Proteome Analysis of Drosophila Mutants Identifies a Regulatory Role

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A proteome analysis of Drosophila mutants identifies a regulatory role for 14-3-3# in metabolic pathways Yeap Shen Ng, Alexandra Sorvina, Christie Ann Bader, Florian Weiland, Angel F. Lopez, Peter Hoffmann, Tetyana Shandala, and Douglas A. Brooks J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b01032 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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Journal of Proteome Research

A proteome analysis of Drosophila mutants identifies a regulatory role for 14-3-3ε in metabolic pathways Yeap S. Ng,† Alexandra Sorvina,† Christie A. Bader,† Florian Weiland,‡ Angel F. Lopez,§ Peter Hoffmann,‡ Tetyana Shandala,⊥ Douglas A. Brooks†,⊥,* ⊥Equal

†

last authors

Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University

of South Australia, Adelaide, SA 5001, Australia ‡

Adelaide Proteomics Center, School of Molecular and Biomedical Sciences, University of

Adelaide, Adelaide, SA 5005, Australia §

Centre for Cancer Biology, Adelaide, SA 5000, Australia

*

E-mail: [email protected] Tel: (618) 8302 1229

KEYWORDS: 14-3-3ε mutants, proteomics, Ecdysone Receptor, Fat body protein 1, Alcohol dehydrogenase, Lamin.

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ABSTRACT: The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here, we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle and lipid metabolic pathways. The Ecdysone Receptor (EcR), which is responsible for initiating the larval-to-adult transition, co-localized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1 and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or through direct interaction with metabolic proteins.


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INTRODUCTION The 14-3-3 family of scaffolding proteins is highly conserved from yeast to man, and has been linked to a variety of vital cellular pathways, including the regulation of signal transduction, transcription, protein trafficking, cell-cycle control, apoptosis, stress response, oxidative stress and metabolism.1 Altered expression of 14-3-3 proteins has been associated with many neurodegenerative diseases (e.g. Alzheimer, Parkinson and Huntington diseases),2 epilepsy,3 malignant transformations4 and metabolic disorders such as diabetic cardiomyopathy and hypokalemic nephropathy.5, 6 However, the specific role of 14-3-3 proteins in these cellular processes and debilitating diseases is yet to be fully elucidated. 14-3-3 proteins can regulate protein function by either altering the active state of a protein, modifying the subcellular localization of binding partners or by mediating protein-protein interactions. This often involves the dimeric form of 14-3-3 proteins, which provides an internal binding pocket to enable protein-protein interaction via the recognition of preferential phosphorylated serine or threonine residues.7 Recently, over 200 target proteins were identified for 14-3-3 in HeLa cells,8, 9 and similarly 170 protein interactions were associated with 14-3-3 isoforms in HEK293 cells.10, 11 In vivo, murine 14-3-3γ and ζ were respectively found to associate with 147 proteins in the brain12, 13 and 135 proteins in the testis.14 A comparison of different interactomes has identified only a moderate overlap between 14-3-3 protein binding partners, suggesting potential tissue-specific differences; and this included proteins involved in core cellular processes such as cytoskeleton remodeling, nutrient sensing and metabolic regulation. The diversity of the 14-3-3 protein binding partners in various cell signaling pathways suggests a high degree of complexity and possible hierarchy of 14-3-3 mediated integrative events. This 14-3-3 protein network may enable precise translation of upstream signals and coordination of downstream responses, which could facilitate both transient and organ specific responses. 3 ACS Paragon Plus Environment


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Given the critical position of 14-3-3 proteins at the nexus of diverse physiological functions, targeting 14-3-3 proteins to affect specific branches of the interactome is being considered as a promising therapeutic approach to address pathophysiology.15 However, there is a risk associated with this approach, since little is known about the inter-dependence of specific intracellular pathways and protein networks, particularly in the context of the loss of scaffolding function for individual 14-3-3 isoforms in vivo. The first attempt to analyze the proteome of murine 14-3-3γ knock-out in brain tissues revealed significant changes in proteins involved in regulating growth and development (growth hormone), metabolic signaling (Glucose-6-phosphate isomerase), oxidation (1-Cys peroxiredoxin), chaperone mediated protein folding (CCT-zeta) and vesicular membrane traffic (alpha-SNAP).13 Despite these changes, there was no apparent phenotypic manifestation in the knock-out mice, possibly due to functional redundancy and the presence of six other highly conserved isoforms. However, 14-3-3ε knock-out mice exhibited a pronounced post-natal lethality due to severe defects in neuronal migration and brain development.16 The analysis of changes in 14-3-3 mutant proteomes is of great interest to reveal 14-3-3 function and might underpin the mechanism of lesions in some human 14-3-3 protein related disorders. However, this analysis is hampered by the presence of multiple 14-3-3 isoforms (i. e. β, ε, γ, η, σ, τ and ζ) in mammals.17 In contrast, Drosophila has only two 14-3-3 homologues, 14-3-3ζ and 14-3-3ε, which are highly similar to their respective mammalian homologues (88% and 82% identity at the amino acid level and functional conservation).18 Similar to its murine orthologue,19 Drosophila 14-3-3ζ is indispensable for embryonic development and its loss results in embryonic lethality,20 whereas 14-3-3ε mutant larvae are viable,21 but show reduced survival during larval-to-adult transition (shown herein). These 14-3-3 protein isoforms can form homo- and heterodimers and exhibit functional redundancy,22 raising more questions about which combinatory pair of 14-3-3 isoforms may exert specific effects on a particular cellular 4 ACS Paragon Plus Environment

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function. The larval-to-adult transition is known to depend on energy supply coming from the histolysis and recycling of larval tissues such as the fat body, which is analogous to vertebrate adipose and liver tissues. Thus, we have undertaken a proteomic analysis of fat body tissue from 14-3-3ε mutants with the aim of identifying molecular pathways that depend on this isoform. Using this approach, we have found that the loss of 14-3-3ε affects proteins involved in protein biosynthesis, glycolysis, tricarboxylic acid cycle and lipid metabolic pathways. There was a significant deregulation of Fat body protein 1 (Fbp1), Alcohol dehydrogenase (Adh) and Lamin, which are each linked to the function of Ecdysone Receptors; the steroid hormone ligand-dependent nuclear receptors that trigger major developmental transitions.23 These findings implicate 14-3-3ε in biosynthetic and metabolic pathways, which may in part function through the regulation of the steroid hormone receptor pathway.

EXPERIMENTAL PROCEDURES Fly Stocks All stocks were maintained in standard medium in plastic vials at 25° C, with a 12 hour light/dark cycle.21 For targeted expression of genes of interest, the yeast GAL4-UAS system was used.24 Fat body specific expression of transgenes from the UAS was driven by CGGAL4.25 RNA silencing stocks UAS-14-3-3ζRNAi (v48724), UAS-14-3-3εRNAi (v108129) and two independent insertion lines of UAS-laminRNAi (v107419 and v45635) were obtained from the Vienna Drosophila RNAi Centre (Austria).26 Transgenic stock UAS-EcR-B1RNAi was obtained from the Bloomington Drosophila Stock Center (Indiana University, USA). The protein-null allele 14-3-3εj2B10 (a P/SUPor-P/insertion at position +2 of the 5-UTR) was as 5 ACS Paragon Plus Environment


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previously described.17 The fat body restricted expression of full length 14-3-3εj2B10 rescued the survival of 14-3-3ε mutants to adulthood,21 indicating the lack of any second site mutations in these flies. The survival assay was performed by scoring the number of adult flies eclosed from pupal cases (n = 500), using a previously described protocol.21 The stages of larval development were assessed by the blue-gut method.27 The larvae were reared at 25° C, with a 12 hour light/dark cycle, on standard medium supplemented with 0.05% bromophenol blue.27, 28 The wandering larvae with guts containing bromophenol blue were determined as early 3rd larval instar (-8 hours of Puparium Formation, PF), while larvae with half-clear guts were referred to as late 3rd larval instar (-4 hours PF). The immobile white pupae that began to pupariate were referred to as 0 hours PF. Preparation of Protein Samples and 2D-DIGE Differential Proteomics Analysis Fat body tissues were isolated from wild-type (w1118) control and 14-3-3ε j2B10 mutant larvae (-4 hours PF); with the expectation of detecting primary proteomic changes in the context of relatively normal tissue morphology as previously characterized.21 Four biological samples for each genotype included fat body tissues from 30 larvae. Samples were resuspended in TUC4 (7 M Urea, 2 M Thiourea, 30 mM Tris, 4% (w/v) CHAPS, 5% Protease Inhibitor PefablocSC (Roche, Germany), 5% (w/v) PSC protector reagent (Roche, Germany)), mechanically homogenized and lyzed for 1 hour on ice. The samples were then centrifuged at 14,000 g for 30 minutes and the supernatants were collected into fresh sample tubes. The proteins were precipitated using a 2D Clean-up kit (Bio-Rad Laboratories, USA) according to the manufacturer’s protocol and resuspended in TUC4. Protein concentrations were determined by an EZQ protein quantitation assay (Invitrogen, USA) using an ovalbumin standard curve according to the manufacturer’s instructions. Proteins from each sample were labeled with Cy Dyes (Cy2, Cy3 and Cy5) according to the manufacturer’s instructions (GE 6 ACS Paragon Plus Environment

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Healthcare, Australia). Briefly, 100 µg of protein from each sample was labeled with 200 pmol of either Cy3 or Cy5, and a reverse-labeling approach was used to avoid dye-labeling bias. The internal pooled standard was labelled with Cy2, which was used for the normalization and quantitation of the Cy3- and Cy5-labeled proteins. Four samples of the wild-type control and 14-3-3εj2B10 mutant fat body tissues were coresolved in 2D-DIGE gels. Isoelectric focusing (IEF) was performed on immobilized nonlinear pH 3-11 gradients of 24 cm length (GE Healthcare, Australia) using an Ettan IPGphor II system (GE Healthcare, Australia) until 27,000 Volt hours were reached. The strips were equilibrated in equilibration buffer (Gel Company, USA) with 6 M urea added, containing 10 mg/mL of DTT for 15 minutes, followed by the exchange of solution for equilibration buffer that contained 25 mg/mL of iodoacetamide in place of DTT. SDS-PAGE in the second dimension was carried out using 2D gel DALT NF precast polyacrylamide gels (T=12.5%) (Gel Company, USA). Electrophoretic separation was performed using an Ettan DALT 12 Separation Unit (GE Healthcare, Australia) in the electrophoresis buffer provided with the pre-cast gels. The gels were scanned using an Ettan DIGE Imager (GE Healthcare, Australia) at 100 µm resolution with exposure times set at 2.5 seconds for Cy2, 0.6 seconds for Cy3 and 0.7 seconds for Cy5. Images were analyzed using DeCyder 2D software (version 7.0; GE Healthcare, Australia) according to a previously published protocol.29 After scanning, the gels were fixed (40% ethanol, 10% acetic acid) overnight, washed twice for 10 minutes in aqua bidest, and then stained overnight using coomassie brilliant blue dye (20% methanol, 8% ammonium sulphate, 1.6% phosphoric acid, 0.08% coomassie brilliant blue G-250). The wild-type control and 14-3-3εj2B10 mutant fat body samples were compared using a twotailed Student’s t-test to define proteins that were differentially expressed. A power of 80% was calculated for a 2.5 fold change in protein abundance, whereas the power calculated for the acceptance criterion of a 2 fold change in abundance was 58% (Table 1). The false 7 ACS Paragon Plus Environment


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discovery rate for the differentially expressed protein spots was calculated to be 23.9%. Protein spots that exhibited an absolute fold change ≥ 2 (20 protein spots; Table 1) were excised from the gel and analyzed by Liquid Chromatography-Electrospray Ionization IonTrap Tandem Mass Spectrometry (LC-ESI-IT MS/MS). In the case of several proteins being identified per spot, the protein exhibiting the largest exponentially modified protein abundance index (emPAI) was selected.30 The power31 and false-discovery rate calculations32 were performed according to a previously published protocols.29 Spots were excised from the gel automatically using the Ettan Spot Cutting Robot (GE Healthcare, Australia), washed in 500 µL of 50 mM ammonium bicarbonate (NH4HCO3) and processed as follows. Destained with 50 mM ammonium bicarbonate in 30% (v/v) acetonitrile, digested with 100 ng of sequencing grade modified trypsin (Promega, USA) in 5 mM ammonium bicarbonate, 10% (v/v) acetonitrile (ACN); and the resulting peptides were extracted with three washes of 1% (v/v) formic acid (FA) in water, 1% (v/v) FA in 50% (v/v) ACN and 100% ACN, respectively. The volumes of the resulting peptide extracts were reduced by vacuum centrifugation. Protein Identification by LC-ESI-IT MS/MS Vacuum concentrated samples were resuspended with 0.1% (v/v) FA in 2% (v/v) ACN to total volume of ~ 8 µL. LC-ESI-IT MS/MS was performed using an online 1100 series HPLC system (Agilent Technologies, USA) and HCT Ultra 3D-Ion-Trap mass spectrometer (Bruker Daltonics, USA). The LC system was interfaced to the MS using an Agilent Technologies Chip Cube operating with a ProtID-Chip-150 (II), which integrated the enrichment column (Zorbax 300SB-C18, 4 mm, 40 nL), analytical column (Zorbax 300 SB-C18, 150 mm x 75 µm) and nanospray emitter. Sample was loaded on the enrichment column at a flow rate of 4 µL/minute in Mobile Phase A (0.1% FA in 2% (v/v) ACN) and resolved with 1-30% gradient 8 ACS Paragon Plus Environment

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of Mobile Phase B (0.1% FA in 98% (w/v) ACN) over 32 minutes at 300 nL/min. Ionizable species (300 < m/z < 1,200) were trapped and the two most intense ions eluting at the time were fragmented by collision-induced dissociation. Active exclusion was used to exclude a precursor ion for 30 seconds following the acquisition of two spectra. MS and MS/MS spectra were subjected to peak detection and deconvolution using DataAnalysis (Version 3.4, Bruker Daltonics, USA). Compound lists were exported to mascot generic format (mgf) then submitted to Mascot (Version 2.3) using Mascot Daemon. Drosophila and Mammalia entries (71,821 sequences) from SwissProt (release 2014_02) were searched with the following parameters: monoisotopic mass values, peptide mass tolerance +/- 0.3 Da, fragment mass tolerance +/- 0.4 Da, enzyme specificity Trypsin (up to 2 missed cleavages), fixed modification Carbamidomethyl (C) and variable modification Oxidation (M). Protein identifications were made on the basis of having at least two matching unique peptides with individual ion scores above the mascot homology threshold. If no homology threshold could be derived, the identity threshold was used. Immunostaining and Confocal Microscopy Antibodies used for immunofluorescence were: rabbit anti-14-3-3ε antibody (two independent batches, A8 and A10, from two inoculated rabbits; a gift from Cheng-Ting Chien, Institute of Molecular Biology, Taiwan).33 Antibodies included mouse anti-EcR DDA2.7, Ag10.2, AD4.4, 15G1A, anti-Lamin ADL101 (Developmental Studies Hybridoma Bank, USA),34, 35 Secondary anti-IgG antibody conjugates with Cy3 and Cy5 labels were obtained from Jackson Immuno Research Laboratories (USA). The Alexa Fluor® 488 Phalloidin and Hoechst 33258 DNA stain were obtained from Invitrogen (USA). Drosophila fat body tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) and stained according to a standard protocol.21 The imaging was performed using a Zeiss LSM710 9 ACS Paragon Plus Environment


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NLO confocal microscope equipped with Argon gas, 543 nm and 633 nm solid-state lasers (Zeiss, Germany) and a two-photon Mai-Tai®, tunable Ti:Sapphire femtosecond pulse laser (Spectra-Physics, USA). Each confocal micrograph represented 1.5 µm thin optical sections. Protein and Gene Expression Analysis Western blotting was used to determine protein expression and was performed as previously described,36 using the antibodies listed above. A total protein extract was prepared from the fat body tissues using a previously described protocol.21 The stages of larval development were assessed as previously described.37 The goat anti-Gapdh (Imgenex, USA) was used as a loading control, as there were no changes in the total amount of Gapdh protein detected by Western blotting. For the analysis, three biological samples were used for each genotype, with each sample containing 10-20 larvae. For quantitative real-time PCR (qRT-PCR) analysis, RNA was isolated from the fat body tissues of 30 larvae per sample using an RNAqueous® kit (Ambion, USA) according to the manufacturer’s protocol. Three independent biological samples were analyzed for each genotype. cDNA was synthesized using a High Capacity RNA-to-cDNA kit (Applied Biosystems, USA). qRT-PCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, USA) using Fast SYBR Green Master Mix kit (Applied Biosystems, USA). The mRNA expression levels were normalized against the endogenous control gene rp49 using the ∆∆C method. PCR primers were obtained from GeneWorks, Australia. The T

primers used for the qRT-PCR were: fbp1 (CG17285) forward, 5’GCGTTGTGGCTGCTGGAAGGA-3’, and reverse, 5’CTCCAAGGACATGCGGTCTGCG-3’; adh (CG3481) forward, 5’TGCAGTTCAGCAGACGGGCT-3’, and reverse 5’-CCTCCCAGACCGGCAACGAAA-3’; lamin (CG6944) forward, 5’-GCGCTCCACTCGTGTGCGTA-3’, and reverse, 5’10 ACS Paragon Plus Environment

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ATCCGCGCATTGAGGTCGGC-3’; rp49 (CG7939, used as an endogenous control) forward, 5’-CGAGTTGAACTGCCTTCAAGATGACCA-3’, reverse 5’GCTTGGTGCGCTTCTTCACGATCT-3’. FLIM System and Imaging A Mai-Tai® femtosecond pulse laser (Spectra-Physics, USA), a time correlated single photon-counting (TCSPC) and lifetime photomultiplier tube (Becker & Hickl GmbH, Germany), were used to acquire lifetime images. The images were acquired with a 920 nm excitation wavelength with a laser power of 100 mW. A Plan-Apochromat 63x Oil (NA 1.40) objective lens was used to collect the emitted fluorescence, which was transmitted through a dichroic mirror and optical filter for detection by photon-multiplier tubes. FLIM images were acquired with a scan speed of 512×512 pixels/13.6 s at a 9.0 µm x 9.0 µm size. FLIM data was analyzed with the software data analysis package SPCImage (Becker & Hickl GmbH, Germany). The iterative convolution with double-exponential model was used to calculate the decay parameters for each individual pixel within the scan. The τ1 and τ2 of the doubleexponential fit was used to analyze the photon decay profiles from the scanned images of the fat body samples, which were fixed and immune-stained with (i) mouse anti-EcR/anti-mouseAlexa 488 and (ii) chicken anti-14-3-3ε/anti-chicken-Alexa 555 (Invitrogen, USA). Alexa 488 immune labelled anti-EcR was used as the energy donor and Alexa 555 immune labelled anti-14-3-3ε was used as the energy acceptor.

Statistical Analysis Data was presented as the mean ± SEM. The difference between group means was assessed by one-way analysis of variance (ANOVA), with individual group variance assessed by a Bartlett’s test. Where the level of significance was P < 0.05, post-hoc tests were performed using a Tukey’s multiple comparison test (GraphPad Prism version 5.00 for Windows, 11 ACS Paragon Plus Environment


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GraphPad Software, USA).

RESULTS Changes in the Fat Body Tissue Proteome Upon the Loss of 14-3-3εε Drosophila 14-3-3εj2B10 homozygous mutants were employed (herein referred to as 14-3-3ε) for the analysis of the 14-3-3ε-dependent protein network. The loss of 14-3-3ε protein was confirmed in fat body tissues by Western blotting at three consecutive developmental stages: early 3rd larval instar (-8 hours of Puparium Formation, PF), late 3rd larval instar (-4 hours PF) and white pupa (0 hours PF), and compared to wild-type control (Figure 1A). The expression of 14-3-3ε protein appeared to be crucial for larval-to-adult transition (metamorphosis), since its loss led to 56 ± 5% 14-3-3ε mutants failing to reach adulthood (Figure 1B). A 2D-DIGE proteomic approach was employed to identify proteins affected by the loss of 14-3-3ε at the stage of metamorphosis. There were 879 protein spots detected on the master gel (repeat gels detected 496, 356 and 472 spots in common), 72 of which exhibited significantly different abundance for wild-type control and 14-3-3ε mutants. From these 72 spots, 20 spots, representing 16 proteins, had an absolute fold change ≥ 2 (Figure S-1, Tables 1 and S-1). In the 14-3-3ε mutant proteome, there were changes in structural constituents of the 60S large ribosomal subunit (60S ribosomal protein L7) and 40S small subunit (40S ribosomal protein S3a); along with the regulator of translational elongation (Elongation factor 2). There was a significant increase in molecular chaperone (Heat shock protein 26), which is essential for protein folding in the endoplasmic reticulum. There were two proteins involved in cellular architecture (Lamin and Cofilin) that had altered abundance following 14-3-3ε loss. The changes in protein expression may have been related to a response to metabolic stress as 12 ACS Paragon Plus Environment

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proteins (Glycogen phosphorylase and Enolase) involved in glucose metabolism showed altered levels in 14-3-3ε mutants. Importantly, Enolase showed increased abundance in 14-33ε mutants. The glycolytic pathway proteins have significant connections to the activity of the tricarboxylic acid cycle, and there was also altered expression of Probable citrate synthase and Succinate dehydrogenase (ubiquinone) flavoprotein subunit in 14-3-3ε mutants. Moreover, there was deregulation in protein of the electron transport/respiratory chain and oxidative phosphorylation, ATP synthase subunit d. In addition, a protein with proposed functions in lipid metabolism, Glycerol-3-phosphate dehydrogenase (NAD+), showed altered abundance in 14-3-3ε mutants. Increased protein abundance was observed for Alcohol dehydrogenases (Adh) and Fat body protein 2, which are thought to be required for alcohol catabolic processes during both carbohydrate and lipid metabolism. 14-3-3ε loss also resulted in the altered abundance of Fat body protein 1 (Fbp1), which is proposed to function as a protein transporter. There was some overlap in protein signatures of fly 14-3-3ε and murine 14-3-3γ mutant proteomes,13 with both showing altered abundance of proteins involved in metabolic pathways. These findings suggested that the loss of 14-3-3ε might exert an effect on both anaerobic and aerobic energy metabolism.



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Figure 1. 14-3-3ε mutants have reduced survival rates. (A) Total protein extracts from fat body tissues of wild-type (WT) control, EcRRNAi and 14-3-3ε j2B10 mutants were assessed for 14-3-3ε protein expression by Western blotting with either anti-14-3-3ε or anti-Gapdh antibodies. Samples were collected at three developmental stages: early 3rd larval instar (-8 hours PF), late 3rd larval instar (-4 hours PF) and white pupa (0 hours PF). (B) Histograms showing the percentage of larval-to-adulthood survival of WT, 14-3-3εj2B10 mutants, EcRRNAi, and EcRRNAi silencing in 14-3-3ε j2B10 mutant background (EcRRNAi; 14-3-3εj2B10). For each genotype, n ≥ 500 larvae were analyzed and results are presented as mean ± SEM. One-way ANOVA analysis of variances showed significant differences between group means (P < 0.05). Tukey's multiple comparison test showed significant differences between the means of genotypes depicted by the different letters on the bars (P < 0.05). 14 ACS Paragon Plus Environment

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Table 1. Combined 2D-DIGE gel and LC-ESI-IT MS MASCOT search results summary. aSpot number corresponding to Figure S-1, bAverage ratio of protein abundance of wild-type control to 14-3-3ε mutant samples in 2D-DIGE experiment, cUniprot Accession, d

Number of unique, significant peptides identified by MASCOT. Entries in bold depict

protein with the highest emPAI within the spot.

a

№

1

Av. ratiob

8.49

P

0.006

2

2.71

0.042

3

3.37

0.015

4

3.06

0.007

5

3.98

0.025

6

12.56

0.004

7

11.23

0.006

8

10.29

0.002

9

10

-2.06

-4.27

0.003

0.001

11

3.19

0.013

12

-2.19

0.001

13

-2.62

Proteome Analysis of Drosophila Mutants Identifies a Regulatory Role

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