrate lyase of the glyoxylate cycle confirmed a metabolic shift associated with larval development.44 Whereas the glyoxylate pathway seems to have high activity during embryonic phases, it decreases during the first larval (L1) stage. However, findings on expression levels of mRNA and proteins of the glyoxylate cycle are contradictory. Although mRNA levels of the bifunctional protein GEI-7 remained constant during development,6,34 we observed an increase in this protein between 20 and 40 h corresponding to L3/L4 stages. This matches with the finding that GEI-7 protein levels peak during larval stages.45 Our protein data grossly confirm enhanced capacity of TCA cycle metabolism during larval development as proposed by Wadsworth and Riddle (1989).44 TCA cycle proteins such as F23B12.5, CST-1 and ACO-1 and those of oxidative phosphorylation (CCO-2 and H28O16.1) increasing substantially. In particular the TCA cycle proteins showed a very similar regulation with an around 3-fold increase. It is intuitive that these changes may cause the increased respiration rates and oxidative phosphorylation capacity as shown by Cuyper and Vanfleteren (1982)46 for later developmental stages of C. elegans. Most interestingly, these changes also match with data from mitochondrial biogenesis,47 that drastically increases from the L4 stage toward adulthood. Although we could not identify proteins encoded by mt-DNA, the increased levels of various metabolic proteins of mitochondrial origin may be taken as an indication for the enhanced mitochondrial biogenesis in this developmental phase.

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CONCLUSION We here present the first LC−MS/MS based quantitative analysis of changes of the proteome during the development of C. elegans and one of the few examples of proteome analysis after metabolic labeling in higher eukaryotes. We show that stable isotope labeling with 15N ammonium chloride as a costeffective alternative to commercially available media can be employed for protein quantification. We were able to identify and quantify 245 proteins in total, and identified subgroups of proteins undergoing major alterations in steady-state levels during development with most prominent changes occurring between 20 and 40 h after hatching. Of the 245 proteins, 61 revealed differential expression between the larval stages. Whereas entities related to protein biosynthesis including ribosomal proteins and aminoacyl tRNA synthetases increased substantially in concentration, extracellular as well as muscle proteins declined in status. Although a large number of ribosomal proteins displayed synchronous changes, individual ribosomal components behaved differently suggesting that they could have additional and possibly extra-ribosomal functions. In addition, a variety of proteins related to citric acid cycle and respiratory chain activity increased in levels toward adulthood of C. elegans, which seems to correlate with increased mitochondrial biogenesis.

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Proteins with Functions in Energy Metabolism Show Marked Changes

ASSOCIATED CONTENT

S Supporting Information *

Alterations in energy metabolism during the larval development of C. elegans were described more than 20 years ago by phosphorus nuclear magnetic resonance analysis. This revealed high contents of AMP in L1 larvae accompanied by ATP levels close to the detection limit. While AMP levels decreased during the progression of development, ATP levels increased. Measurements of enzymatic activity of the TCA cycle isocitrate

Supplementary Table S1: p-values of Tukey Posthoc test and fold changes of 61 significantly changed proteins. Supplementary Table S2: Proteins identified for each sample with corresponding L/H ratios. Supplementary Table S3: Overrepresented gene ontology categories for 61 significantly changed proteins determined 4602



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(12) Venable, J. D.; Dong, M. Q.; Wohlschlegel, J.; Dillin, A.; Yates, J. R., III Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra. Nat. Methods 2004, 1 (1), 39−45. (13) Gouw, J. W.; Tops, B. B. J.; Mortensen, P.; Heck, A. J. R.; Krijgsveld, J. Optimizing identification and quantitation of N-labeled proteins in comparative proteomics. Anal. Chem. 2008, 80 (20), 7796−7803. (14) Dong, M. Q.; Venable, J. D.; Au, N.; Xu, T.; Park, S. K.; Cociorva, D.; Johnson, J. R.; Dillin, A.; Yates, J. R. Quantitative mass spectrometry identifies insulin signaling targets in C. elegans. Science 2007, 317 (5838), 660−663. (15) Tops, B. B. J.; Gauci, S.; Heck, A. J. R.; Krijgsveld, J. Worms from Venus and Mars: Proteomics profiling of sexual differences in Caenorhabditis elegans using in vivo 15N isotope labeling. J. Proteome Res 2010, 9 (1), 341−351. (16) Fredens, J.; Engholm-Keller, K.; Giessing, A.; Pultz, D.; Larsen, M. R.; Højrup, P.; Møller-Jensen, J.; Færgeman, N. J. Quantitative proteomics by amino acid labeling in C. elegans. Nat. Methods 2011, 8 (10), 845−847. (17) Larance, M.; Bailly, A. P.; Pourkarimi, E.; Hay, R. T.; Buchanan, G.; Coulthurst, S.; Xirodimas, D. P.; Gartner, A.; Lamond, A. I. Stableisotope labeling with amino acids in nematodes. Nat. Methods 2011, 8 (10), 849−851. (18) Stiernagle, T. Maintenance of C. elegans. In WormBook; The C. elegans Research Community, February 11, 2006; doi: 10.1895/ wormbook.1.101.1, http://www.wormbook.org. (19) Benner, J.; Daniel, H.; Spanier, B. A glutathione peroxidase, intracellular peptidases and the TOR complexes regulate peptide transporter PEPT-1 in C. elegans. PLoS ONE 2011, 6 (9), e25624. (20) Martin, F. P. J.; Spanier, B.; Collino, S.; Montoliu, I.; Kolmeder, C.; Giesbertz, P.; Affolter, M.; Kussmann, M.; Daniel, H.; Kochhar, S.; et al. Metabotyping of Caenorhabditis elegans and their culture media revealed unique metabolic phenotypes associated to amino acid deficiency and insulin-like signaling. J. Proteome Res. 2011, 10 (3), 990−1003. (21) Wiese, S.; Reidegeld, K. A.; Meyer, H. E.; Warscheid, B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 2007, 7 (3), 340−350. (22) Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 203551−3567. (23) Bindschedler, L. V.; Cramer, R. Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes. Rapid Commun. Mass Spectrom. 2011, 25 (11), 1461− 1471. (24) Kanehisa, M.; Goto, S.; Sato, Y.; Furumichi, M.; Tanabe, M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012, 40 (D1), 109−114. (25) Lyssenko, N. N.; Miteva, Y.; Gilroy, S.; Hanna-Rose, W.; Schlegel, R. A. An unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the C. elegans putative aminophospholipid translocases. BMC Dev. Biol. 2008, 8 (1), 96. (26) Haegler, K.; Mueller, N. S.; Maccarrone, G.; Hunyadi-Gulyas, E.; Webhofer, C.; Filiou, M. D.; Zhang, Y.; Turck, C. W. QuantiSpec Quantitative mass spectrometry data analysis of 15N-metabolically labeled proteins. J. Proteomics 2009, 71 (6), 601−608. (27) Spieth, J.; Blumenthal, T. The Caenorhabditis elegans vitellogenin gene family includes a gene encoding a distantly related protein. Mol. Cell. Biol. 1985, 5 (10), 2495−2501. (28) Kamath, R. S.; Fraser, A. G.; Dong, Y.; Poulin, G.; Durbin, R.; Gotta, M.; Kanapin, A.; Le Bot, N.; Moreno, S.; Sohrmann, M.; et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003, 421 (6920), 231−237. (29) Kerins, J. A.; Hanazawa, M.; Dorsett, M.; Schedl, T. PRP-17 and the pre-mRNA splicing pathway are preferentially required for the proliferation versus meiotic development decision and germline sex

using the Cytoscape plugin BiNGO-2.44. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: ++49-8161-712367. Fax: ++49-8161-713999. Author Contributions #

These authors contributed equally to the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Magdalena Pawlas, Katrin Lasch, and Irmgard Sperrer for excellent technical assistance and Dr. Culotta for supplying α-SOD-1 antibody. We thank other members of our working groups for valuable comments on the manuscript and fruitful discussion. Part of this project, especially M.E., was funded by PURE (Protein Research Unit Ruhr within Europe), a project of Nordrhein-Westfalen, a federal state of Germany. We want to thank Prof. Dr. Turck for providing the QuantiSpec software tool.

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REFERENCES

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