Worms from Venus and Mars: Proteomics Profiling ... - ACS Publications

Male and female worms (C. elegans) were differentially labeled in vivo with 14N and 15N, allowing a quantitative analysis of protein expression levels...
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Worms from Venus and Mars: Proteomics Profiling of Sexual Differences in Caenorhabditis elegans Using in Vivo 15N Isotope Labeling Bastiaan B. J. Tops, Sharon Gauci, Albert J. R. Heck,* and Jeroen Krijgsveld*,# Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands Received July 31, 2009

Hermaphrodites of the nematode Caenorhabditis elegans produce both sperm and oocytes in the same germline. To investigate the process underlying spermatogenesis and oogenesis separately, we used a quantitative proteomics approach applied to two mutant worm lines (fem-3(q20) and fem-1(hc17)) developing only male and female germlines, respectively. We used stable isotopic labeling of whole animals by feeding them either 14N or 15N labeled Escherichia coli. This way, we could confidently identify and quantify 1040 proteins in two independent experiments. Of these, ∼400 proteins showed significant differential expression between female-like and male-like animals. As expected, proteins linked to oogenesis were found to be highly upregulated in the feminized worms, whereas proteins involved in spermatogenesis were found to be highly upregulated in the masculinized worms. This was complemented by many proteins strongly enriched in either mutant. Although the function of the majority of these proteins is unknown, their expression profile indicates that they have an as yet unrecognized role in the development and/or function of the female- and male germline in C. elegans. We show that members of several protein complexes as well as functionally similar proteins show comparable abundance ratios, indicating coregulation of protein expression. Additional analysis comparing our protein data to a previously published microarray data set shows that mRNA and protein expression are poorly correlating. We provide one of the first examples of a large-scale quantitative proteomics experiment in C. elegans and show the potential and feasibility of an approach enabling system-wide accurate quantitative proteomics experiments in this model organism. Keywords: fem-1 • fem-3 • male • female •

15

N • isotope • sex • proteomics

Introduction Gametogenesis is the process by which precursor cells divide and specialize to form mature haploid sex cells (gametes). Species that reproduce sexually have two forms of gametogenesis: spermatogenesis (sperm production) in males and oogenesis (oocyte production) in females. This restriction to gamete formation in the two sexes is not observed in Caenorhabditis elegans, which generates males and hermaphrodites instead of males and females, thus, being capable of sexual reproduction by self-fertilization. Although both spermatogenesis and oogenesis use meiosis to produce haploid cells from diploid precursor cells, the end products are distinct and functionally specialized. C. elegans hermaphrodites produce sperm and oocytes in a temporally regulated process in the same gonad. Spermatogenesis in the developing hermaphrodite starts at the third larval stage, storing the sperm in organs called the spermatheca. Around the fourth larval stage, the germline ceases sperm production * To whom correspondence should be addressed. E-mails: [email protected] (A.J.R.H.) or [email protected] (J.K.). # Current address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 10.1021/pr900678j

 2010 American Chemical Society

and switches to oogenesis, and continues producing oocytes throughout the hermaphrodite’s adult life. Forward genetic screens aimed to identify genes involved in this process have revealed a complex pathway that ensures proper germline development [reviewed in ref 1]. In this respect, the fem genes (fem-1/2/3) have been shown to be crucial as transcriptional regulators at several levels.2 Although the precise function of these genes is still unknown, the fem gene products inactivate TRA-1, a transcription factor that negatively regulates spermatogenesis.3-5 FEM protein activity therefore actively promotes spermatogenesis, and lossof-function alleles of the fem genes consequently result in animals exclusively producing oocytes (feminized animals). In wild-type animals, sperm production has to be terminated eventually and switched to oocyte production. One of the processes essential for this switch is the active repression of fem-3 activity. This is probably realized by the binding of a protein complex to a “point mutation element” (PME) in the 3′ UTR of the fem-3 mRNA, resulting in decreased translation of fem-3 messengers. This is likely the reason why mutations affecting the PME (gain-of-function alleles of fem-3) result in Journal of Proteome Research 2010, 9, 341–351 341 Published on Web 11/16/2009

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masculinized animals that only produce sperm. The availability of fem-1 and fem-3 mutants provides an ideal system to study the differences between oogenesis and spermatogenesis in C. elegans: fem-1 (loss-of-function)4 and fem-3 (gainof-function)9 animals only differ in germline development (oocyte vs sperm production, respectively) without the interference of sexual differences (e.g., due to the number of sex chromosomes), since both strains are cytogenetically identical. To profile proteins that are specific in germline differentiation, that is, involved in germline development and/or sperm and oocytes production, we use here a quantitative proteomics approach comparing protein expression profiles between induced feminized fem-1(hc17) and masculinized fem-3(q20) animals. We specifically chose these mutants since fem-1 and fem-3 mutants have previously been compared at the transcript level using microarrays, allowing us to compare transcript and protein expression profiles.10 Using state-of-the-art liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), in combination with stable isotopic labeling of whole animals by feeding on either 14 N or 15N labeled bacteria, we provide one of the first examples of a large-scale quantitative proteomics experiment in C. elegans.11,12 Comparing young adult fem-1(hc17) and fem3(q20) animals at the protein level, we could confidently identify and quantify 1040 proteins (with at least 2 peptides) in two independent experiments. Of these, ∼400 proteins showed significant differential expression between fem-1(hc17) and fem-3(q20) animals. Of the proteins with annotations, proteins functionally related and proteins associating in distinct protein complexes were found to be similarly regulated. As expected, proteins linked to oogenesis were found to be highly upregulated in the feminized worms, whereas proteins involved in spermatogenesis were found to be highly upregulated in the masculinized worms. We also compared our proteomic data to the previously published transcriptome data set,10 showing that mRNA expression profiles are poor indicators for protein expression levels in this system. In general, this study shows the potential and feasibility of a proteomics approach to identify and quantify proteins in a proteome-wide manner at the organismal level in the model organism C. elegans.

Results and Discussion In this study, we set out to profile the differences between C. elegans animals with male and female germlines. To study these differences, we used fem-1(hc17) and fem-3(q20) mutants. Both these alleles are temperature-sensitive, meaning that animals carrying these mutations are wild-type at 15 °C (permissive temperature), while they display the mutant phenotype at 25 °C (restrictive temperature). The fem-1(hc17) animals cultured at 25 °C only develop the female germline and thus only produce oocytes, while fem-3(q20) animals cultured at 25 °C only develop the male germline and thus exclusively produce sperm (Figure 1). We refer to this as femalelike and male-like worms, respectively. The approach we used to identify and differentially quantify proteins expressed in fem1(hc17) and fem-3(q20) animals is schematically outlined in Figure 2A and adopted from an approach that we have described previously.13 Our quantitation method is based on the differential labeling of the two worm strains with light (14N) and heavy (15N) nitrogen isotopes, by providing them a diet of isotope labeled bacteria.13 Full incorporation of 15N into proteins leads to a mass increase compared to nonlabeled 342

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Figure 1. Germline of fem-1(hc17) and fem-3(q20) animals at 25 °C. Shown are Normarski and DAPI images of germlines of adult fem-1(hc17) and fem-3(q20) animals grown for approximately 2 days at 25 °C. Germ cells progressively differentiate in the germline to mature gametes. Because of their size, fully differentiated oocytes and their chromosomal content are clearly visible in fem-1(hc17) animals. Individual sperm cells are hard to distinguish using Nomarski contrast microscopy, but their DNA content is readily visualized using DAPI staining. Pictures are taken with a 400× magnification.

proteins (containing the natural isotope 14N), while other physicochemical characteristics remain identical. In mixtures of labeled and unlabeled peptides, mass spectrometry is subsequently used to identify individual peptides from their fragmentation spectra, while the intensity ratio of 14N and 15N peak pairs in the MS spectra reflect the relative peptide abundance (Figure 2B). To differentially label induced feminized and masculinized animals, worms were cultured for several generations on either 14 N or 15N labeled bacteria at 15 °C (permissive temperature). Worm cultures were subsequently synchronized and allowed to grow to adulthood at 25 °C (restrictive temperature). After preparation of protein extracts, the protein concentrations of the 14N and 15N labeled protein extracts were determined and mixed in a 1:1 ratio. Sample complexity was reduced by first separating the proteins on a SDS-PAGE gel, after which the gel was sliced into 24 pieces that were individually subjected to in-gel trypsin digestion. Each of these 24 samples was subsequently analyzed by nano-LC MS/MS and the obtained MS/MS spectra were searched against a C. elegans protein database. We performed a biological replicate of this experiment, including a “label swap” now growing fem-1(hc17) on 15 N and fem-3(q20) on 14N. In total, more than 380 000 spectra were generated. This resulted in 1871 and 2161 protein identifications in the biological replicates, respectively. Next, proteins were quantified by integrating all MS peak areas of the identified peptides using MSQuant.14 Requiring a minimum of two quantifiable peptides per protein, a Mascot peptide cutoff score of 20 and a protein cutoff score of 60, we quantified 1516 proteins in the first experiment (14N fem-1 vs 15N fem-3; Supplemental Table 1a) and 1540 proteins in the biological replicate (15N fem-1 vs 14N fem-3; Supplemental Table 1b). In total, we quantified 2016 unique proteins, of which 1040 were common to both experiments (i.e., 66% overlap between the two experiments). Peptides that could not be quantified represent mainly low-abundance peptides that disappear in the noise or that produce an insufficient number of data points for proper peak integration. This core of 1040 proteins quantified in both experiments (Supplemental Table 2) was taken further for detailed analysis. This by no means disqualifies the 976 proteins that were only identified and quantified in one of the screens, since they all meet the same stringent criteria for identification and quantitation (minimum of 2 peptides per

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Figure 2. (A) Outline of the approach used in this study to quantitate differences in protein expression levels between masculinized and feminized worms. The fem-1(hc17) and fem-3(q20) animals were fed on 14N and 15N-labeled bacteria, and vice versa for the biological duplicate experiment. Combined extracts of light and heavy nitrogen labeled worms were analyzed by LC-MS/MS for peptide identification and quantification. (B) Examples of MS spectra of peptides with differential expression levels between 14N labeled fem1(hc17) and 15N labeled fem-3(q20) animals are shown.

protein, peptide score >20, protein score >60). The fact that these proteins were observed in only one experiment may be mainly due to under sampling, a well-known phenomenon in shotgun proteomics.15 Yet, this subset (Supplemental Table 3) may still contain valuable and biologically relevant differential protein expression between male-like and female-like animals.

We estimated the False Discovery Rate (FDR) by searching our spectra against a reversed C. elegans database.16 Using the same criteria as for the forward database (Mascot peptide cutoff score of 20 and a protein cutoff score of 60) and only allowing proteins with a minimum of 2 peptides, we established an FDR of about 0.4%. In the set of quantified peptides, the actual FDR Journal of Proteome Research • Vol. 9, No. 1, 2010 343

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Figure 3. Expression profiles of all quantified proteins. Scatter plot representation of protein data from the two independent biological experiments. The correlation coefficient for the linear regression analysis is indicated (R2 ) 0.80). Proteins that significantly change in abundance (based on the duplicate experiment) are indicated as black diamonds. Proteins in the lower left quadrant are upregulated in female-like animals and proteins in the upper right quadrant are upregulated in male-like animals.

is probably much lower than this, since we required two or more quantifiable peptides. This is testified by the fact that we did not observe a single “reversed” hit among the quantified proteins meeting this criterion, suggesting that the FDR in the set of quantified proteins is close to zero. Protein Quantitation. Protein quantitation was of high quality, reflected by the low overall average coefficient of variation (CV, the error between peptides of the same protein) for both experiments, that is, 13.6% and 15.6%. We next tested which proteins in the entire data set had significantly different expression levels between female-like and male-like animals. Therefore, we employed a two-tailed t test and calculated the probability of a significant (p e 0.05) difference between the ratio of individual proteins and the arithmetic mean of all protein ratios.17 This turned out to be the case for ∼370 of the 1040 proteins that were identified in the two independent experiments. This does not imply however that all of these ∼370 differentially regulated proteins represent biologically relevant differences, but states that these proteins are significantly regulated in our experiment based on our statistical analysis. The reproducibility of the data was high, indicated by the correlation coefficient of 0.71 when all 1040 quantified proteins was used, and a correlation coefficient of 0.80 when this was restricted to the 370 proteins that showed a significant change (Figure 3). To further validate our approach, we performed immunoblots for some of the differentially expressed proteins for which good antibodies were available (UNC-54, LIN-5 and GLH-1). This confirmed the differential protein expression levels between the female-like and male-like animals as determined by quantitative mass spectrometry (Figure 4). We also determined if our normalization was correct by determining the ratios of known nongermline expressed proteins between female- and male-like animals. For example, CLIC-1 is a vesicle clathrin light chain that is expressed in the head 344

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Figure 4. Quantitation of proteins by Western blotting. (A) fem1(hc17) and fem-3(q20) extracts were immunoblotted and probed with antibodies against LIN-5, UNC-54 and GLH-1. (B) Coomassie staining was used as a loading control. (C) Fold changes for the same proteins as in panel A as identified by MS. n.i. means not identified.

region and in neurons, and is expressed at similar levels in female- and male-like animals (2log ratios of -0.07 and -0.13

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Worms from Venus and Mars Table 1. Proteins Showing a > 4-fold Upregulation in fem-1(hc17) and fem-3(q20) Animals

a

2

Wormbase ID

log ratio fem-1/fem-3

experiment 1

NASP-2 Y18D10A.11 CEY-2 MCM-4

4.52 4.31 3.33 2.75

CAR-1

2.65

CEY-3 CGH-1

2.46 2.38

PQN-45 F21D5.1 F25D7.4

2.26 2.23 2.21

T21G5.4 C39H7.1 Y59H11AM.2/3 ZK550.5 SPE-15 ZC434.8 F15E11.1 CPZ-1

experiment 2

upregulated in

description

Proteins Upregulated in fem-1(hc17) Animals 4.54 oogenesis Cell cycle-regulated histone H1-binding protein 5.06 oogenesis Caenorhabditis-specific hypothetical protein 3.97 oogenesis Y-box domain-containing protein 2.72 oogenesis similarity to human MCM4 minichromosome maintenance deficient 4 protein 3.31 oogenesis putative RNA-binding protein, inhibits physiological apoptosis in oocytes 3.01 oogenesis Y-box domain-containing protein 3.07 oogenesis putative DEAD-box RNA helicase, inhibits physiological apoptosis in oocytes 2.64 oogenesis nematode-specific hypothetical protein 2.84 oogenesis putative phosphoacetylglucosamine mutase 2.71 oogenesis Caenorhabditis-specific hypothetical protein

5.29 >5.3 3.92 4.26 3.45

>9.1 6.1 6.4 5.9 4.08

2.39 1.47 1.57

2.29 3.31 2.6

Proteins Upregulated in spermatogenesis spermatogenesis nd spermatogenesis spermatogenesis spermatogenesis spermatogenesis spermatogenesis

fem-3(q23) Animals nematode-specific hypothetical protein Casein kinase thiosulfate sulfurtransferase peroxisomal phytanoyl-CoA hydroxylase unconventional myosin, required during the terminal stages of spermatogenesis creatine kinase nematode-specific hypothetical protein cathepsin Z-like cysteine protease

a The ratios from the two independent label-swapped experiments are shown. Also indicated is if the transcripts of the identified proteins are upregulated in oogenesis or spermatogenesis. This distinction is based on data from ref 10. Nd means not done.

in experiment 1 and 2, respectively). DIM-1, expressed in body wall muscles, is required for normal muscle organization (2log ratios of -0.06 and 0.0) as is UNC-89, a protein expressed in pharyngeal and body wall muscles (2log ratios of -0.10 and -0.01). SOD-2 is a superoxide dismutase specifically expressed in the head and tail region (2log ratios of 0.05 and -0.09). On the basis of our statistical analysis, the above-mentioned proteins are also not significantly differentially regulated. All these experiments therefore further reinforce the validity of our data set, providing a solid foundation for further analyses. Differentially Regulated Proteins. Of the ∼370 proteins that were differentially regulated with statistical significance, ∼200 and ∼170 proteins were upregulated in female-like and malelike animals, respectively. A summary of the proteins that were found to be exceptionally differentially regulated between female- and male-like animals (2log ratio >2) is listed in Table 1. Functionality ascribed to these proteins, for example, in oogenesis or spermatogenesis, largely coincides with enrichment of male and female-specific genes, as determined by microarray analysis using the same system (fem-1/fem-3 mutants).10 In addition, several of these proteins have known functions in the germline. One of these proteins is NASP-2, a predicted cell cycle-regulated histone H1-binding protein >22fold upregulated (2log ratio is 4.5) in female-like animals. NASP-2 RNAi knockdown experiments have shown that this protein promotes female development.18 Although its precise function is unclear, it has been suggested that NASP-2 (partially redundant with NASP-1) is involved in the transcriptional repression of TRA-1 target genes, consistent with our data indicating that the protein is overexpressed in feminized fem1(hc17) animals. Other proteins with a higher abundance in female-like animals include CGH-1, CEY-2/3 and CAR-1. These proteins associate in P granules and other cytoplasmic foci.19-21 During early embryogenesis, P granules segregate asymmetri-

cally into those cells that eventually give rise to the germ line.22 Because of the correlation between P granule distribution and the development of the germ line, P granules are thought to function in germ line specification or differentiation (for example, see refs 23 and 24). Among the ∼170 proteins upregulated in male-like animals is SPE-15 (2log ratio is 3.8- to 13-fold upregulated), a protein that is required during the terminal stages of spermatogenesis.25,26 Although several proteins have assigned functions based on sequence homology and/or experimental data, other differentially regulated proteins are only hypothetical and have as yet unknown functions. Some of these have extremely different abundance levels in female- and male-like animals (see Table 1). For instance, Y18D10A.11 is more than 25 times upregulated (2log ratio is 4.7) in fem-1(hc17) animals. The protein appears to be C. elegans-specific with no homology to other proteins, and with unknown function. Unfortunately, also no mutants or phenotypes have been described in genome-wide RNAi screens for this protein. The same is true for T21G5.4, C39H7.1, Y59H11AM.2 and ZK550.5 which are proteins that are >30-fold upregulated in male-like animals (2log ratio >5). T21G5.4 is supposedly a nematode-specific Casein kinase with unknown function. Y59H11AM.2 contains an N-terminal inactive thiosulfate sulfurtransferase domain. The role of this domain is uncertain, but it is believed to be involved in protein-protein interactions. ZK550.5 contains a phytanoyl-CoA dioxygenase domain and is homologous to human PAHX, which causes Refsum disease, a defect affecting the myelin sheaths around nerve cells.27 No mutant data or RNAi phenotypes have been described for these proteins, making it difficult to speculate on their function in fem-3(q20) animals. Although not much is known about these proteins, our protein expression data is in agreement with microarray data on the same proteins (Table 1 and see below). Journal of Proteome Research • Vol. 9, No. 1, 2010 345

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Figure 5. (A) Expression profiles of proteins belonging to the same protein complex or protein family. Members of these complexes show similar abundance ratios in the comparison between male-like and female-like animals, suggesting coregulation. This applies to proteins belonging to the CGH-1/CEY complex (CGH-1, CAR-1, CEY-2/3/4), T-complex (CCT-1/2/3/4/5/6/7/8), Vitellogenins (VIT-1/2/3/4/ 5/6), Nucleopore complex (NPP-9/10/16/21) and the Aspartyl proteases (ASP-2/4/5/6). (B) Expression profiles of homologous proteins, or proteins derived from the same transcriptional unit. Indicated are the homologues of the mammalian centromere protein-F (HCP-1 and -2), Heat Shock proteins (HSP-1/3/4/6/16.1/17/25/43, SIP-1, DAF-21, DNJ-12, C30C11.4 and T14G8.3), homologues of the mRNA cap-binding protein eIF4E (IFE-1/2/3), Splice-variants of F02A9.4 (a and b), and orthologs of the human MAPRE proteins (EBP-2/3). The complete set of protein complexes and families identified in both screens is listed in Supplemental Table 4.

Some of the proteins with less extreme abundance ratios are known to be involved in germline function/development. One example is NOS-3, which we find to be ∼2.5-fold upregulated in female-like animals. It is required in development of the hermaphrodite germline and promotes the switch from sperm to oocyte production, probably by repressing translation of the fem-3 mRNA.28,29 Protein Classes/Complexes. During the analysis of our data, we noticed that subunits of several protein complexes could be identified, like CGH-1, CEY-2/3 and CAR-1. We also observed that several of these proteins show a quite similar differential expression level between female- and male-like animals. We therefore reasoned that proteins belonging to the same complex may be similarly regulated. The same could be argued for proteins that act in the same biological pathway/process, although differences might be observed due to redundancy or functionality in multiple pathways. To test this hypothesis, we systematically screened our data for proteins that have similar functions or belong to the same protein complex (Supplemental Table 4). Indeed, proteins that are found to associate in vivo clearly display a similar expression level in female- and malelike animals (Figure 5A). The T-complex is a hexadecameric chaperonin, consisting of 8 different subunits (CCT-1-8), that binds unfolded polypeptides and mediates their folding and release in an ATPdependent manner.30 We identified all 8 subunits and all show an almost identical distribution comparing expression levels in female- and male-like animals (Figure 5A). This may be explained by the fact that the T-complex binds ∼10% of newly synthesized proteins,31 and that we enrich for proteins involved in protein synthesis in female-like animals. As mentioned before, we identified CGH-1, CEY-2/3 and CAR-1, which all associate in P granules and are specifically expressed in femalelike animals. Strikingly, CGH-1, CEY-2/3 and CAR-1 are all significantly higher expressed in female-like animals (2log ratios < -2.7). In addition, we identified CEY-4 that has also been found to associate with CGH-1 and CEY-2/3 in cytoplasmic particles. However, CEY-4 clearly displays a different pattern 346

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(2log ratio of ∼ -0.3), thus being hardly enriched in femalelike animals. This could indicate that CEY-4, besides acting in the female-germline with CEY-2/3, may have an additional function that is sex-independent or even male-specific. Similarly, CEY-1, a Y-box protein highly similar to CEY-2/3/4, but not found to be associated with CGH-1, CEY-2/3/4 and CAR1, was expressed at similar levels in female- and male-like animals (2log ratio ∼ -0.1). Proteins with similar biological functions, but that do not associate in complexes, show more diverse distribution levels. An example of proteins with a similar function and acting in the same pathway are the vitellogenins. Vitellogenins are produced in the hermaphrodite intestine and bind lipids, forming lipoprotein particles (yolk). These are ultimately taken up into vesicles within the growing oocytes and processed to provide essential nutrients for the embryo.32,33 Since male-like animals do not produce any oocytes, the yolk cannot be taken up and processed.34 This may explain why we find vitellogenins to be overexpressed in male-like animals (Figure 5A). Another example is the group of heat shock proteins (Figure 5B). While most heat shock proteins are not preferentially expressed in female- or male-like animals, HSP-16.1 is >3-fold higher expressed in female-like animals (2log ratio is -1.7), while HSP-4 is more abundant in male-like animals (2log ratio is 1.4), suggesting that individual heat shock proteins may have sex-specific germline-related functions. We also identified the two proteins encoded by the two splice-variants of F02A9.4. Both transcripts share the first two exons, and presumably are under the transcriptional control of the same promoter. However, the two splice-variants are functionally different, and show a different abundance profile in our study (Figure 5B). F02A9.4a encodes a carboxyl transferase, predicted to be involved in lipid metabolism, and is enriched in female-like animals (1.4-fold enriched). F02A9.4b, on the other hand, encodes an RNase III enzyme that was enriched in male-like animals (1.2-fold enriched). RNase III cuts double-stranded RNA and is involved in the processing of rRNA precursors. Although these two proteins originate from the

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Table 2. Gene Ontology (GO) Enrichment for Proteins Upregulated in fem-1(hc17) and fem-3(q20) Animalsa GO term

0032553: 0009790: 0000003: 0051082: 0016070: 0006418: 0006412: 0003006:

# proteins

Benjamini-Hochberg p value

GO-Enrichment for fem-1(hc17) Animals ribonucleotide binding 4.71 embryonic development 4.53 reproduction 4.53 unfolded protein binding 3.35 metabolic process 3.32 tRNA aminoacylation for protein translation 3.32 translation 2.38 reproductive developmental process 2.31

46 101 83 11 25 13 36 24

1.40 × 10-3 6.50 × 10-4 5.10 × 10-4 2.70 × 10-2 5.10 × 10-4 6.30 × 10-3 7.20 × 10-3 2.60 × 10-2

GO-Enrichment for fem-3(q23) Animals 1.53

10

4.70 × 10-3

0005783: endoplasmic reticulum a

fold enrichment

The top 85% of proteins upregulated in fem-1(hc17) and fem-3(q20) animals was analyzed for GO-enrichment using the bioinformatics tool DAVID.47

same transcriptional unit, their expression is differentially regulated either in time (different stages) or space (different cells). Since spermatogenesis and oogenesis are temporally, but not spatially, regulated in the wild-type hermaphrodite germline, this suggests that either splicing or translation of F02A9.4a and F02A9.4b is temporally regulated in the C. elegans germline. Two other proteins that show an opposite expression pattern are HCP-1 and its paralog HCP-2. Both proteins encode a homologue of the mammalian centromere protein-F (CENPF) and are involved in mitotic chromosome segregation. Although it has been suggested that HCP-1 and HCP-2 act redundantly,35,36 their upregulation in female-like and malelike animals, respectively, suggests that these two proteins may have specific functions in the germline. More recent reports studying the synthetic effects of hcp-1 or hcp-2 RNAi knockdowns in animals with defects in the spindle-checkpoint pathway also suggest that both proteins have nonredundant functions.37,38 Knockdown of hcp-1 or -2 by RNAi in fem1(hc17) and fem-3(q20) animals, respectively, does not result in a loss of oocytes or sperm, suggesting that these proteins do not function exclusively in the female or male germline (results not shown). Similarly, two microtubule End Binding Proteins, EBP-2 and -3 also show opposing differential expressions. EBP-2/3 are orthologous to human MAPRE2/3 and bind the plus ends of growing microtubules.39,40 EBP-2 and -3 have a distinct distribution pattern, the former being overexpressed in femalelike animals and the latter in male-like animals (Figure 5B). Another protein with a remarkable distribution pattern is the translation initiation factor IFE-3. Although all translation initiation factors we identified show a minor upregulation in female-like animals (Supplemental Figure 1), IFE-3 stands out with a ∼4-fold upregulation (2log ratio is 2). IFE-3 encodes one of five C. elegans homologues of the mRNA cap-binding protein eIF4E. It is the one most similar to human eIF4E and is the only isoform required for viability. In C. elegans, ∼70% of the mRNAs receive a 5′ 22 nt noncoding spliced-leader (SL) RNA by trans-splicing.41,42 The SL RNA contains a 2,2,7-trimethylguanosine (TMG) cap43 that is retained on the mature mRNA.44 IFE-3, however, only binds monomethylated guanosine (MMG) caps, and does not bind TMG caps, which suggests that IFE-3 likely mediates translation of those mRNAs that do not contain a spliced-leader sequence.45,46 Although not much is known about the function of MMG versus TMG caps in mRNA translation or stability, it is tempting to speculate that the combination of cap type (MMG or TMG) and eIF4E isoform in cells or tissues (e.g., germline) drives translation of

specific pools of mRNAs. We also identified two other eIF4E homologues, IFE-1 and IFE-2, which are not clearly differentially regulated (Figure 5B). Functional Classification. To gain insight in protein function of the proteins preferentially expressed in fem-1(hc17) or fem3(q20) animals, we classified the proteins based on gene ontology (GO) using the DAVID classification tool.47 To minimize the contribution of proteins with only marginally different abundance ratio, we used the top 85 percentile of the proteins that were significantly differentially expressed in males and females, respectively. We determined GO-enrichment by comparing the proteins in the top 85 percentiles against the population of all 1040 identified proteins. Proteins overexpressed in male-like animals are associated with very diverse GO-terms, illustrated by the fact that we hardly enrich for specific GO-terms that are associated with these proteins (Table 2). The only GO-term associated with male-like proteins that is slightly enriched above background is ‘endoplasmatic reticulum’ (GO:0005783), suggesting that secreted and/or transmembrane proteins may be relatively more produced in the male germline. GO-term enrichment for proteins preferentially expressed in female-like animals (Table 2) results in more distinct classes. Not unexpectedly, female-like proteins are associated with reproduction and embryogenesis (GO:0003006, reproductive developmental process; GO:0000003, reproduction; and GO: 0009790, embryonic development). However, a substantial number of proteins enriched in the female germline appear to be involved in protein biogenesis (GO:0051082, unfolded protein binding; GO:0006418, tRNA aminoacylation for protein translation; and GO:0006412, translation). The latter observation probably reflects the surplus of proteins that is generated and incorporated into the developing oocytes. These maternal proteins are essential for the early embryo to sustain development, until zygotic production takes over. Protein versus mRNA. To further analyze our data, we compared our total set of 1040 proteins to a previously published microarray experiment comparing fem-1(hc17) and fem-3(q20) animals at the transcript level.10 We could match 690 proteins from our data set to their corresponding transcripts in the microarray experiment. As illustrated in Figure 6, the correlation coefficient between the protein and transcript data sets is only 0.41, indicating that mRNA expression profiles are on average weak indicators for protein expression profiles. Still, in general, most of the transcripts-protein pairs showed a similar trend, clustering along the axis from the lower left (female-enriched) to the upper right quadrant (male-enriched). Journal of Proteome Research • Vol. 9, No. 1, 2010 347

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Figure 6. Correlation of transcript and protein expression. Scatter plot representing differential expression levels of transcripts10 and their corresponding proteins between fem-1(hc17) and fem-3(q20) animals (this study). The correlation coefficient for the linear regression analysis is indicated (R2 ) 0.41, weak correlation). The left encircled region is enriched (compared to the right encircled region) for data points where transcripts levels are differentially regulated while protein levels are not.

Yet, overall, more pronounced differential expression levels had been reported at the mRNA level than we see here at the protein level, indicated by the average slope of 0.6 of the trend line. This is most evident for genes that cluster left of the y-axis in Figure 6 (compare the circles left and right of the y-axis). These transcripts are highly expressed in female-like animals, but change at the protein level only to a limited extent. A likely explanation is that these transcripts are maternal RNAs, which are translationally inactive in the female germline and thus do not produce new protein. In zebrafish embryos, maternal mRNAs are targeted and promoted for clearance by miRNA MiR-430 upon the maternalto-zygotic transition in the developing embryo.48 Analysis of our pool of maternal transcripts has not revealed such enrichment for a miRNA target-site (data not shown). This could indicate that either the miRNA responsible for this action has not been identified in C. elegans (and thus does not show up in our analysis), that C. elegans does not contain such a mechanism to clear maternal RNAs, or that our data set is simply too small to detect enrichment above background levels. Genes for which their transcripts are upregulated in malelike animals also show an upregulation at the protein level. Notable exceptions are the genes F17C8.3, Y53G8AR.6 and T08H10.1. These genes display a marked increase of their transcript levels in male-like animals (∼18-, ∼16-, ∼7-fold upregulated, respectively), while the corresponding proteins show hardly any differential expression between female- and male-like animals. Not much is known, however, about these genes and their potential function in the germline. F17C8.3 is a gene with unknown function, Y53G8AR.6 is homologous to human Hcc-1, a nucleic acid-binding protein, and T08H10.1 is an aldo/keto reductase that possibly serves as a substrate for the apoptotic protein CED-3.49 In general, we observe a modest correlation of the differential expression levels between 348

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transcript and protein. This is largely due to more pronounced differential expression at the transcript level than at the protein level, although exceptions are observed.

Conclusion Here we presented data on one of the first high-throughput quantitative proteomics experiments at the organismal level in C. elegans. Stable isotope labeling provides an easy and reliable way of labeling whole proteins and minimizes the chance of technical errors that can be introduced with chemical labeling methods. On the basis of the reproducibility, the low CV value, immunoblot data, the distribution of proteins with known biological functions and proteins that function in the same complex, quantification is also very reliable. This opens the possibility to combine the powerful genetics of C. elegans with large-scale quantitative protein data, for example, to study microRNA function.50-54 We charted the differences in protein expression between worms differing in their germline development (fem-1(hc17) and fem-3(q20) animals). While at 25 °C fem-1(hc17) animals develop a “female” germline that only produces oocytes, fem3(q20) animals develop a “male” germline that only produces sperm. This means that our data set of differentially regulated proteins includes both proteins involved in germline development, like NOS-3 and NASP-2, and proteins associated with the end products (i.e., sperm or oocytes), like SPE-15. Besides these proteins that have a clear biological function in the C. elegans germline, our data set contains numerous hypothetical proteins or proteins with unknown functions. Nevertheless, many of these proteins are clearly differentially regulated between fem-1(hc17) and fem-3(q20) animals, with many proteins expressed at similar or even higher levels as proteins known to function in oogenesis or spermatogenesis.

Worms from Venus and Mars Some even appear to be exclusively expressed in either fem1(hc17) or fem-3(q20) animals, for example, Y18D10A.11 or T21G5.4, respectively. Additional genetic and biochemical experiments should reveal the role for these proteins in the C. elegans germline.

Experimental Procedures Worm Culture and Sample Preparation. The BA17 strain was ontained from the Caenorhabditis Genetics Center and JK816 was kindly provided by Dr. J. Kimble. Strains were cultured for several generations on either 14N (unlabeled) or 15 N medium at 15 °C. Unlabeled worms were cultured on NGM plates, containing 1% agarose instead of agar, 0.3% (w/v) NaCl, and 25% (v/v) Spectra 9-U neutral medium (Spectra Stable Isotopes), seeded with Escherichia coli OP50 grown in Spectra 9-U medium. Strains grown for 15N enrichment were grown on plates, containing 1% agarose, 0.3% (w/v) NaCl, and 25% (v/v) Spectra9-N 15N medium (Spectra Stable Isotopes), seeded with E. coli OP50 grown in Spectra9-N 15N medium. Animals cultured at 15 °C were bleached and eggs were allowed to hatch overnight at 25 °C in M9 buffer. L1 animals were subsequently cultured on the appropriate culture medium for approximately 2 days at 25 °C. Synchronized young adults grown at 25 °C were harvested in M9 buffer on ice. The resulting pellet was subsequently dried by vacuum centrifugation and extracted in 8 M urea. After determining the protein concentrations, extracts were mixed in a 1:1 ratio. The worm protein mixture was resolved by an SDS-PAGE gel (10%) and Coomassie-stained. The gel lane was cut into 24 pieces and subjected to in-gel reduction, alkylation, and tryptic digestion. The peptides were extracted from the gel in 25 µL of 0.1 M acetic acid. Nanoflow-HPLC-MS. Half of the tryptic digest was used for further analysis. All samples were analyzed by nanoflow liquid chromatography using an Agilent 1200 HPLC system (Agilent Technologies) coupled online to a LTQ-Orbitrap mass spectrometer (Thermo Electron). The liquid chromatography part of the system was operated in a setup essentially as described previously.55 Aqua C18, 5 µm, (Phenomenex) resin was used for the trap column, and ReproSil-Pur C18-AQ, 3 µm, (Dr. Maisch, GmbH) resin was used for the analytical column. Peptides were trapped at 5 µL/min in 100% solvent A (0.1 M acetic acid in water) on a 2 cm trap column (100 µm i.d., packed in-house) and eluted to a 40 cm analytical column (50 µm i.d., packed in-house) at 100 nL/min in a 90 min gradient from 10 to 40% solvent B (0.1 M acetic acid in 8/2 (v/v) acetonitrile/ water). The eluent was sprayed via standard coated emitter tips (New Objective), butt-connected to the analytical column. A 33 MΩ resistor was introduced between the high voltage supply and the electrospray needle to reduce ion current. The LTQOrbitrap mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Fullscan MS spectra (300-1500 m/z) were acquired with a resolution of 60 000 at 400 m/z after accumulation to a target value of 500 000. The five most intense peaks above a threshold of 500 were selected for collision induced dissociation in the linear ion trap at normalized collision energy of 35% after accumulation to a target value of 30 000. Peptide Identification and Quantitation. All MS2 spectra were converted to single dta files using Bioworks Browser 3.1 (Thermo) and merged into a Mascot generic format file which was searched using an in-house licensed Mascot v2.2.04 search engine (Matrix Science) against a C. elegans database (downloaded April 9, 2007; containing 22 622 sequences). In both

research articles cases, carbamidomethyl cysteine was set as a fixed modification and oxidized methionine as a variable modification. The search was performed with the option of ‘15N metabolic labeling’ checked, to identify both labeled and unlabeled peptides. Trypsin was specified as the proteolytic enzyme, and one missed cleavage was allowed. The mass tolerance of the precursor and fragment ions was set at 5 ppm and 0.8 Da, respectively. Peptide and protein quantitation was performed using the open source program MSQuant.14,55 Briefly, peptide ratios were obtained by calculating the extracted ion chromatograms (XIC) of the unlabeled and labeled forms of the peptide using the monoisotopic peaks only. The total XIC for each of the peptide forms was obtained by summing the XIC in consecutive MS cycles for the duration of their respective LC-MS peaks in the total ion chromatogram using FTMS scans. These total XICs were then used to compute the peptide ratio. Determination of the 15N Enrichment Level and Correction. All MS scans of a 15N-labeled peptide were combined, and the resulting isotope distribution was manually correlated against theoretical isotope patterns varying in degree of 15N enrichment. In this way, the 15N enrichment of 10 randomly selected peptides of each data set was determined, reflecting the isotope enrichment in a particular experiment (data not shown). On the basis of this value, abundance ratios of all proteins were corrected to compensate for the additional isotope intensities below the monoisotopic peak.56 To correct for minor errors in mixing of the two extracts, we subsequently normalized our data by determining the median ratio observed after MS analysis of the mixture, and adjusted the ratios accordingly. False Discovery Rate (FDR). The FDR was essentially determined as described in Beausoleil, et al.16 In short, we identified the FDR by searching our MS/MS data against a “reverse” C. elegans protein database at 50 ppm (with the above-mentioned cutoff scores and a minimum of 2 peptides). Next, we plotted the distribution of the number of identified peptides against the ppm and focused only on the peak area (around -5 to +5 ppm). The FDR was subsequently calculated using to following formula: (2 × number “reverse” proteins)/ number of “forward” proteins.

Acknowledgment. We would like to thank Robert Kerkhoven for bioinformatic analyses. We would like to thank Dr. Kimble for supplying strain JK816, Dr. Van den Heuvel for anti-LIN-5, Dr. Epstein for anti-UNC-54 and Dr. Bennett for anti-GLH-1. This project was financially supported by The Netherlands Proteomics Centre (NPC). Supporting Information Available: Supplemental Figure 1, expression profiles of proteins belonging to the same protein complex or protein family. Supplemental Table 1, proteins and peptides that were identified and quantified in biological replicates, including label-swap experiments. Supplemental Table 2, the relative abundance of all proteins identified and quantified in feminized and masculinized worms in the two independent label-swapped experiments. Supplemental Table 3, the relative abundance of all proteins identified and quantified in feminized and masculinized worms in a single experiment. Supplemental Table 4, protein complexes and proteins with similar functionality quantified in fem-1(hc17) and fem-3(q20) animals. This material is available free of charge via the Internet at http://pubs.acs.org. Journal of Proteome Research • Vol. 9, No. 1, 2010 349

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