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Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with... Background: During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results: Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions: Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ. Background of the organism’smassand areusedfor generation of Ionic gradients across cell membranes (bioelectricity) are electric shocks for predation or protection [2]. utilized by all organisms. Some fish have developed Developmental studies have shown most electric extreme adaptations of bioelectricity with the evolution organs are derived from muscle anlage tissue; the excep- of electric organ systems. It is thought that electric tion is the neurogenic development of the Sternarcus organs have evolved independently six or seven times in electric organ. Several basic differences exist amongst fish and can be classified as either weak or strong, which myogenic-derived electric organs. The location of the is reflective of the size and function of the organs within myogenic-derived electric organs varies from gill (Tor- the fish. For example, Gymnotids are weakly electrogenic pedo), tail (Raja, Gnathonemus, Gymnarchus, Gymnotus), and ocular muscle (Astroscopus). Strong electrogenic and only possess accessory electric organs used for elec- troreception and electrolocation [1]. In contrast, Torpedi- organs lose the characteristic myofibrils and sarcomeres nid and Electrophorous arestronglyelectrogenicand during transdifferentiation of the organ. In contrast, possess organs that account for approximately one-third weakly electrogenic Gymnarchids and Mormyrids main- tain the myofibrillar structures into adulthood [3]. Organs differinthe abilitytoinitiateand propagatean action potential. Generally, marine fish possess organs * Correspondence: ehoffman@cnmcresearch.org Department of Biochemistry and Molecular Genetics, IBS, George with electrically inexcitable membranes (lacking voltage- Washington University, Washington DC, USA sensitive sodium channels), whereas fresh water fish have Full list of author information is available at the end of the article © 2011 Mate et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mate et al. Skeletal Muscle 2011, 1:20 Page 2 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 organs that are electrically excitable (have voltage-sensi- look at how the innervated membrane of Torpedo elec- tive sodium channels). Succinctly put, the degree of mus- trocytes relates to the NMJ. cle likeness of precursor cells differs among electrogenic From a developmental perspective, Torpedo electroblasts fish families. These anatomical differences may represent are derived from the mesodermal layer that gives rise to an evolutionary divergence required for the performance branchial arches from which the electric organ and gill of strong and weak electric organs. musculature form. The primordial electric organ first gen- erates ‘muscle-like’ cells that are multinucleated and have The research presented here focuses on Torpedo cali- a single striated myofibril, reminiscent of myotubes in ske- fornica (Pacific electric ray), a cartilaginous fish within letal muscle. At this stage, meromyosin is expressed at the Chondrichthyes class and Torpedinidae family. This species evolved an electric organ capable of generating high levels and the single striated myofibril has a similar approximately 45-50 V (electron motive force 110 mV), diameter to actin-myosin myofibrilar structures compos- released in 414 monophasic discharges that last 3-5 ms ing sarcomeres [26]. As the electroblast transforms into an each, with a total power output up to 1 kW [4-6]. An electrocyte at the onset of electromotor neuron synapto- electrocyte from the electric organ of Torpedo nobiliana genesis, Z-disc-like structures disassemble and degenerate (Atlantic Torpedo with similar length but twice the completely [27]. It is thought that the electromotor neuron weight of T. californica) measures 5-7 mm in diameter sends signals that induce the degeneration of the myofibril by 10-30 μm thick and 500-1,000 electrocytes are stacked structures, allowing the elongated cells to flatten into thin into columns, all with ventrally innervated and dorsally electrocytes [28]. Desmin, or a light intermediate filament, non-innervated membranes aligned [5]. Approximately replaces the myofibril following disassembly, but keratin, a 50 A of current has been measured from the parallel protein typically associated with epithelium, dominates the stacks composing the electric organ of T. nobiliana,and intracellular architecture [26,29]. Upon denervation, myo- about 1 A measured from the series-aligned electrocytes fibril-like structures reappear near the synapse but are of Electrophorous [6]. The postsynaptic membrane of the highly disorganized and short lived [28]. In addition, tran- electric organ in Torpedo is rich in nicotinic acetylcholine script evidence was shown for myoblast determination receptors (AChR) and is multi-innervated with dendrites protein and myogenic factor 5 expression in adult Torpedo from four large, heavily myelinated neurons descending electric organ without evidence of protein expression, sug- from the electric lobe of the brain. The non-innervated gesting strong post-transcriptional regulation of messenger membrane is extensively invaginated into structures RNA translation and maintenance of a muscle-like pro- gramming [30]. No synapse is observed until late phase of called caniculi that may be reminiscent of skeletal muscle electric organ development when the ventral face of elec- T tubules [5]. The electrocytes are multinucleated and filled with a gelatinous cytoplasm with an extensive fila- troblasts develop subneural arches that have increased mentous network. The electrocyte itself has low internal levels of acetylcholinesterase (AChE) and AChRs that resistance with low resistance across the non-innervated reach 300 times the level in skeletal muscle [27,28,30]. membrane [7]. Insulating septa, extracellular matrix com- From an anatomical perspective, post-transdifferentia- ponents, blood vessels, nerves, and amoeboid cells have tion, the electroneuroelectrocyte synapse (electroplate) also been described in intercellular regions [8]. appears to maintain characteristic synaptic folds and a Proteins that were originally identified in the Torpedo high density of membrane particles as revealed by elec- electric organ and subsequently studied in higher verte- tron microscopy and freeze-etch replicas of electric brates include agrin, dynein, chloride channel, and rapsyn organ tissue [5,31,32]. However, the extensive nerve [9-12]. Also identified in the electric organ are a, b, δ, terminal network, formed by four or five electromotor and g AChR subunits, a and b dystroglycan, dystrophin, neurons covers nearly the entire postsynaptic mem- syntrophin, dystrobrevin, receptor tyrosine kinase, tyro- brane, differs from the minute motor neuron connection sine protein kinase fyn, protein tyrosine kinase fyk, and with a single mammalian myofiber [5,18]. desmin [13-23]. The electric organ has been used to Despite the electric organ being used as a model for the define the structure and function of creatine kinase and mammalian NMJ, current literature describes a number of AChR pore [24,25]. These proteins also are characterized NMJ-associated proteins that have not been characterized at the mammalian neuromuscular junction (NMJ) or are in the electric organ. One such protein is low-density lipo- components of skeletal muscle, which is consistent with protein receptor-related protein 4 (Lrp4), which forms a the Torpedo electric organ representing an extreme adap- complex with muscle-specific tyrosine-protein kinase receptor (MuSK) to facilitate neuronal agrin binding and tation of muscle tissue and the NMJ. Thus, the electric subsequent initiation of downstream signaling for tran- organ has served as a model to study the NMJ. However, scriptional activation of synaptic genes or AChR clustering the number of NMJ proteins described in current mouse, cell culture, and Drosophila studies demands a closer [33,34]. It is likely that agrin plays a similar role in the Mate et al. Skeletal Muscle 2011, 1:20 Page 3 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 electric organ as in the NMJ, transferring communication Results between the nerve and postsynaptic tissue, but its down- Validation of Torpedo-specific protein identification stream target, MuSK, is loosely defined. The published Tissue fractionation, gel electrophoresis, in-gel tryptic sequence for a tyrosine kinase receptor transcript digestion, and mass spectrometry (MS) analysis of the extracted from the Torpedo electric organ not only electric organ provided a global proteomic profile com- encodes extracellular Ig and frizzled domains and intracel- prising 435 proteins (count includes the different subu- lular C-terminal tyrosine kinase domains like human nits, subunit isoforms, isoforms, and types of proteins MuSK but also encodes a kringle-like domain that is with unique identifiers and does not include identical encoded in proteases and Ror receptor tyrosine kinases proteins found in different spots). Fractionating the elec- [20,35,36]. The orthology of the Torpedo tyrosine kinase tric organ decreases the complexity of its protein consti- receptor with mammalian MuSK was demonstrated by tuents and improves detection of low-abundant proteins inducing AChR clustering in the presence and absence of and protein digestion by decreasing the number of pro- agrin [37]. Furthermore, the cytoplasmic domain of MuSK teins resolved through electrophoresis in a single lane. binds directly to the tetratricopeptide repeat domain of Confidently identified proteins were determined by a rapsyn, supporting the presence of MuSK and possibly its combination of nanospray ESI-LTQ MS/MS spectral downstream effectors in the electric organ [38]. searches of our in silico translated cDNA library, MALDI Aside from this knowledge, electrocyte components TOF/TOF MS and MS/MS spectra from two-dimen- are undefined mainly because studying the proteome of sional gel spots, and through cross-species spectral T. californica is limited. A map of its genome does not matches to UniProtKB/Swiss-Prot amino acid sequences currently exist to computationally derive a hypothetical (Additional file 1) [40]. For peptides processed by ESI- protein profile and public databases contain sparse LTQMS/MS andsubsequentidentificationby sequence data for this species. While the Torpedo gen- SEQUEST, our threshold for positive protein identifica- ome has not yet been reported, the genome sequence tion was two independent peptides, ΔCn >0.1, a variable likely would be a relatively blunt instrument to under- threshold of Xcorr versus charge state: Xcorr = 1.9 for stand the highly specialized structure and function of z = 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, the Torpedo electric organ. For this reason, we sought protein Xcorr >40, and a peptide probability based score to understand the molecular components of the electric with a P value <0.01. For peptides processed by MALDI- organ using a combined mRNA (expressed sequence tag TOF/TOF MS and subsequent identification by MAS- (EST)) and proteomics approach. COT http://www.matrixscience.com/search_form_select. We have previously reported a preliminary proteome html, our threshold for positive criteria were protein based on two-dimensional matrix-assisted laser deso- score CI >95%, protein score >69, and proteins with iso- rption/ionization - time of flight/time of flight mass spec- electric points (PI) and molecular weights (MW) that trometry (MALDI-TOF/TOF MS) of soluble proteins and match the gel spot. To represent a concise proteome for shotgun proteomics of insoluble electric organ fractions in the electric organ, all accepted protein identifications which mass spectral mapping was based on a preliminary were further processed by selecting the highest scoring library composed of 607 cDNA sequences [39]. More identification amongst redundant proteins and the recently, we reported sequencing the transcriptome of T. removal of lower scoring duplicates. Isoforms and sub- californica to assemble a Torpedo cDNA library composed types of proteins were treated as unique identifications of 10,326 sequences assembled into 4,243 non-overlapping and are included in the 435 proteins listed in Additional contigs [40]. Here, we present a comprehensive electric file 1. organ proteome as defined by one-dimensional SDS- As an initial step to validate our data from SDS-PAGE, PAGE followed by nanospray electrospray ionization we performed mass spectral mapping using known Tor- quadrupole linear ion-trap tandem mass spectrometry pedo proteins. We created a validation database consist- (ESI-LTQ MS/MS) and two-dimensional isoelectric focus- ing of all Torpedo protein sequences found on public ing (IEF) SDS-PAGE followed by MALDI-TOF/TOF MS- access databases (GenBank and UniProtKB) to search based approaches of electric organ fractions in which mass raw spectra. We identified 20 out of 44 Torpedo proteins spectral mapping was performed using sequences from listed on the public access databases (Figure 1, Additional 10,326 Torpedo cDNA sequences and The Universal Pro- file 2). The Torpedo AChR (a, b, g, δ) and sodium/potas- + + tein Resource (UniProtKB/Swiss-Prot). Our results sium-transporting ATPase (Na /K -ATPase) subunits (a, demonstrate concordance between skeletal muscle, NMJ, b) were amongst the most abundant proteins identified. and electric organ proteomes. In addition, the electric These two protein complexes are functionally related in organ expresses several uncharacterized proteins that may that the binding of a ligand to one complex also influ- + + function at a synapse. ences the activity of the other [41]. The Na /K -ATPase Mate et al. Skeletal Muscle 2011, 1:20 Page 4 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 1 Identification of Torpedo proteins listed in public access databases. To validate tandem mass spectrometry (MS/MS) data against species-specific sequences, spectra acquired via MS/MS analysis of electric organ fractions were analyzed by the SEQUEST algorithm in BioWorks 3.3.1 software, crossreferencing known and characterized Torpedo proteins listed in GenBank. Peptide acceptance criteria was set at ΔCn >0.1, a variable threshold of Xcorr versus charge state: Xcorr = 1.9 for z = 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr >40, and a peptide probability based score with a P value <0.01. Protein identifications were compared with a search against UniProtKB (Swiss-Prot and TrEMBL) release 14.0, all species, to maintain consistency with databases used and protein accession numbers reported. Proteins identified are categorized by the likelihood and appropriateness of detection based on protein subcellular location or on the quality of data on public access databases. is essential for maintaining the electrochemical potential of the mammalian NMJ and these were also identified of electrocytes, and activity of this ion exchanger is in our Torpedo electric organ proteome (14-3-3 protein required for generating electric pulses that Torpedo uses g, heat shock protein (HSP)90b,HSP 70 kDaprotein, for predation. Of the remaining 24 proteins, we do not laminin subunit a-2, laminin subunit b-2, laminin subu- expect to identify 13 proteins that are expressed in brain, nit g-1, stress-induced phosphoprotein 1, dynamin 1, in neurons, or in immune cells. We did not identify 11 vesicle-fusing ATPase, Ras-related C3 botulinum toxin listed Torpedo proteins that were previously character- substrate 1, prostaglandin E synthase 3, guanine nucleo- ized through study of the electric organ (Additional file tide-binding protein G(I)/G(S)/G(T) subunit b-1, G sub- 2). However, all but one of these sequences are unre- unit b-1, subunit b-2-like1, G(s) subunit a,and Rho viewed by the UniProtKB consortium. In addition, the GDP-dissociation inhibitor 1, Ras-related protein R-Ras2 sequence information for a and b dystroglycan was (TC21)) [43,44]. Additionally, several presynaptic pro- extremely limited on UniProtKB (a single peptide) and teins localized to both synaptic vesicles (synaptic vesicle this would, by definition, fall below our two peptide mini- membrane protein VAT-1, synaptotagmin-B, choline mum requirement for identification from our MS/MS transporter-like protein 1), and the electromotor neuron scans. Another protein, Torpedo receptor tyrosine kinase, membrane were identified, showing representation of was translated in UniProtKB/Swiss-Prot from 2 small the presynaptic apparatus of the electric organ. overlapping ESTs and was in low abundance in the cDNA library previously described (2 in 90,000 clones), Torpedo cDNA sequences with both nucleotide and suggesting that the very low abundance underlies our protein sequence similarity to human ORFs inability to identify it by either MS/MS or via our cDNA Electric organ peptides mapped to the uncharacterized sequencing [20]. Lastly, dystrophin has multiple isoforms human ORFs C1orf123 and C6orf130, which aligned with and we identified two of the isoforms (Additional file 2) high sequence similarity to Torpedo cDNA sequences, [42]. Thus, we identified 16 of 17 listed Torpedo proteins supporting their expression in the electric organ. Human that have reviewed sequences and are known to be nucleotide and protein sequences were obtained from expressed in the electric organ, demonstrating the quality GenBank for alignment with translated Torpedo cDNA and scope of our data. sequence (Expasy translate tool) using EBI ClustalW Our proteome profile included two uncharacterized (default parameters with gonnet matrix). Sequence align- open reading frames (ORFs; C1orf123 and C6orf130) ments between human ORF nucleotide and protein and several well characterized mammalian NMJ pro- sequences and Torpedo cDNA nucleotide and translated teins, including AChR and AChR-associated proteins. nucleotide sequences are shown in Figure 2. In all, 79% Recent publications have characterized new components of amino acids in the translated Torpedo cDNA sequence Mate et al. Skeletal Muscle 2011, 1:20 Page 5 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 A. >gi|8923540|ref|NM_017887.1|C1orf123, mRNA; >sp|Q9NWV4|CA123_HUMAN UPF0587 protein C1orf123 >Contig[3573] Torpedo cDNA; >Contig[3573] Torpedo cDNA frame3 translation Experimental Peptides aacggcaagggcggcagccagcaccgggcggagagggctaccatggggaaaatcgcgctg CLUSTAL 2.0.12 multiple sequence alignment N G K G G S Q H R A E R A T M G K I A L ------------------------------------------------------------ SeqA Name Len(aa) SeqB Name Len(aa) Score caactcaaagccacgctggagaacatcaccaacctccggcccgtgggcgaggacttccgg =================================================================================== Q L K A T L E N I T N L R P V G E D F R 1 sp|Q9NWV4|CA123_HUMAN 160 2 Contig[3573]5'3'Frame3 512 76 cagttgaaagcgactttggaaaatatcagcaagttgcggccggacggagaggatttccgc =================================================================================== Q L K A T L E N I S K L R P D G E D F R tggtacctgaagatgaaatgtggcaactgtggtgagatttcggacaagtggcagtacatc sp|Q9NWV4|CA123_HUMAN MGKIALQLKATLENITNLRPVGEDFRWYLKMKCGNCGEISDKWQYIRLMD 50 W Y L K M K C G N C G E I S D K W Q Y I Contig[3573]5'3'Frame3 ---FGLQLKATLENISKLRPDGEDFRWYLKLKCQNCGEVSDKWQYVTLMN 47 tggtacctgaagttgaaatgtcagaattgcggtgaagtttccgataaatggcagtatgtc :.**********::*** *********:** ****:******: **: W Y L K L K C Q N C G E V S D K W Q Y V cggctgatggacagtgtggcactgaaggggggccgtggcagtgcttccatggtccagaag sp|Q9NWV4|CA123_HUMAN SVALKGGRGSASMVQKCKLCARENSIEILSSTIKPYNAEDNENFKTIVEF 100 R L M D S V A L K G G R G S A S M V Q K Contig[3573]5'3'Frame3 SAPLKGGRGSANMIQRCKLCSRENSIDILKNTIKPYNAEDSERFKTIVHF 97 acattaatgaacagcgccccactcaaaggtgggagaggaagtgccaacatgatacaaagg *..********.*:*:****:*****:**..*********.*.*****.* T L M N S A P L K G G R G S A N M I Q R tgcaagctgtgtgcaagagaaaattccatcgagattttaagcagcaccatcaagccttac sp|Q9NWV4|CA123_HUMAN ECRGLEPVDFQPQAGFAAEGVESGTAFSDINLQEKDWTDYDEKAQESVGI 150 C K L C A R E N S I E I L S S T I K P Y Contig[3573]5'3'Frame3 ECRGLEPVDFQPQAGFAAEGTESGTKFDEINLLEKDWNEYDEKIQESVGI 147 tgcaagttatgctcaagagagaactccattgatattctgaagaataccatcaagccatac ********************.**** *.:*** ****.:**** ****** C K L C S R E N S I D I L K N T I K P Y aatgctgaagacaatgagaacttcaagacaatagtggagtttgagtgccggggccttgaa sp|Q9NWV4|CA123_HUMAN YEVTHQFVKC---------------------------------------- 160 N A E D N E N F K T I V E F E C R G L E Contig[3573]5'3'Frame3 YDVTHKFVKI-TSLQLIPQPST-MDKSSDQ-TNSSSLSGICLPMQLHCVI 194 aatgctgaagacagtgaaagatttaagaccattgtacatttcgaatgtcggggattggag *:***:*** N A E D S E R F K T I V H F E C R G L E ccagttgatttccagccgcaggctgggtttgctgctgaaggtgtggagtcagggacagcc P V D F Q P Q A G F A A E G V E S G T A ccagttgattttcaaccgcaggctggatttgctgcagaaggaacagaatccggaacaaaa P V D F Q P Q A G F A A E G T E S G T K ttcagtgacattaatctgcaggagaaggactggactgactatgatgaaaaggcccaggag F S D I N L Q E K D W T D Y D E K A Q E tttgatgaaattaatctgctggaaaaggactggaatgaatatgatgagaaaatccaagaa F D E I N L L E K D W N E Y D E K I Q E tctgtgggaatctatgaggtcacccaccagtttgtgaagtgctga S V G I Y E V T H Q F V K C - tcggtgggaatctatgacgtcactcataagtttgttaaaatatga S V G I Y D V T H K F V K I - B. >gi|34147711|ref|NM_145063.2| C6orf130, mRNA; >sp|Q9Y530|CF130_HUMAN Uncharacterized protein C6orf130 >TFI_1_F6.T3 Torpedo cDNA; >TFI_1_F6.T3 Torpedo cDNA frame2 translation Experimental Peptides ggtgacttggctgaagaaacacttaaattctggaaatagcgactcagtatcatggccagc CLUSTAL 2.0.12 multiple sequence alignment G D L A E E T L K F W K - R L S I MA S ctgaagacgaacacccaacgaaaggacgaacaaacggaaaaactaaacaaaatgactagc SeqA Name Len(aa) SeqB Name Len(aa) Score L K T N T Q R K D E Q T E K L N K M T S ================================================================================= Agccttaatgaagatccagaaggaagc------agaatcacttatgtgaaaggagacctt 1 sp|Q9Y530|CF130_HUMAN 152 2 TFI_1_G6.T3 218 55 S L N E D P E G S - - R I T Y V K G D L ================================================================================= tctgcagacaagccactagagggcaatacctttgagatctgttatgtgcaaggtgatctg S A D K P L E G N T F E I C Y V Q G D L sp|Q9Y530|CF130_HUMAN -------------------------------------------------- tttgcatgcccgaaaacagactctttagcccactgtatcagtgaggattgtcgcatgggc TFI_1_G6.T3 CFTNLKIINCLSPSILKDPPHPSHALFSLLPSGRRYRSLKTNTQRKDEQT 50 F A C P K T D S L A H C I S E D C R M G ttctcatgcccagagaaggaagcactggcacattgcatcagcgaagactgcaaaatgaaa F S C P E K E A L A H C I S E D C K M K sp|Q9Y530|CF130_HUMAN -----MASSLNEDPEGS--RITYVKGDLFACPKTDSLAHCISEDCRMGAG 43 gctgggatagctgtcctctttaagaagaaatttggaggggtgcaagaacttttaaatcaa TFI_1_G6.T3 EKLNKMTSSADKPLEGNTFEICYVQGDLFSCPEKEALAHCISEDCKMKAG 100 A G I A V L F K K K F G G V Q E L L N Q *:** :: **. .* **:****:**:.::*********:* ** gcagggatagcagtcttgttcaagaagaaatatggatgtgtcgaggaactacagaatcag A G I A V L F K K K Y G C V E E L Q N Q sp|Q9Y530|CF130_HUMAN IAVLFKKKFGGVQELLNQQKKSGEVAVLKRDGRYIYYLITKKRASHKPTY 93 caaaagaaatctggagaagtggctgttctgaagagagatgggcgatatatatattacttg TFI_1_G6.T3 IAVLFKKKYGCVEELQNQKKKVGDVAVLQKDQRCIYYLITKSLAADKPTY 150 Q K K S G E V A V L K R D G R Y I Y Y L ********:* *:** **:** *:****::* * *******. *:.**** aaaaaaaaagttggggatgttgcagtactacagaaagatcagagatgcatctattacttg K K K V G D V A V L Q K D Q R C I Y Y L sp|Q9Y530|CF130_HUMAN ENLQKSLEAMKSHCLKNGVTDLSMPRIGCGLDRLQWENVSAMIEEVF-EA 142 attacaaagaaaagggcttcgcacaagccaacttatgaaaacttacagaagagtttagag TFI_1_G6.T3 DDLQKSLKAMRDHCLDNGILKISXPKIGCGLDXLXWDKVSAIIXEVFXKX 200 I T K K R A S H K P T Y E N L Q K S L E ::*****:**:.***.**: .:* *:****** * *::***:* *** : attaccaaatcattagcagcagataagcctacttatgacgatctgcagaagagcctcaag I T K S L A A D K P T Y D D L Q K S L K sp|Q9Y530|CF130_HUMAN TDIKITVYTL-------- 152 gcaatgaagtctcattgtctgaagaatggagtcactgacctctccatgcccaggattgga TFI_1_G6.T3 KYLQFTVXSFVEEXLWLP 218 A M K S H C L K N G V T D L S M P R I G . :::** :: gccatgagggaccactgcctggataatggaattttgaagatctcantgccgaagattgga A M R D H C L D N G I L K I S X P K I G tgtggtcttgatcgtctgcaatgggaaaatgtatctgcgatgatcgaggaggtatttgag C G L D R L Q W E N V S A M I E E V F - tgtggactggacnacctgnagtgggacaaagtttctgccataattcnagaagtctttnaa C G L D X L X W D K V S A I I X E V F X Gcaacagacatcaaaattactgtgtacacactctgatga--------------------- E A T D I K I T V Y T L -- --- - - - aagcncaaatatttacaatttactgtgtnctcttttgttgaagaatntctgtggttaccg K X K Y L Q F T V X S F V E E X L W L P Figure 2 Sequence alignments between uncharacterized human open reading frames (ORF) and Torpedo cDNA. Two human ORFs were identified by tandem mass spectrometry (MS/MS) analysis of electric organ fractions by the SEQUEST algorithm in BioWorks 3.3.1 software, crossreferencing our in-house Torpedo californica cDNA library translated into six reading frames. Comprehensive alignments of nucleotide and protein sequences between uncharacterized human ORFs (blue text) and Torpedo cDNA (black text) were compiled from individual ClustalW alignments (default parameters with gonnet matrix) for C1orf123 (a) and C6orf130 (b). ClustalW protein alignment is shown separately to highlight protein sequence similarity with the translated cDNA sequence (Expasy translate tool) and peptides identified by mass spectral mapping (highlighted in red). Start and stop amino acids are highlighted in yellow. Mate et al. Skeletal Muscle 2011, 1:20 Page 6 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Contig [3573] are identical to C1orf123 and 59% of membrane proteins receptor expression-enhancing TF_1_F6.T3 with C6orf130. TF_1_F6.T3 cDNA sequence protein 5, MIP18 family protein FAM96A, WD repeat- is from a single insert and not a contig such that coverage containing protein 1, and matrix-remodeling-associated may be reduced by sequencing errors that were not cor- protein 7. rected for by a consensus of multiple reads like Contig [3573]. However, peptide SLAADKPTYDDLQK is unique Electric organ proteome compared to skeletal muscle to C6orf130 when queried in blastp (word size 2, PAM30 proteome to assess the degree of ‘muscle likeness’ matrix, Homo sapiens) supporting the identification of Electric organ literature claims that a ‘muscle-like’ phe- this ORF. These ORFs demonstrate that further investi- notype is maintained after transdifferentiation. In our gation of the electric organ transcriptome may advance profile, several proteins are considered highly expressed our knowledge of the human proteome. in skeletal muscle or are muscle specific to include AChR subunits a,b,δ,and g, rapsyn, syntrophin, L-lactate dehy- Global proteomic profile classified according to drogenase A chain, phosphoglycerate mutase 2, creatine UniProtKB/Swiss-Prot annotation kinase M-type, cofilin 2, sorcin, 14-3-3 protein g, myosin To obtain a preliminary identification for each Torpedo 11, actin, aortic smooth muscle, transgelin, dystrophin, cDNA sequence identified in the spectral data, all dystrobrevin a, desmin, plectin 1, HSP90b, laminin subu- 2+ sequences were queried in blastx (Swiss-Prot sequence, nit b-2, and SR Ca( )-ATPase 1. As a further step to word size 3, BLOSSUM80 matrix) across all species and compare the skeletal muscle versus the electric organ then against human (See Additional file 1 for a full list of repertoire of proteins, we compared the proteins identi- cDNA sequences with blastx results). Only the top ranking fied in the electric organ presented in this paper to a aligned sequence was accepted for identification of the mouse skeletal muscle proteome produced in our labora- cDNA sequence. The blastx identification allowed cDNA tory using similar methods. Plotting the number of pep- sequences to be grouped with the UniProtKB list of identi- tides for each protein composing the electric organ or fications for classifying the proteins as NMJ, muscle, likely skeletal muscle proteome not only visually displays the in muscle, and metabolic proteins according to Uni- overlap in proteins in both tissues but more importantly ProtKB/Swiss-Prot annotation (Figure 3 andAdditional file displays the detectable proteins unique to each tissue, 3). A total of 33% of proteins are known muscle proteins, those lying on the × and y axis corresponding to tissue 3% of which are located at the NMJ. A total of 36% are type (Figure 6). Analysis showed the distribution of these involved in metabolism and 3% are known to be electric proteins differed in biological processes and molecular organ specific. Ingenuity Pathway Analysis (IPA version function (Table 1). Proteins composing the myofibrillar 8.8-3204) of all UniProtKB and Torpedo cDNA identifica- apparatus or are involved in calcium transport are pre- tions classified 40 molecules (P value 2.93E-09 to 1.18E- sent in theskeletalmuscleproteomeand absent in the 02) involved in skeletal and muscular system development electric organ proteome, as expected given that the elec- and function, the top physiological system designated trocytes are non-contractile cells. However, no common from our list of identifications. NMJ proteins were identified in the skeletal muscle pro- To summarize the electric organ proteome, we used teome but are amongst the highest expressed proteins in IPA Path Designer tool to map the annotated subcellular the electric organ. This was also expected for an analysis location of each protein identified (Figure 4). This also based on total muscle extract given the limited size and provides a virtual model of the electrocyte to assess how number of endplates in skeletal muscle. it may relate to skeletal muscle and the NMJ. The virtual electrocyte revealed several proteins believed to be mus- Discussion cle specific or highly abundant in muscle, confirming the T. californica proteome and defining the NMJ proteome muscle-like identity of the organ (Additional file 3). It for accurate comparison also depicted relatively intact pathways for energy meta- Our goal was to generate a proteomic profile of the bolism (oxidative phosphorylation and glycolysis), protein T. californica electric organ, both to assess its similarity processing (translation initiation, elongation, trafficking, to the mammalian NMJ proteome and to provide novel and proteasome degradation) and several components candidate proteins for localization to the NMJ. We and involved in redox reactions and caveolar endocytosis. A others have carried out microdissection of the NMJ prominent feature is an abundance of cytoskeletal pro- region and messenger RNA profiling to characterize the teins to include molecular motors, capping and folding NMJ constituents, but these have proven technically proteins, and focal adhesion molecules. Also notable are challenging and have fallen short in describing a broader a number of proteins that interact with known NMJ pro- proteome [45,46]. teins (Figure 5). Finally, the virtual electrocyte reveals A key resource for our one-dimensional ESI-LTQ MS/ several relatively uncharacterized proteins such as MS and two-dimensional MALDI-TOF/TOF MS Mate et al. Skeletal Muscle 2011, 1:20 Page 7 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 3 Classification of proteins identified in electric organ fractions by tissue association or function as determined by UniProtKB annotation. Electric organ fractions were separated one dimensionally and analyzed by nanospray electrospray ionization quadrupole linear ion- trap tandem mass spectrometry (ESI-LTQ MS/MS). Mass spectral matching of raw spectra against UniProtKB and Torpedo cDNA library was performed in BioWorks 3.3.1 in which the peptide acceptance criteria was set at ΔCn >0.1, a variable threshold of Xcorr versus charge state: Xcorr = 1.9 for z = 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr >40, and a peptide probability based score with a P value <0.01. All cDNA sequences were queried in blastx (standard genetic code, Swiss-Prot, default algorithm parameters except for BLOSSUM80 scoring matrix) for identification via sequence similarity with a known protein, first across all species and then against Homo sapiens selected database. Cytosolic proteins were separated two dimensionally, analyzed via matrix-assisted laser desorption/ionization - time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS), and identified by MASCOT. Identification criteria was set at a protein score CI >95%, protein score >69, and proteins with isoelectric points (PI) and molecular weights (MW) that match the gel spot. Each identification was queried in UniProtKB for annotation of tissue expression and or function then categorized by the sections composing the pie chart. (See Additional file 3 for a list of proteins composing the pie chart.) profiles were cDNA sequences from the electric organ well as transdifferentiation from muscle precursor cells that enabled mass spectral mapping [40]. Of 435 pro- into the electric organ. Additionally, we identified sev- teins we identified in the electric organ, 300 (69%) eral proteins that are expressed by non-electrocyte cells composing the electric organ, such as the electromotor showed ≥2 peptides that mapped to our Torpedo cDNA sequences while the remaining 135 (31%) were charac- neuron proteins, Schwann cell proteins, and proteins of terized via cross-species peptide spectral mapping to the immune and circulatory systems. mammalian proteins. We found that 48% of identified To compare our Torpedo data to previous studies of proteins were highly expressed in skeletal muscle or are the mammalian NMJ, we scanned the literature for muscle specific, which supports the ‘muscle-like’ lineage known NMJ proteins, grouped these into three cate- of the electric organ. The proteome includes cytoskele- gories, and overlaid our Torpedo proteome with these tal, glycolytic, translational, and degradative proteins. groups. The first category was limited to proteins in The high prevalence of glycolytic enzymes likely is which experimental knockout (loss of function) data necessary to support the high metabolic load of the suggested an important functional role in postsynaptic organ that is required for establishing and maintaining architecture and function (for example, disruption of the membrane potential. The abundance of proteasome morphology) (Additional file 4; see also references cited and degradative enzymes is in line with high protein therein). The second category included protein-protein turnover and degradation during synapse renewal as networks nucleated by the key functional candidates in Mate et al. Skeletal Muscle 2011, 1:20 Page 8 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 4 Virtual Torpedo electrocyte. All identifications from UniProtKB/Swiss-Prot and Torpedo cDNA searches of fractions analyzed by nanospray electrospray ionization quadrupole linear ion-trap tandem mass spectrometry (ESI-LTQ MS/MS) and matrix-assisted laser desorption/ ionization - time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) were mapped to cellular regions based on UniProtKB annotations using the Path Designer tool in Ingenuity IPA 8.8-3204. category 1. Most category 2 proteins were shown to molecules listed). The presence of these molecules sug- attenuate AChR clusters when mutated, inhibited, or gests the neuromuscular protein machinery supporting deleted. Finally, the remaining category contained pro- thecholinergicendplatecoincides with theelectric teins strictly concentrated at the endplate but do not organ and may serve as a model NMJ to study these alter AChR clusters or synapse morphology (category 3). proteins. We identified rapsyn, b-spectrin, Ras-related C3 botuli- In addition to the few Torpedo proteins characterized num toxin substrate 1, and laminin subunit b-2 from at the cholinergic synapse (AChR subunits a, b, δ,and category 1, HSP90b, HSP 70 kDa protein, a syntrophin, g,ACHE,rapsyn,14-3-3 g, syntrophin) we identified 14-3-3 protein g, dynamin, vesicle-fusing ATPase, a- several uncharacterized proteins in the electric organ known to play a role in maintaining AChR clustering actinin, utrophin, and Ras from category 2, and ankyrin, desmin, and dystrobrevin from category 3 (16/38 and in transducing signals between the membrane and Mate et al. Skeletal Muscle 2011, 1:20 Page 9 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 5 Torpedo electrocyte proteins in context of the mammalian neuromuscular junction (NMJ). Electric organ identifications are displayed in context of the mammalian NMJ paradigm. Red shapes indicate proteins we identified in our Torpedo electric organ proteome. White shapes are proteins we did not identify. This image was created using Path Designer in Ingenuity IPA 8.8-3204. nucleus (Figure 5). These proteins include laminin subu- interacts with extracellular matrix (integrins and agrin) nits a-2, b-2, and g-1, HSP90b, HSP 70 kDa protein, and postsynaptic membrane components (basal cell stress-induced-phosphoprotein 1, dynamin 1 and vesi- adhesion molecule (Bcam), a dystroglycan, and AChR) to cle-fusing ATPase, a-actinin, prostaglandin E synthase link the extracellular regions with the intracellular cytos- 3, Ras-related C3 botulinum toxin substrate 1, guanine keleton and to regulate the release of intracellular cal- nucleotide-binding protein G, guanine nucleotide-bind- cium directed at AChR cluster formation [48-50]. ing protein subunit b-2-like1, Rho GDP-dissociation Subunit b2 also assists in the development of synaptic inhibitor 1, and Ras-relatedprotein R-Ras2. Below,we folds and Schwann cell placement at the synapse [51]. describe each of these electric organ components as The laminin receptors characterized at the synapse, they relate to the mammalian NMJ. Bcam and dystroglycan, were not identified but dystro- glycan was previously characterized in the electric organ T. californica proteome related to AChR clustering [13,52]. However, we identified laminin receptor 1 A key event in the formation of the neuromuscular junc- (LamR1 or RPSA), a known binding partner for the lami- tion is the clustering of AChRs to focal points underlying nin complex in the electric organ (a2, b2, and g1; also motor neuron synapses. At the developing synapse, a key called S-merosin or laminin 2/4). Interestingly, LamR1 protein complex involved in clustering is the laminins: has not been previously reported at the NMJ [53,54]. multisubunit glycoprotein complexes consisting of a, b, Recent evidence supports the role of HSP90b and HSP 70 and g chains, each with multiple isoforms, assembled in a kDa protein (HSP70) as stabilizing chaperones of NMJ pro- trimer of equal stoichiometry. Laminin subunits a2, b2, teins. HSP90b was shown to interact directly with rapsyn at and g1 are seen most frequently in mature NMJs where its tetratricopeptide repeat (TPR) domain following its binding to surface AChR clusters. Recruitment of HSP90b they form the laminin 4 complex (also called S-merosin); we identified each of these subunits in the T. californica is believed to stabilize AChR-rapsyn binding to influence proteome. Subunit g1 facilitates the interaction between AChR stability and maintenance and also may associate AChR and a7b1 integrins to prime cluster formation with a dystrobrevin and a syntrophin [44]. HSP70 may be prior to neuronal agrin release or when agrin levels are a cochaperone of HSP90b along with DnaJ homolog sub- low [47,48]. At the mature synapse, the laminin complex family C member 7, HSP40, and prostaglandin E synthase 3 Mate et al. Skeletal Muscle 2011, 1:20 Page 10 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 6 Electric organ proteome overlaps with mouse skeletal muscle proteome but shows tissue-specific protein expression. Mouse skeletal muscle (tibialis anterior muscle or gastrocnemius muscle) and the Torpedo electric organ were fractionated and processed under similar conditions as stated under Figure 1. Mouse skeletal muscle proteins were identified by BioWorks 3.3.1 referencing only UniProtKB/Swiss-Prot. Electric organ (EO) and skeletal muscle proteins were compared and graphed in Microsoft Excel 2007 based on the number of peptides per protein identified in each tissue. EO proteins are mapped (#peptides/protein) on the × axis and mouse skeletal muscle on the y axis. The lower two graphs represent zoomed sections for visual clarity. (p23). p23 is involved in stabilizing the ATP-bound confor- (Rac1), G proteins) involved in agrin-dependent MuSK mation of HSP90, permitting the release of activated inter- activation and subsequent AChR clustering and synaptic acting partners [55]. We identified p23 in our Torpedo gene transcription (Figure 5). In this process, agrin cDNA library, suggesting its role as a cochaperone with binds Lrp4 to activate MuSK and its subsequent interna- HSP90 in the electric organ. We also identified stress- lization via clathrin-mediated endocytosis and to activate induced phosphoprotein 1, which facilitates the interaction expression of MuSK interacting proteins. Dynamin 1 between HSP90b and HSP70 [56]. and NSF are involved in receptor-mediated endocytosis, vesicle transport, and protein trafficking. NSF is essen- Intracellular signal transduction tial for agrin-induced receptor-mediated endocytosis of We identified proteins (vesicle-fusing ATPase (NSF), MuSK and activation of its downstream signaling mole- dynamin, Ras-related C3 botulinum toxin substrate 1 cules Abl kinase and Rac1 in C2C12 cells, which Mate et al. Skeletal Muscle 2011, 1:20 Page 11 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Table 1 T. californica electric organ proteome shows Table 1 T. californica electric organ proteome shows tis- tissue-specific proteins when compared to mouse sue-specific proteins when compared to mouse skeletal skeletal muscle proteome muscle proteome (Continued) Unique to skeletal muscle Unique to electric organ KINH TBB1 proteome proteome PBIP1 TBB2 Development (myogenesis): Myofibrillar: STIM1 TBB5 UN45B Cytoskeleton, actin M-band TBB2A Intermediate filaments Myofibrillar: ANK1 TBB2C K1C9 Cytoskeleton, actin Light chain part of A-band Sarcoplasmic reticulum/calcium K1H1 TITIN MYL9 pathways: Z-disk Neuromuscular junction (NMJ): AT2A2 NFH ACTN2 ACES AT2A3 NFL ACTN3 ACHA CALU NFM MYOTI ACHB JPH2 Sarcoplasmic reticulum/calcium pathways: PP2BA ACHD SRCA SORCN Class II myosins (A-band) ACHG CASQ1 Ion channels: MYBPH DTNA CASQ2 Chloride channel MYH1 HSP70 KPB1 CICH MYH3 HSP90B Ion channels: Calcium ATPase MYH4 RAPSN Voltage-sensitive calcium channels AT2B1 MYH6 NMJ-ECM CA2D1 AT8A1 MYH7 LAMA2 CAC1S Hydrogen-potassium ATPase MYH8 LAMB2 CACB1 AT12A MYPC2 LAMC1 Potassium channel AT1A Light chain part of A-band Cytoskeleton: TM38A Sodium-potassium ATPase MYL1 Cytoskeleton, actin Sodium-potassium ATPase AT1A3 M-band SEPT6 AT1A2 AT1B1 MYOM1 sept7 AT1B2 Extracellular matrix (ECM): OBSCN ACTG Oxygenation (muscle): NID2 Contraction ADDG MYG FINC PHKG1 ACTC ECM: CO1A1 MYLK2 ANK2 CO6A1 CO1A2 TNNC2 ARPC2 COEA1 CO6A3 TNNI2 ARPC4 ITB1 BGH3 TNNT3 CCDC6 NID1 HPLN1 TPM2 COF1 PEPD Neuronal: ACTN4 COF2 PGS2 AINX Cytoskeleton: DNJC7 Neurogenesis: VAMP3 Cytoskeleton, actin FERM2 NDKA ACTS PROF2 NDRG2 ML12B SLMAP Proteins on the × and y axis of Figure 6, indicating unique identification in MLRS TCPA the corresponding tissue, were listed based on biological process, molecular MLRV TLN2 function, and cellular localization. See Additional File 1 for expanded names of all abbreviated proteins. Actin capping-binding Actin capping-binding CAPZB CAZA2 RADI SPTB1 XIRP1 Cytoskeleton, microtubule promote AChR clustering [57]. In addition, dynamin supports clathrin-coated vesicles formed upon agrin- Cytoskeleton, microtubule DYN1 induced endocytosis of MuSK, which is translocated CLIP1 SIRT2 into lipid rafts for activation and signaling. Mate et al. Skeletal Muscle 2011, 1:20 Page 12 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Several studies support the ‘signaling endosome Lrp4/Src/Rapsyn network involved in AChR cluster for- hypothesis’ in which neurotrophic factors initiate ligand- mation and stabilization (Figure 5). The second is the mediated endocytosis of receptor tyrosine kinases into Agrin-MuSK/NRG-ErbB/MAPK/GABP network for clathrin-coated vesicles that contain activators such as G transcriptional activation of synaptic genes (Figure 5). proteins and downstream effector molecules involved in Absent molecules involved in these pathways include Ras-mitogen-activated protein kinase (MAPK) signaling downstream of tyrosine kinase 7 (Dok7), dishevelled [58-61]. In the electric organ, we identified guanine (Dvl), PAK, RAF-1, and extracellular signal-regulated nucleotide binding proteins and inhibitors that act on kinase (ERK). In addition, several Torpedo proteins with Rho family of Ras-related G proteins that may be UniProtKB/TrEMBL annotation that are expressed at involved in signaling endosomes. These include G subu- the NMJ were not detected by tandem mass spectrome- nit b-1 (GNB1), subunit b-2-like1 (RACK1), G(s) subu- try analysis of subcellular fractions. These include a and nit a (GNAS1), and Rho GDP-dissociation inhibitor 1 b dystroglycan homologs, the receptor tyrosine kinase (ARHGDIA). GNB1 composes part of the catalytic similar to MuSK, and protein tyrosine kinases Fyn and machinery of GTPases and provides docking regions for Fyk. However, we did identify dystrophin, dystrobrevin, interacting proteins. ARHGDIA prevents the release of and syntrophin that compose the dystroglycan complex GDP from Rho proteins (Rho, Rac, cdc42, TC10). and we did show several molecules that may be up and RACK1 is the receptor of protein kinase C (PKC), which downstream of receptor tyrosine-protein kinase ErbB is known to inactivate Rho; PKC also phosphorylates (neuroplastin (NPTN), Ras-related protein R-Ras2 serine residues of AChR δ subunit to promote receptor (RRAS2) or Ras-related protein Rap-1A (RAP1A), desensitization and disassembly [62-64]. Identification of HSP90b). several proteins involved in ligand-mediated endocytosis, We failed to identify relatively well characterized activators and inhibitors of GTPases, signaling, lysoso- mammalian NMJ proteins in our survey, including Lrp4, mal and proteasomal degradation support the mainte- MuSK, Dok7, Src and Fyn Kinase, Dvl, ErbB2, PKC nance of protein function across myogenic-derived cell (category 1), and agrin, laminin subunits a4, a5, PAK1, types. Rho, cyclin-dependent kinase 5 (cdk5), ephexin1, neure- Interestingly, Rac1 is involved in clathrin/dynamin- gulin, ETS transcription factor, Raf, MEK, MKK4, c-Jun independent endocytosis of AChR following binding N-terminal kinase (JNK), and c-Jun (category 2). This with bungarotoxin [65]. Rac1 functions in actin polymer may reflect technical issues with the sensitivity of our rearrangement to create compartments for AChR sur- proteomics methods and parameters (for example, false face sequestration and, most likely, polymerization of negative and low maximum mass range for glycosylated the cytoskeletal network involved in vesicle transport to peptides), challenges in mapping peptide spectral data to the lysosome for degradation. Rac is a key mediator of the partial cDNA sequence coverage or to cross-species receptor surface sequestration in addition to its role in transcript units, or significant differences in the struc- actin polymerization and rearrangement, which controls ture and function of the electric organ compared to the the number and arrangement of receptors at the synapse mammalian NMJ. Our study is based on non-targeted to modulate synaptic transmission. proteomics and it may be possible to identify these spe- Our proteome also includes an inhibitory protein of cific proteins in the electric organ using a more targeted synaptic gene expression. The 14-3-3 g (YWHAG), approach. The literature on the Torpedo electric organ extracted from Torpedo electric organ, reduced the supports the presence of agrin, a and b dystroglycan, expression of MuSK, AChR subunits ε and a,utrophin, MuSK, and Src kinases, which strengthens the organ’s and rapsyn and resulted in aberrant NMJ morphology use as a model NMJ. [43]. It is known that 14-3-3 g interacts with the N-ter- minus of Raf-1, HSP90 interacts with the C-terminus of Conclusions Raf-1, and Ras (RRAS2 (TC21)) binds to the Raf-1- The virtual electrocyte revealed that the Torpedo electric HSP90-p50 complex, causing the complex to translocate organ is a resource for several uncharacterized proteins to the plasma membrane and become an active kinase whose function may be clarified in future studies. for phosphorylating mitogen-activated protein kinase Knockout and reporter assays of C6orf130, C1orf123, kinase (MEK) [66-68]. PKC also is a target of 14-3-3 g. matrix-remodeling-associated protein 7, protein NipS- nap homolog 2, septin-6, prohibitin 2, GATS-like pro- T. californica proteome: limits as a NMJ model tein 2, SH3 domain-binding glutamic acid-rich protein, Several critical NMJ proteins are absent from our data. and 14-3-3 protein ζ/δ in mouse skeletal muscle may Most notable are proteins within the two major net- clarify their subcellular roles, which may reveal novel works responsible for postsynaptic stabilization and components involved in AChR expression and mainte- gene expression. The first network is the Agrin-MuSK- nance. The electric organ will continue to serve as a Mate et al. Skeletal Muscle 2011, 1:20 Page 13 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 model of membrane excitability and electrogenesis as it filtered through a 100 μm nylon filter (BD, Franklin + + is abundant in AChR and the Na /K -ATPase channels Lakes, NJ,USA)toremoveconnectivetissue. Thefil- and may be used as a model to design a prototype trate was centrifuged at 627 g for 15 min at 4°C. This biobattery. pellet was resuspended in 2 M STM buffer (2 M Based on our identification of electric organ proteins sucrose, 50 mM Tris-HCl pH 8.0, 5 mM MgCl ,Roche that match proteins in the three categories of our Mini Complete Protease Inhibitor and PhosStop Phos- defined ‘NMJ proteome’ and the persistent similarity to phatase Inhibitor) and placed in an ultracentrifuge at skeletal muscle, the electric organ can serve as a reposi- 80,000 g for 35 min at 4°C. The resultant pellet was tory of these NMJ molecules that are in low abundance resuspended in EBC buffer (50 mM Tris-HCl pH 8.0, in skeletal muscle. However, the absence of several NMJ 120 mM NaCl, 1% Triton-X 100, Roche Mini Complete components involved in synaptic gene expression and Protease Inhibitor and PhosStop Phosphatase Inhibitor). AChR clustering in our model limits our ability to con- After 15 min incubation at 4°C, the suspension was clude that it indeed represents the mammalian NMJ passed through a 20-gauge needle ten times to lyse any that is maintained similarly. This study offers a more cells. Soluble (S1) and insoluble fractions were separated detailed understanding of the electrocyte protein reper- by centrifugation at 9,000 g for 30 min at 4°C. The pel- toire with insight into the presence and absence of pro- let (P1.1) was resuspended in EBC buffer. teins between these two related tissues. It reflects their P2 was resuspended in 0.5 ml HDP buffer (10 mM 4- unique tissue-function specializations and insight into (2-hydroxyethyl)-1-piperazineethanesulfonic acid evolutionary conservation and divergence between (HEPES), 1 mM dithiothreitol (DTT), Roche Mini Com- synaptic gene expression, maintenance, and regulation. plete Protease Inhibitor and PhosStop Phosphatase Inhi- The data raises questions whether the pathways respon- bitor). After 30 min incubation on ice, the suspension sible for AChR clustering are required in the electrocyte was sonicated (Sonifier Cell Distributer 350, Branson given its dense innervations and high AChR expression Scientific Danbury, CT) on ice for five initial pulses, or whether electromotor neurons support the postsy- paused for 30 s, then a final ten pulses (50% Duty Cycle, napse with different neurotrophic or signaling molecules Pulsed-Hold, Output Control Limit 3). The lysate was than mammalian motor neurons such that the neuregu- centrifuged at 9,000 g for 30 min at 4°C. The superna- lin-ErbB pathway is unnecessary. tant was saved as S2. P2.1 was resuspended in ME buf- fer (20 mM Tris-HCl, 0.4 M NaCl, 15% glycerol, 1 mM Methods DTT, 1.5% TritonX-100, Roche Mini Complete Protease Sequencing and mass spectral database indexing of T. Inhibitor and PhosStop Phosphatase Inhibitor), incu- bated for 30 min at 4°C with rocking, then centrifuged californica cDNA library The 10,326 cDNA sequences utilized for proteomics at 9,000 g for 30 min at 4°C. The supernatant of solu- mass spectral mapping database have been previously blized P2.1 was saved as P2.1. described [40]. All T. californica sequences were saved S1 and S3 fractions were concentrated in a speed as a Fasta database and indexed in BioWorks 3.3.1 SP1 vacuum. P3 was suspended in EBC buffer. Each fraction (Thermo Fisher Scientific, Waltham, MA) as trypsin except P2.1 and S2 was desalted by passing the sample digested protein sequences from the translation across through a BioSpin6 column before protein quantitation all six reading frames. using the DC Protein Assay (BioRad, Hercules, CA, USA). Protein extracts were stored at 80°C until Fractionation of the electric organ electrophoresis. T. californica electric organ was fractionated by grinding and homogenizing electroplax in lysis buffer (0.25 M Protein isolation and identification sucrose, 20 mM Tris pH 8.0, 25 mM KCl, 5 mM Protein separation MgCl , Roche Mini Complete Protease Inhibitor and Proteins within each fraction were resolved using one- PhosStop Phosphatase Inhibitor (Roche, Branchburg, NJ, dimensional SDS-PAGE on Novex NuPage 3% to 8% USA) [69]. Tissue homogenate was centrifuged at 627 g Tris-Acetate MidiGel and 4% to 12% Bis-Tris MiniGel (2,500 rpm) for 15 min at 4°C. The pellet (P1) was Systems (Invitrogen, Carlsbad, CA, USA) according to saved for further purification and the supernatant was manufacturer’s directions such that 2.5-400 kDa pro- centrifuged at 10,000 g for 20 min at 4°C. The pellet teins may be isolated and prepared for ESI-MS/MS ana- (P2) was saved for further purification and the superna- lysis. In addition, 250 μg of cytosolic proteins were tant was ultracentrifuged at 100,000 g for 60 min at 4°C resolved by two-dimensional electrophoresis as pre- resulting in pellet P3 and supernatant S3. viously described with minor differences [70]. The P1 was processed further by homogenizing the iso- immobilized pH gradient (IPG) strip was rehydrated for lated pellet in 2-3 ml lysis buffer. The homogenate was 12 h at 20°C and was focused at 20°C using the Mate et al. Skeletal Muscle 2011, 1:20 Page 14 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 following conditions: 250 V for 15 min, 8,000 V for 2.5 15.0) by MASCOT using the following parameters: MS h, 500 V hold. Following isoelectric focusing (IEF), the peak filtering mass range 800-4,000 Da, minimum S/N IPG strip was incubated in equilibration buffer (6 M 10, peak density filter 50 per 200 Da, maximum num- urea, 50 mM Trizma preset crystals (pH 8.8), 2% SDS ber peaks 65; MS/MS peak filtering: mass 60 Da to 20 (w/v), 30% glycerol (w/v), 0.002% bromophenol blue) Da below precursor mass, lowest precursor 707.46 Da, with 1% DTT for 20 min followed by a 20 min incuba- peak density filter 50 per 200 Da, maximum number tion in equilibration buffer with 2% iodoacetamide. Each peaks 65, fixed modification carbamidomethyl (C), gel was fixed for 30 min in 5% acetic acid, 45% metha- variable modification oxidation (M), fragment ion tol- nol solution, stained with Bio-Safe Coomassie (Bio-Rad) erance 0.3, precursor tolerance 0.5. Proteins identified for 60 min, and destained in distilled water overnight. by the MASCOT algorithm were filtered based on pro- Protein digestion teins identified with MS/MS spectra, protein score CI Multiple molecular weight bands and spots were manu- >95%, protein score >69, proteins with PI and MW ally excised from the gel (Additional file 5) and pro- that match the gel spot. All .dat files of spectral data cessed for in-gel digestion with 12.5 ng/μl Trypsin Gold were also uploaded to the PRIDE database under the (reconstituted according to manufacturer’sdirections, same project title as ESI-LTQ-MS/MS data stated Promega, Madison, WI, USA) in 50 mM NH HCO as above. 4 3 previously described [71]. Lipid raft assay of fraction Protein identification: ESI-LTQ-MS/MS To isolate membrane proteins localized to lipid rafts, Recovered peptides from SDS-PAGE were analyzed using membrane was isolated from 3 g of electric organ nanospray ESI-LTQ MS/MS as previously described, according to the above procedure (protein separation) with minor differences [72]. Peptides were loaded onto a with minor modifications. Tissue homogenate was cen- C18 reverse-phase column for 10 min at a flow rate of 5 trifuged twice at 627 g for 15 min at 4°C and the super- nl/min then separated at a flow rate of 250 nl/min. A 65 natant passed through a 40 μm filter to clear cellular min linear gradient eluted peptides. The LTQ operated debris. The supernatant was ultracentrifuged at 100,000 in data-dependent mode to perform one full MS scan g for 60 min at 4°C to collect an insoluble pellet rich in (300-2,000 m/z) to select the five most intense peaks membrane proteins. Lipid rafts were isolated from elec- through dynamic exclusion for MS/MS analysis via colli- tric organ membrane fraction following previously pub- sion-induced dissociation (CID) with helium at 35% nor- lished guidelines with the following modifications: the malized energy. Raw spectra were analyzed by the gradient was ultracentrifuged at 100,000 g [77]. Visible SEQUEST algorithm in BioWorks 3.3.1 software, crossre- bands were collected and centrifuged at 14,000 g for 30 ferencing our T. californica cDNA library translated into min. The resultant pellets were resuspended in EBC buf- six reading frames and The Universal Protein Resource fer. Intermediate solutions were also collected and con- (UniProtKB/Swiss-Prot) release 14.0 [73]. Peptide accep- centrated by vacuum centrifugation. All collected tance criteria was set at ΔCn >0.1, a variable threshold of fractions were subjected to one-dimensional SDS-PAGE Xcorr versus charge state: Xcorr = 1.9 for z = 1, Xcorr = on Novex NuPAGE 4% to 12% Bis-Tris MiniGel using 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr NuPAGE MES SDS Running Buffer (Invitrogen, Carls- >40, and a peptide probability based score with a P value bad, CA) according to the manufacturer’sinstructions. <0.01. Spectral data (.raw files) were first converted into Protein bands were manually excised and processed for MS2 file format (.ms2 files) using pXtract, default set- ESI-LTQ-MS/MS analysis as described earlier. tings, and then into PRIDE XML format using PRIDE Converter for upload onto the PRIDE database [74-76]. Additional material Data can be found under the project name ‘Torpedo cali- fornica Electric Organ Proteome’, accession numbers: Additional file 1: Torpedo californica electric organ proteome. All 435 proteins, identified across different sample processing and mass 16,474-16,476. spectral data acquisition techniques, representing the T. californica Protein identification: MALDI-TOF/TOF MS proteome are listed in separated tabs based on the method of Two-dimensional IEF SDS-PAGE separated cytosolic identification. Electric organ fractions were separated by SDS-PAGE and analyzed by nanospray electrospray ionization quadrupole linear ion-trap peptides were processed and analyzed for protein iden- tandem mass spectrometry (ESI-LTQ MS/MS) or matrix-assisted laser tification as previously described with the following desorption/ionization - time of flight/time of flight mass spectrometry additional details. Data was acquired using the follow- (MALDI-TOF/TOF MS). For ESI-LTQ MS/MS, mass spectral matching of raw spectra against UniProtKB and Torpedo cDNA library was performed in ing parameters: mass range 500-4,000 Da, minimum S/ BioWorks 3.3.1 in which the peptide acceptance criteria was set at ΔCn N 20, mass tolerance ± 2 m/z, minimum peak match >0.1, a variable threshold of Xcorr versus charge state: Xcorr = 1.9 for z = 4, maximum outlier error 10 ppm, monoisotopic mass 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr >40, and a peptide probability based score with a P value <0.01. All cDNA [70]. MS and MS/MS spectra of peptides were sequences were queried in blastx (standard genetic code, Swiss-Prot, searched against the UniProtKB/Swiss-Prot (release Mate et al. Skeletal Muscle 2011, 1:20 Page 15 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Acknowledgements default algorithm parameters except for BLOSSUM80 scoring matrix) for We would like to acknowledge Aaron Lorsong for his work in generating identification via sequence similarity with a known protein, first across all the mouse skeletal muscle proteome and providing the proteomic data for species and then against Homo sapiens selected database (column C: all comparison with the Torpedo electric organ. In addition, we thank the PRIDE species/Homo sapiens). Score of blastx alignments: black ≥200, pink = 80- support team for their overwhelming assistance and technical support for 200, green = 50-80. For MALDI-TOF/TOF MS of cytosolic proteins the conversion and submission of spectral data. Supported by the National resolved two dimensionally, acquired data were searched against Center for Medical Rehabilitation Research (NIH 5R24HD050846-06), the UniProtKB/Swiss-Prot (release 15.0) by MASCOT using the following Intellectual and Developmental Disabilities Research Center (IDDRC) (NIH parameters: MS peak filtering mass range 800-4,000 Da, minimum S/N 10, P30HD40677), the WM Keck Foundation, and the Erynn Godla Family via the peak density filter 50 per 200 Da, maximum number peaks 65; MS/MS Juvenile ALS Foundation http://www.juvenileals.org/. peak filtering: mass 60 Da to 20 Da below precursor mass, lowest precursor 707.46 Da, peak density filter 50 per 200 Da, maximum number Author details peaks 65, fixed modification carbamidomethyl (C), variable modification 1 Department of Biochemistry and Molecular Genetics, IBS, George oxidation (M), fragment ion tolerance 0.3, precursor tolerance 0.5. 2 Washington University, Washington DC, USA. Department of Pediatrics, Proteins identified by the MASCOT algorithm were filtered based on 3 George Washington School of Medicine, Washington DC, USA. Research proteins identified with MS/MS spectra, protein score CI >95%, protein Center for Genetic Medicine, Children’s National Medical Center, Washington score >69, proteins with isoelectric points (PI) and molecular weights 4 DC, USA. Department of Integrative Systems Biology, George Washington (MW) that match the gel spot. School of Medicine, Washington DC, USA. Additional file 2: Validation of identified Torpedo proteins in spectral data using Public Access Databases. Torpedo protein Authors’ contributions sequences listed in GenBank were collected in a single database to SEM carried out the proteomic profiling of the Torpedo californica electric directly search Torpedo electric organ fractions with a Torpedo-specific organ, data comparison with mouse skeletal muscle proteome, the protein database. Results include Torpedo proteins also identified by a development of images projecting the electrocyte proteome as well as the search against UniProtKB, all species. UniProtKB accessions are used for proteome within the context of the NMJ, and preparation of the manuscript. consistency. The chart categorizes proteins positively identified, not KJB helped design and execute proteomic profiling and data analysis as well identified that are expected to be found with a possible explanation, and as assisting in the write-up of methodology and manuscript editing. EPH proteins not identified that are not expected to be identified. Reviewed provided the conceptual design of the project and insight into data analysis sequences are from the UniProtKB/Swiss-Prot database and unreviewed as well as drafting of the manuscript. All authors have read and approved sequences from UniProtKB/TrEMBL. this manuscript. SEM is a predoctoral student in the Biochemistry and Molecular Genetics Program of the Institute for Biomedical Sciences at the Additional file 3: Torpedo californica electric organ proteome George Washington University. This work is from a dissertation to be classified according to tissue expression or associated function. All presented to the above program in partial fulfillment of the requirements proteins identified by mass spectral mapping (listed in Additional file 1) for the PhD degree. were queried in UniProtKB for annotation of tissue expression and or function then categorized as electric organ (EO) specific, neuromuscular Competing interests junction (NMJ) specific, muscle specific or highly expressed in muscle, The authors declare that they have no competing interests. likely expressed in muscle based on function, expressed in neurons, or functions in metabolism and energy production. Torpedo cDNA Received: 20 December 2010 Accepted: 4 May 2011 sequences were queried by blastx (standard genetic code, Swiss-Prot, Published: 4 May 2011 default algorithm parameters except for BLOSSUM80 scoring matrix) to obtain a protein identification with high sequence similarity. References Additional file 4: Neuromuscular junction (NMJ) proteins from the 1. Bennett MVL: Comparative physiology: electric organs. Annu Rev Physiol literature categorized by the degree of influence on synapse 1970, 32:471-528. architecture. The NMJ proteome was defined by searching the current 2. Connolly JA, St John PA, Fischbach GD: Extracts of electric lobe and literature and categorizing the influence of proteins on the synaptic electric organ from Torpedo californica increase the total number as well structure and function. 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Vizcaíno JA, Côté R, Reisinger F, Barsnes H, Foster JM, Rameseder J, and take full advantage of: Hermjakob H, Martens L: The Proteomics Identifications database: 2010 update. Nucleic Acids Res 2010, 38:D736-742. • Convenient online submission 77. Williamson CD, Zhang A, Colberg-Poley AM: The human cytomegalovirus UL37 exon1 protein associates with internal lipid rafts. J Virol 2011, • Thorough peer review 85:2100-2111. • No space constraints or color figure charges • Immediate publication on acceptance doi:10.1186/2044-5040-1-20 Cite this article as: Mate et al.: Integrated genomics and proteomics of • Inclusion in PubMed, CAS, Scopus and Google Scholar the Torpedo californica electric organ: concordance with the • Research which is freely available for redistribution mammalian neuromuscular junction. Skeletal Muscle 2011 1:20. Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

Skeletal Muscle , Volume 1 (1) – May 4, 2011

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results: Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions: Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ. Background of the organism’smassand areusedfor generation of Ionic gradients across cell membranes (bioelectricity) are electric shocks for predation or protection [2]. utilized by all organisms. Some fish have developed Developmental studies have shown most electric extreme adaptations of bioelectricity with the evolution organs are derived from muscle anlage tissue; the excep- of electric organ systems. It is thought that electric tion is the neurogenic development of the Sternarcus organs have evolved independently six or seven times in electric organ. Several basic differences exist amongst fish and can be classified as either weak or strong, which myogenic-derived electric organs. The location of the is reflective of the size and function of the organs within myogenic-derived electric organs varies from gill (Tor- the fish. For example, Gymnotids are weakly electrogenic pedo), tail (Raja, Gnathonemus, Gymnarchus, Gymnotus), and ocular muscle (Astroscopus). Strong electrogenic and only possess accessory electric organs used for elec- troreception and electrolocation [1]. In contrast, Torpedi- organs lose the characteristic myofibrils and sarcomeres nid and Electrophorous arestronglyelectrogenicand during transdifferentiation of the organ. In contrast, possess organs that account for approximately one-third weakly electrogenic Gymnarchids and Mormyrids main- tain the myofibrillar structures into adulthood [3]. Organs differinthe abilitytoinitiateand propagatean action potential. Generally, marine fish possess organs * Correspondence: ehoffman@cnmcresearch.org Department of Biochemistry and Molecular Genetics, IBS, George with electrically inexcitable membranes (lacking voltage- Washington University, Washington DC, USA sensitive sodium channels), whereas fresh water fish have Full list of author information is available at the end of the article © 2011 Mate et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mate et al. Skeletal Muscle 2011, 1:20 Page 2 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 organs that are electrically excitable (have voltage-sensi- look at how the innervated membrane of Torpedo elec- tive sodium channels). Succinctly put, the degree of mus- trocytes relates to the NMJ. cle likeness of precursor cells differs among electrogenic From a developmental perspective, Torpedo electroblasts fish families. These anatomical differences may represent are derived from the mesodermal layer that gives rise to an evolutionary divergence required for the performance branchial arches from which the electric organ and gill of strong and weak electric organs. musculature form. The primordial electric organ first gen- erates ‘muscle-like’ cells that are multinucleated and have The research presented here focuses on Torpedo cali- a single striated myofibril, reminiscent of myotubes in ske- fornica (Pacific electric ray), a cartilaginous fish within letal muscle. At this stage, meromyosin is expressed at the Chondrichthyes class and Torpedinidae family. This species evolved an electric organ capable of generating high levels and the single striated myofibril has a similar approximately 45-50 V (electron motive force 110 mV), diameter to actin-myosin myofibrilar structures compos- released in 414 monophasic discharges that last 3-5 ms ing sarcomeres [26]. As the electroblast transforms into an each, with a total power output up to 1 kW [4-6]. An electrocyte at the onset of electromotor neuron synapto- electrocyte from the electric organ of Torpedo nobiliana genesis, Z-disc-like structures disassemble and degenerate (Atlantic Torpedo with similar length but twice the completely [27]. It is thought that the electromotor neuron weight of T. californica) measures 5-7 mm in diameter sends signals that induce the degeneration of the myofibril by 10-30 μm thick and 500-1,000 electrocytes are stacked structures, allowing the elongated cells to flatten into thin into columns, all with ventrally innervated and dorsally electrocytes [28]. Desmin, or a light intermediate filament, non-innervated membranes aligned [5]. Approximately replaces the myofibril following disassembly, but keratin, a 50 A of current has been measured from the parallel protein typically associated with epithelium, dominates the stacks composing the electric organ of T. nobiliana,and intracellular architecture [26,29]. Upon denervation, myo- about 1 A measured from the series-aligned electrocytes fibril-like structures reappear near the synapse but are of Electrophorous [6]. The postsynaptic membrane of the highly disorganized and short lived [28]. In addition, tran- electric organ in Torpedo is rich in nicotinic acetylcholine script evidence was shown for myoblast determination receptors (AChR) and is multi-innervated with dendrites protein and myogenic factor 5 expression in adult Torpedo from four large, heavily myelinated neurons descending electric organ without evidence of protein expression, sug- from the electric lobe of the brain. The non-innervated gesting strong post-transcriptional regulation of messenger membrane is extensively invaginated into structures RNA translation and maintenance of a muscle-like pro- gramming [30]. No synapse is observed until late phase of called caniculi that may be reminiscent of skeletal muscle electric organ development when the ventral face of elec- T tubules [5]. The electrocytes are multinucleated and filled with a gelatinous cytoplasm with an extensive fila- troblasts develop subneural arches that have increased mentous network. The electrocyte itself has low internal levels of acetylcholinesterase (AChE) and AChRs that resistance with low resistance across the non-innervated reach 300 times the level in skeletal muscle [27,28,30]. membrane [7]. Insulating septa, extracellular matrix com- From an anatomical perspective, post-transdifferentia- ponents, blood vessels, nerves, and amoeboid cells have tion, the electroneuroelectrocyte synapse (electroplate) also been described in intercellular regions [8]. appears to maintain characteristic synaptic folds and a Proteins that were originally identified in the Torpedo high density of membrane particles as revealed by elec- electric organ and subsequently studied in higher verte- tron microscopy and freeze-etch replicas of electric brates include agrin, dynein, chloride channel, and rapsyn organ tissue [5,31,32]. However, the extensive nerve [9-12]. Also identified in the electric organ are a, b, δ, terminal network, formed by four or five electromotor and g AChR subunits, a and b dystroglycan, dystrophin, neurons covers nearly the entire postsynaptic mem- syntrophin, dystrobrevin, receptor tyrosine kinase, tyro- brane, differs from the minute motor neuron connection sine protein kinase fyn, protein tyrosine kinase fyk, and with a single mammalian myofiber [5,18]. desmin [13-23]. The electric organ has been used to Despite the electric organ being used as a model for the define the structure and function of creatine kinase and mammalian NMJ, current literature describes a number of AChR pore [24,25]. These proteins also are characterized NMJ-associated proteins that have not been characterized at the mammalian neuromuscular junction (NMJ) or are in the electric organ. One such protein is low-density lipo- components of skeletal muscle, which is consistent with protein receptor-related protein 4 (Lrp4), which forms a the Torpedo electric organ representing an extreme adap- complex with muscle-specific tyrosine-protein kinase receptor (MuSK) to facilitate neuronal agrin binding and tation of muscle tissue and the NMJ. Thus, the electric subsequent initiation of downstream signaling for tran- organ has served as a model to study the NMJ. However, scriptional activation of synaptic genes or AChR clustering the number of NMJ proteins described in current mouse, cell culture, and Drosophila studies demands a closer [33,34]. It is likely that agrin plays a similar role in the Mate et al. Skeletal Muscle 2011, 1:20 Page 3 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 electric organ as in the NMJ, transferring communication Results between the nerve and postsynaptic tissue, but its down- Validation of Torpedo-specific protein identification stream target, MuSK, is loosely defined. The published Tissue fractionation, gel electrophoresis, in-gel tryptic sequence for a tyrosine kinase receptor transcript digestion, and mass spectrometry (MS) analysis of the extracted from the Torpedo electric organ not only electric organ provided a global proteomic profile com- encodes extracellular Ig and frizzled domains and intracel- prising 435 proteins (count includes the different subu- lular C-terminal tyrosine kinase domains like human nits, subunit isoforms, isoforms, and types of proteins MuSK but also encodes a kringle-like domain that is with unique identifiers and does not include identical encoded in proteases and Ror receptor tyrosine kinases proteins found in different spots). Fractionating the elec- [20,35,36]. The orthology of the Torpedo tyrosine kinase tric organ decreases the complexity of its protein consti- receptor with mammalian MuSK was demonstrated by tuents and improves detection of low-abundant proteins inducing AChR clustering in the presence and absence of and protein digestion by decreasing the number of pro- agrin [37]. Furthermore, the cytoplasmic domain of MuSK teins resolved through electrophoresis in a single lane. binds directly to the tetratricopeptide repeat domain of Confidently identified proteins were determined by a rapsyn, supporting the presence of MuSK and possibly its combination of nanospray ESI-LTQ MS/MS spectral downstream effectors in the electric organ [38]. searches of our in silico translated cDNA library, MALDI Aside from this knowledge, electrocyte components TOF/TOF MS and MS/MS spectra from two-dimen- are undefined mainly because studying the proteome of sional gel spots, and through cross-species spectral T. californica is limited. A map of its genome does not matches to UniProtKB/Swiss-Prot amino acid sequences currently exist to computationally derive a hypothetical (Additional file 1) [40]. For peptides processed by ESI- protein profile and public databases contain sparse LTQMS/MS andsubsequentidentificationby sequence data for this species. While the Torpedo gen- SEQUEST, our threshold for positive protein identifica- ome has not yet been reported, the genome sequence tion was two independent peptides, ΔCn >0.1, a variable likely would be a relatively blunt instrument to under- threshold of Xcorr versus charge state: Xcorr = 1.9 for stand the highly specialized structure and function of z = 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, the Torpedo electric organ. For this reason, we sought protein Xcorr >40, and a peptide probability based score to understand the molecular components of the electric with a P value <0.01. For peptides processed by MALDI- organ using a combined mRNA (expressed sequence tag TOF/TOF MS and subsequent identification by MAS- (EST)) and proteomics approach. COT http://www.matrixscience.com/search_form_select. We have previously reported a preliminary proteome html, our threshold for positive criteria were protein based on two-dimensional matrix-assisted laser deso- score CI >95%, protein score >69, and proteins with iso- rption/ionization - time of flight/time of flight mass spec- electric points (PI) and molecular weights (MW) that trometry (MALDI-TOF/TOF MS) of soluble proteins and match the gel spot. To represent a concise proteome for shotgun proteomics of insoluble electric organ fractions in the electric organ, all accepted protein identifications which mass spectral mapping was based on a preliminary were further processed by selecting the highest scoring library composed of 607 cDNA sequences [39]. More identification amongst redundant proteins and the recently, we reported sequencing the transcriptome of T. removal of lower scoring duplicates. Isoforms and sub- californica to assemble a Torpedo cDNA library composed types of proteins were treated as unique identifications of 10,326 sequences assembled into 4,243 non-overlapping and are included in the 435 proteins listed in Additional contigs [40]. Here, we present a comprehensive electric file 1. organ proteome as defined by one-dimensional SDS- As an initial step to validate our data from SDS-PAGE, PAGE followed by nanospray electrospray ionization we performed mass spectral mapping using known Tor- quadrupole linear ion-trap tandem mass spectrometry pedo proteins. We created a validation database consist- (ESI-LTQ MS/MS) and two-dimensional isoelectric focus- ing of all Torpedo protein sequences found on public ing (IEF) SDS-PAGE followed by MALDI-TOF/TOF MS- access databases (GenBank and UniProtKB) to search based approaches of electric organ fractions in which mass raw spectra. We identified 20 out of 44 Torpedo proteins spectral mapping was performed using sequences from listed on the public access databases (Figure 1, Additional 10,326 Torpedo cDNA sequences and The Universal Pro- file 2). The Torpedo AChR (a, b, g, δ) and sodium/potas- + + tein Resource (UniProtKB/Swiss-Prot). Our results sium-transporting ATPase (Na /K -ATPase) subunits (a, demonstrate concordance between skeletal muscle, NMJ, b) were amongst the most abundant proteins identified. and electric organ proteomes. In addition, the electric These two protein complexes are functionally related in organ expresses several uncharacterized proteins that may that the binding of a ligand to one complex also influ- + + function at a synapse. ences the activity of the other [41]. The Na /K -ATPase Mate et al. Skeletal Muscle 2011, 1:20 Page 4 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 1 Identification of Torpedo proteins listed in public access databases. To validate tandem mass spectrometry (MS/MS) data against species-specific sequences, spectra acquired via MS/MS analysis of electric organ fractions were analyzed by the SEQUEST algorithm in BioWorks 3.3.1 software, crossreferencing known and characterized Torpedo proteins listed in GenBank. Peptide acceptance criteria was set at ΔCn >0.1, a variable threshold of Xcorr versus charge state: Xcorr = 1.9 for z = 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr >40, and a peptide probability based score with a P value <0.01. Protein identifications were compared with a search against UniProtKB (Swiss-Prot and TrEMBL) release 14.0, all species, to maintain consistency with databases used and protein accession numbers reported. Proteins identified are categorized by the likelihood and appropriateness of detection based on protein subcellular location or on the quality of data on public access databases. is essential for maintaining the electrochemical potential of the mammalian NMJ and these were also identified of electrocytes, and activity of this ion exchanger is in our Torpedo electric organ proteome (14-3-3 protein required for generating electric pulses that Torpedo uses g, heat shock protein (HSP)90b,HSP 70 kDaprotein, for predation. Of the remaining 24 proteins, we do not laminin subunit a-2, laminin subunit b-2, laminin subu- expect to identify 13 proteins that are expressed in brain, nit g-1, stress-induced phosphoprotein 1, dynamin 1, in neurons, or in immune cells. We did not identify 11 vesicle-fusing ATPase, Ras-related C3 botulinum toxin listed Torpedo proteins that were previously character- substrate 1, prostaglandin E synthase 3, guanine nucleo- ized through study of the electric organ (Additional file tide-binding protein G(I)/G(S)/G(T) subunit b-1, G sub- 2). However, all but one of these sequences are unre- unit b-1, subunit b-2-like1, G(s) subunit a,and Rho viewed by the UniProtKB consortium. In addition, the GDP-dissociation inhibitor 1, Ras-related protein R-Ras2 sequence information for a and b dystroglycan was (TC21)) [43,44]. Additionally, several presynaptic pro- extremely limited on UniProtKB (a single peptide) and teins localized to both synaptic vesicles (synaptic vesicle this would, by definition, fall below our two peptide mini- membrane protein VAT-1, synaptotagmin-B, choline mum requirement for identification from our MS/MS transporter-like protein 1), and the electromotor neuron scans. Another protein, Torpedo receptor tyrosine kinase, membrane were identified, showing representation of was translated in UniProtKB/Swiss-Prot from 2 small the presynaptic apparatus of the electric organ. overlapping ESTs and was in low abundance in the cDNA library previously described (2 in 90,000 clones), Torpedo cDNA sequences with both nucleotide and suggesting that the very low abundance underlies our protein sequence similarity to human ORFs inability to identify it by either MS/MS or via our cDNA Electric organ peptides mapped to the uncharacterized sequencing [20]. Lastly, dystrophin has multiple isoforms human ORFs C1orf123 and C6orf130, which aligned with and we identified two of the isoforms (Additional file 2) high sequence similarity to Torpedo cDNA sequences, [42]. Thus, we identified 16 of 17 listed Torpedo proteins supporting their expression in the electric organ. Human that have reviewed sequences and are known to be nucleotide and protein sequences were obtained from expressed in the electric organ, demonstrating the quality GenBank for alignment with translated Torpedo cDNA and scope of our data. sequence (Expasy translate tool) using EBI ClustalW Our proteome profile included two uncharacterized (default parameters with gonnet matrix). Sequence align- open reading frames (ORFs; C1orf123 and C6orf130) ments between human ORF nucleotide and protein and several well characterized mammalian NMJ pro- sequences and Torpedo cDNA nucleotide and translated teins, including AChR and AChR-associated proteins. nucleotide sequences are shown in Figure 2. In all, 79% Recent publications have characterized new components of amino acids in the translated Torpedo cDNA sequence Mate et al. Skeletal Muscle 2011, 1:20 Page 5 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 A. >gi|8923540|ref|NM_017887.1|C1orf123, mRNA; >sp|Q9NWV4|CA123_HUMAN UPF0587 protein C1orf123 >Contig[3573] Torpedo cDNA; >Contig[3573] Torpedo cDNA frame3 translation Experimental Peptides aacggcaagggcggcagccagcaccgggcggagagggctaccatggggaaaatcgcgctg CLUSTAL 2.0.12 multiple sequence alignment N G K G G S Q H R A E R A T M G K I A L ------------------------------------------------------------ SeqA Name Len(aa) SeqB Name Len(aa) Score caactcaaagccacgctggagaacatcaccaacctccggcccgtgggcgaggacttccgg =================================================================================== Q L K A T L E N I T N L R P V G E D F R 1 sp|Q9NWV4|CA123_HUMAN 160 2 Contig[3573]5'3'Frame3 512 76 cagttgaaagcgactttggaaaatatcagcaagttgcggccggacggagaggatttccgc =================================================================================== Q L K A T L E N I S K L R P D G E D F R tggtacctgaagatgaaatgtggcaactgtggtgagatttcggacaagtggcagtacatc sp|Q9NWV4|CA123_HUMAN MGKIALQLKATLENITNLRPVGEDFRWYLKMKCGNCGEISDKWQYIRLMD 50 W Y L K M K C G N C G E I S D K W Q Y I Contig[3573]5'3'Frame3 ---FGLQLKATLENISKLRPDGEDFRWYLKLKCQNCGEVSDKWQYVTLMN 47 tggtacctgaagttgaaatgtcagaattgcggtgaagtttccgataaatggcagtatgtc :.**********::*** *********:** ****:******: **: W Y L K L K C Q N C G E V S D K W Q Y V cggctgatggacagtgtggcactgaaggggggccgtggcagtgcttccatggtccagaag sp|Q9NWV4|CA123_HUMAN SVALKGGRGSASMVQKCKLCARENSIEILSSTIKPYNAEDNENFKTIVEF 100 R L M D S V A L K G G R G S A S M V Q K Contig[3573]5'3'Frame3 SAPLKGGRGSANMIQRCKLCSRENSIDILKNTIKPYNAEDSERFKTIVHF 97 acattaatgaacagcgccccactcaaaggtgggagaggaagtgccaacatgatacaaagg *..********.*:*:****:*****:**..*********.*.*****.* T L M N S A P L K G G R G S A N M I Q R tgcaagctgtgtgcaagagaaaattccatcgagattttaagcagcaccatcaagccttac sp|Q9NWV4|CA123_HUMAN ECRGLEPVDFQPQAGFAAEGVESGTAFSDINLQEKDWTDYDEKAQESVGI 150 C K L C A R E N S I E I L S S T I K P Y Contig[3573]5'3'Frame3 ECRGLEPVDFQPQAGFAAEGTESGTKFDEINLLEKDWNEYDEKIQESVGI 147 tgcaagttatgctcaagagagaactccattgatattctgaagaataccatcaagccatac ********************.**** *.:*** ****.:**** ****** C K L C S R E N S I D I L K N T I K P Y aatgctgaagacaatgagaacttcaagacaatagtggagtttgagtgccggggccttgaa sp|Q9NWV4|CA123_HUMAN YEVTHQFVKC---------------------------------------- 160 N A E D N E N F K T I V E F E C R G L E Contig[3573]5'3'Frame3 YDVTHKFVKI-TSLQLIPQPST-MDKSSDQ-TNSSSLSGICLPMQLHCVI 194 aatgctgaagacagtgaaagatttaagaccattgtacatttcgaatgtcggggattggag *:***:*** N A E D S E R F K T I V H F E C R G L E ccagttgatttccagccgcaggctgggtttgctgctgaaggtgtggagtcagggacagcc P V D F Q P Q A G F A A E G V E S G T A ccagttgattttcaaccgcaggctggatttgctgcagaaggaacagaatccggaacaaaa P V D F Q P Q A G F A A E G T E S G T K ttcagtgacattaatctgcaggagaaggactggactgactatgatgaaaaggcccaggag F S D I N L Q E K D W T D Y D E K A Q E tttgatgaaattaatctgctggaaaaggactggaatgaatatgatgagaaaatccaagaa F D E I N L L E K D W N E Y D E K I Q E tctgtgggaatctatgaggtcacccaccagtttgtgaagtgctga S V G I Y E V T H Q F V K C - tcggtgggaatctatgacgtcactcataagtttgttaaaatatga S V G I Y D V T H K F V K I - B. >gi|34147711|ref|NM_145063.2| C6orf130, mRNA; >sp|Q9Y530|CF130_HUMAN Uncharacterized protein C6orf130 >TFI_1_F6.T3 Torpedo cDNA; >TFI_1_F6.T3 Torpedo cDNA frame2 translation Experimental Peptides ggtgacttggctgaagaaacacttaaattctggaaatagcgactcagtatcatggccagc CLUSTAL 2.0.12 multiple sequence alignment G D L A E E T L K F W K - R L S I MA S ctgaagacgaacacccaacgaaaggacgaacaaacggaaaaactaaacaaaatgactagc SeqA Name Len(aa) SeqB Name Len(aa) Score L K T N T Q R K D E Q T E K L N K M T S ================================================================================= Agccttaatgaagatccagaaggaagc------agaatcacttatgtgaaaggagacctt 1 sp|Q9Y530|CF130_HUMAN 152 2 TFI_1_G6.T3 218 55 S L N E D P E G S - - R I T Y V K G D L ================================================================================= tctgcagacaagccactagagggcaatacctttgagatctgttatgtgcaaggtgatctg S A D K P L E G N T F E I C Y V Q G D L sp|Q9Y530|CF130_HUMAN -------------------------------------------------- tttgcatgcccgaaaacagactctttagcccactgtatcagtgaggattgtcgcatgggc TFI_1_G6.T3 CFTNLKIINCLSPSILKDPPHPSHALFSLLPSGRRYRSLKTNTQRKDEQT 50 F A C P K T D S L A H C I S E D C R M G ttctcatgcccagagaaggaagcactggcacattgcatcagcgaagactgcaaaatgaaa F S C P E K E A L A H C I S E D C K M K sp|Q9Y530|CF130_HUMAN -----MASSLNEDPEGS--RITYVKGDLFACPKTDSLAHCISEDCRMGAG 43 gctgggatagctgtcctctttaagaagaaatttggaggggtgcaagaacttttaaatcaa TFI_1_G6.T3 EKLNKMTSSADKPLEGNTFEICYVQGDLFSCPEKEALAHCISEDCKMKAG 100 A G I A V L F K K K F G G V Q E L L N Q *:** :: **. .* **:****:**:.::*********:* ** gcagggatagcagtcttgttcaagaagaaatatggatgtgtcgaggaactacagaatcag A G I A V L F K K K Y G C V E E L Q N Q sp|Q9Y530|CF130_HUMAN IAVLFKKKFGGVQELLNQQKKSGEVAVLKRDGRYIYYLITKKRASHKPTY 93 caaaagaaatctggagaagtggctgttctgaagagagatgggcgatatatatattacttg TFI_1_G6.T3 IAVLFKKKYGCVEELQNQKKKVGDVAVLQKDQRCIYYLITKSLAADKPTY 150 Q K K S G E V A V L K R D G R Y I Y Y L ********:* *:** **:** *:****::* * *******. *:.**** aaaaaaaaagttggggatgttgcagtactacagaaagatcagagatgcatctattacttg K K K V G D V A V L Q K D Q R C I Y Y L sp|Q9Y530|CF130_HUMAN ENLQKSLEAMKSHCLKNGVTDLSMPRIGCGLDRLQWENVSAMIEEVF-EA 142 attacaaagaaaagggcttcgcacaagccaacttatgaaaacttacagaagagtttagag TFI_1_G6.T3 DDLQKSLKAMRDHCLDNGILKISXPKIGCGLDXLXWDKVSAIIXEVFXKX 200 I T K K R A S H K P T Y E N L Q K S L E ::*****:**:.***.**: .:* *:****** * *::***:* *** : attaccaaatcattagcagcagataagcctacttatgacgatctgcagaagagcctcaag I T K S L A A D K P T Y D D L Q K S L K sp|Q9Y530|CF130_HUMAN TDIKITVYTL-------- 152 gcaatgaagtctcattgtctgaagaatggagtcactgacctctccatgcccaggattgga TFI_1_G6.T3 KYLQFTVXSFVEEXLWLP 218 A M K S H C L K N G V T D L S M P R I G . :::** :: gccatgagggaccactgcctggataatggaattttgaagatctcantgccgaagattgga A M R D H C L D N G I L K I S X P K I G tgtggtcttgatcgtctgcaatgggaaaatgtatctgcgatgatcgaggaggtatttgag C G L D R L Q W E N V S A M I E E V F - tgtggactggacnacctgnagtgggacaaagtttctgccataattcnagaagtctttnaa C G L D X L X W D K V S A I I X E V F X Gcaacagacatcaaaattactgtgtacacactctgatga--------------------- E A T D I K I T V Y T L -- --- - - - aagcncaaatatttacaatttactgtgtnctcttttgttgaagaatntctgtggttaccg K X K Y L Q F T V X S F V E E X L W L P Figure 2 Sequence alignments between uncharacterized human open reading frames (ORF) and Torpedo cDNA. Two human ORFs were identified by tandem mass spectrometry (MS/MS) analysis of electric organ fractions by the SEQUEST algorithm in BioWorks 3.3.1 software, crossreferencing our in-house Torpedo californica cDNA library translated into six reading frames. Comprehensive alignments of nucleotide and protein sequences between uncharacterized human ORFs (blue text) and Torpedo cDNA (black text) were compiled from individual ClustalW alignments (default parameters with gonnet matrix) for C1orf123 (a) and C6orf130 (b). ClustalW protein alignment is shown separately to highlight protein sequence similarity with the translated cDNA sequence (Expasy translate tool) and peptides identified by mass spectral mapping (highlighted in red). Start and stop amino acids are highlighted in yellow. Mate et al. Skeletal Muscle 2011, 1:20 Page 6 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Contig [3573] are identical to C1orf123 and 59% of membrane proteins receptor expression-enhancing TF_1_F6.T3 with C6orf130. TF_1_F6.T3 cDNA sequence protein 5, MIP18 family protein FAM96A, WD repeat- is from a single insert and not a contig such that coverage containing protein 1, and matrix-remodeling-associated may be reduced by sequencing errors that were not cor- protein 7. rected for by a consensus of multiple reads like Contig [3573]. However, peptide SLAADKPTYDDLQK is unique Electric organ proteome compared to skeletal muscle to C6orf130 when queried in blastp (word size 2, PAM30 proteome to assess the degree of ‘muscle likeness’ matrix, Homo sapiens) supporting the identification of Electric organ literature claims that a ‘muscle-like’ phe- this ORF. These ORFs demonstrate that further investi- notype is maintained after transdifferentiation. In our gation of the electric organ transcriptome may advance profile, several proteins are considered highly expressed our knowledge of the human proteome. in skeletal muscle or are muscle specific to include AChR subunits a,b,δ,and g, rapsyn, syntrophin, L-lactate dehy- Global proteomic profile classified according to drogenase A chain, phosphoglycerate mutase 2, creatine UniProtKB/Swiss-Prot annotation kinase M-type, cofilin 2, sorcin, 14-3-3 protein g, myosin To obtain a preliminary identification for each Torpedo 11, actin, aortic smooth muscle, transgelin, dystrophin, cDNA sequence identified in the spectral data, all dystrobrevin a, desmin, plectin 1, HSP90b, laminin subu- 2+ sequences were queried in blastx (Swiss-Prot sequence, nit b-2, and SR Ca( )-ATPase 1. As a further step to word size 3, BLOSSUM80 matrix) across all species and compare the skeletal muscle versus the electric organ then against human (See Additional file 1 for a full list of repertoire of proteins, we compared the proteins identi- cDNA sequences with blastx results). Only the top ranking fied in the electric organ presented in this paper to a aligned sequence was accepted for identification of the mouse skeletal muscle proteome produced in our labora- cDNA sequence. The blastx identification allowed cDNA tory using similar methods. Plotting the number of pep- sequences to be grouped with the UniProtKB list of identi- tides for each protein composing the electric organ or fications for classifying the proteins as NMJ, muscle, likely skeletal muscle proteome not only visually displays the in muscle, and metabolic proteins according to Uni- overlap in proteins in both tissues but more importantly ProtKB/Swiss-Prot annotation (Figure 3 andAdditional file displays the detectable proteins unique to each tissue, 3). A total of 33% of proteins are known muscle proteins, those lying on the × and y axis corresponding to tissue 3% of which are located at the NMJ. A total of 36% are type (Figure 6). Analysis showed the distribution of these involved in metabolism and 3% are known to be electric proteins differed in biological processes and molecular organ specific. Ingenuity Pathway Analysis (IPA version function (Table 1). Proteins composing the myofibrillar 8.8-3204) of all UniProtKB and Torpedo cDNA identifica- apparatus or are involved in calcium transport are pre- tions classified 40 molecules (P value 2.93E-09 to 1.18E- sent in theskeletalmuscleproteomeand absent in the 02) involved in skeletal and muscular system development electric organ proteome, as expected given that the elec- and function, the top physiological system designated trocytes are non-contractile cells. However, no common from our list of identifications. NMJ proteins were identified in the skeletal muscle pro- To summarize the electric organ proteome, we used teome but are amongst the highest expressed proteins in IPA Path Designer tool to map the annotated subcellular the electric organ. This was also expected for an analysis location of each protein identified (Figure 4). This also based on total muscle extract given the limited size and provides a virtual model of the electrocyte to assess how number of endplates in skeletal muscle. it may relate to skeletal muscle and the NMJ. The virtual electrocyte revealed several proteins believed to be mus- Discussion cle specific or highly abundant in muscle, confirming the T. californica proteome and defining the NMJ proteome muscle-like identity of the organ (Additional file 3). It for accurate comparison also depicted relatively intact pathways for energy meta- Our goal was to generate a proteomic profile of the bolism (oxidative phosphorylation and glycolysis), protein T. californica electric organ, both to assess its similarity processing (translation initiation, elongation, trafficking, to the mammalian NMJ proteome and to provide novel and proteasome degradation) and several components candidate proteins for localization to the NMJ. We and involved in redox reactions and caveolar endocytosis. A others have carried out microdissection of the NMJ prominent feature is an abundance of cytoskeletal pro- region and messenger RNA profiling to characterize the teins to include molecular motors, capping and folding NMJ constituents, but these have proven technically proteins, and focal adhesion molecules. Also notable are challenging and have fallen short in describing a broader a number of proteins that interact with known NMJ pro- proteome [45,46]. teins (Figure 5). Finally, the virtual electrocyte reveals A key resource for our one-dimensional ESI-LTQ MS/ several relatively uncharacterized proteins such as MS and two-dimensional MALDI-TOF/TOF MS Mate et al. Skeletal Muscle 2011, 1:20 Page 7 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 3 Classification of proteins identified in electric organ fractions by tissue association or function as determined by UniProtKB annotation. Electric organ fractions were separated one dimensionally and analyzed by nanospray electrospray ionization quadrupole linear ion- trap tandem mass spectrometry (ESI-LTQ MS/MS). Mass spectral matching of raw spectra against UniProtKB and Torpedo cDNA library was performed in BioWorks 3.3.1 in which the peptide acceptance criteria was set at ΔCn >0.1, a variable threshold of Xcorr versus charge state: Xcorr = 1.9 for z = 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr >40, and a peptide probability based score with a P value <0.01. All cDNA sequences were queried in blastx (standard genetic code, Swiss-Prot, default algorithm parameters except for BLOSSUM80 scoring matrix) for identification via sequence similarity with a known protein, first across all species and then against Homo sapiens selected database. Cytosolic proteins were separated two dimensionally, analyzed via matrix-assisted laser desorption/ionization - time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS), and identified by MASCOT. Identification criteria was set at a protein score CI >95%, protein score >69, and proteins with isoelectric points (PI) and molecular weights (MW) that match the gel spot. Each identification was queried in UniProtKB for annotation of tissue expression and or function then categorized by the sections composing the pie chart. (See Additional file 3 for a list of proteins composing the pie chart.) profiles were cDNA sequences from the electric organ well as transdifferentiation from muscle precursor cells that enabled mass spectral mapping [40]. Of 435 pro- into the electric organ. Additionally, we identified sev- teins we identified in the electric organ, 300 (69%) eral proteins that are expressed by non-electrocyte cells composing the electric organ, such as the electromotor showed ≥2 peptides that mapped to our Torpedo cDNA sequences while the remaining 135 (31%) were charac- neuron proteins, Schwann cell proteins, and proteins of terized via cross-species peptide spectral mapping to the immune and circulatory systems. mammalian proteins. We found that 48% of identified To compare our Torpedo data to previous studies of proteins were highly expressed in skeletal muscle or are the mammalian NMJ, we scanned the literature for muscle specific, which supports the ‘muscle-like’ lineage known NMJ proteins, grouped these into three cate- of the electric organ. The proteome includes cytoskele- gories, and overlaid our Torpedo proteome with these tal, glycolytic, translational, and degradative proteins. groups. The first category was limited to proteins in The high prevalence of glycolytic enzymes likely is which experimental knockout (loss of function) data necessary to support the high metabolic load of the suggested an important functional role in postsynaptic organ that is required for establishing and maintaining architecture and function (for example, disruption of the membrane potential. The abundance of proteasome morphology) (Additional file 4; see also references cited and degradative enzymes is in line with high protein therein). The second category included protein-protein turnover and degradation during synapse renewal as networks nucleated by the key functional candidates in Mate et al. Skeletal Muscle 2011, 1:20 Page 8 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 4 Virtual Torpedo electrocyte. All identifications from UniProtKB/Swiss-Prot and Torpedo cDNA searches of fractions analyzed by nanospray electrospray ionization quadrupole linear ion-trap tandem mass spectrometry (ESI-LTQ MS/MS) and matrix-assisted laser desorption/ ionization - time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) were mapped to cellular regions based on UniProtKB annotations using the Path Designer tool in Ingenuity IPA 8.8-3204. category 1. Most category 2 proteins were shown to molecules listed). The presence of these molecules sug- attenuate AChR clusters when mutated, inhibited, or gests the neuromuscular protein machinery supporting deleted. Finally, the remaining category contained pro- thecholinergicendplatecoincides with theelectric teins strictly concentrated at the endplate but do not organ and may serve as a model NMJ to study these alter AChR clusters or synapse morphology (category 3). proteins. We identified rapsyn, b-spectrin, Ras-related C3 botuli- In addition to the few Torpedo proteins characterized num toxin substrate 1, and laminin subunit b-2 from at the cholinergic synapse (AChR subunits a, b, δ,and category 1, HSP90b, HSP 70 kDa protein, a syntrophin, g,ACHE,rapsyn,14-3-3 g, syntrophin) we identified 14-3-3 protein g, dynamin, vesicle-fusing ATPase, a- several uncharacterized proteins in the electric organ known to play a role in maintaining AChR clustering actinin, utrophin, and Ras from category 2, and ankyrin, desmin, and dystrobrevin from category 3 (16/38 and in transducing signals between the membrane and Mate et al. Skeletal Muscle 2011, 1:20 Page 9 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 5 Torpedo electrocyte proteins in context of the mammalian neuromuscular junction (NMJ). Electric organ identifications are displayed in context of the mammalian NMJ paradigm. Red shapes indicate proteins we identified in our Torpedo electric organ proteome. White shapes are proteins we did not identify. This image was created using Path Designer in Ingenuity IPA 8.8-3204. nucleus (Figure 5). These proteins include laminin subu- interacts with extracellular matrix (integrins and agrin) nits a-2, b-2, and g-1, HSP90b, HSP 70 kDa protein, and postsynaptic membrane components (basal cell stress-induced-phosphoprotein 1, dynamin 1 and vesi- adhesion molecule (Bcam), a dystroglycan, and AChR) to cle-fusing ATPase, a-actinin, prostaglandin E synthase link the extracellular regions with the intracellular cytos- 3, Ras-related C3 botulinum toxin substrate 1, guanine keleton and to regulate the release of intracellular cal- nucleotide-binding protein G, guanine nucleotide-bind- cium directed at AChR cluster formation [48-50]. ing protein subunit b-2-like1, Rho GDP-dissociation Subunit b2 also assists in the development of synaptic inhibitor 1, and Ras-relatedprotein R-Ras2. Below,we folds and Schwann cell placement at the synapse [51]. describe each of these electric organ components as The laminin receptors characterized at the synapse, they relate to the mammalian NMJ. Bcam and dystroglycan, were not identified but dystro- glycan was previously characterized in the electric organ T. californica proteome related to AChR clustering [13,52]. However, we identified laminin receptor 1 A key event in the formation of the neuromuscular junc- (LamR1 or RPSA), a known binding partner for the lami- tion is the clustering of AChRs to focal points underlying nin complex in the electric organ (a2, b2, and g1; also motor neuron synapses. At the developing synapse, a key called S-merosin or laminin 2/4). Interestingly, LamR1 protein complex involved in clustering is the laminins: has not been previously reported at the NMJ [53,54]. multisubunit glycoprotein complexes consisting of a, b, Recent evidence supports the role of HSP90b and HSP 70 and g chains, each with multiple isoforms, assembled in a kDa protein (HSP70) as stabilizing chaperones of NMJ pro- trimer of equal stoichiometry. Laminin subunits a2, b2, teins. HSP90b was shown to interact directly with rapsyn at and g1 are seen most frequently in mature NMJs where its tetratricopeptide repeat (TPR) domain following its binding to surface AChR clusters. Recruitment of HSP90b they form the laminin 4 complex (also called S-merosin); we identified each of these subunits in the T. californica is believed to stabilize AChR-rapsyn binding to influence proteome. Subunit g1 facilitates the interaction between AChR stability and maintenance and also may associate AChR and a7b1 integrins to prime cluster formation with a dystrobrevin and a syntrophin [44]. HSP70 may be prior to neuronal agrin release or when agrin levels are a cochaperone of HSP90b along with DnaJ homolog sub- low [47,48]. At the mature synapse, the laminin complex family C member 7, HSP40, and prostaglandin E synthase 3 Mate et al. Skeletal Muscle 2011, 1:20 Page 10 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Figure 6 Electric organ proteome overlaps with mouse skeletal muscle proteome but shows tissue-specific protein expression. Mouse skeletal muscle (tibialis anterior muscle or gastrocnemius muscle) and the Torpedo electric organ were fractionated and processed under similar conditions as stated under Figure 1. Mouse skeletal muscle proteins were identified by BioWorks 3.3.1 referencing only UniProtKB/Swiss-Prot. Electric organ (EO) and skeletal muscle proteins were compared and graphed in Microsoft Excel 2007 based on the number of peptides per protein identified in each tissue. EO proteins are mapped (#peptides/protein) on the × axis and mouse skeletal muscle on the y axis. The lower two graphs represent zoomed sections for visual clarity. (p23). p23 is involved in stabilizing the ATP-bound confor- (Rac1), G proteins) involved in agrin-dependent MuSK mation of HSP90, permitting the release of activated inter- activation and subsequent AChR clustering and synaptic acting partners [55]. We identified p23 in our Torpedo gene transcription (Figure 5). In this process, agrin cDNA library, suggesting its role as a cochaperone with binds Lrp4 to activate MuSK and its subsequent interna- HSP90 in the electric organ. We also identified stress- lization via clathrin-mediated endocytosis and to activate induced phosphoprotein 1, which facilitates the interaction expression of MuSK interacting proteins. Dynamin 1 between HSP90b and HSP70 [56]. and NSF are involved in receptor-mediated endocytosis, vesicle transport, and protein trafficking. NSF is essen- Intracellular signal transduction tial for agrin-induced receptor-mediated endocytosis of We identified proteins (vesicle-fusing ATPase (NSF), MuSK and activation of its downstream signaling mole- dynamin, Ras-related C3 botulinum toxin substrate 1 cules Abl kinase and Rac1 in C2C12 cells, which Mate et al. Skeletal Muscle 2011, 1:20 Page 11 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Table 1 T. californica electric organ proteome shows Table 1 T. californica electric organ proteome shows tis- tissue-specific proteins when compared to mouse sue-specific proteins when compared to mouse skeletal skeletal muscle proteome muscle proteome (Continued) Unique to skeletal muscle Unique to electric organ KINH TBB1 proteome proteome PBIP1 TBB2 Development (myogenesis): Myofibrillar: STIM1 TBB5 UN45B Cytoskeleton, actin M-band TBB2A Intermediate filaments Myofibrillar: ANK1 TBB2C K1C9 Cytoskeleton, actin Light chain part of A-band Sarcoplasmic reticulum/calcium K1H1 TITIN MYL9 pathways: Z-disk Neuromuscular junction (NMJ): AT2A2 NFH ACTN2 ACES AT2A3 NFL ACTN3 ACHA CALU NFM MYOTI ACHB JPH2 Sarcoplasmic reticulum/calcium pathways: PP2BA ACHD SRCA SORCN Class II myosins (A-band) ACHG CASQ1 Ion channels: MYBPH DTNA CASQ2 Chloride channel MYH1 HSP70 KPB1 CICH MYH3 HSP90B Ion channels: Calcium ATPase MYH4 RAPSN Voltage-sensitive calcium channels AT2B1 MYH6 NMJ-ECM CA2D1 AT8A1 MYH7 LAMA2 CAC1S Hydrogen-potassium ATPase MYH8 LAMB2 CACB1 AT12A MYPC2 LAMC1 Potassium channel AT1A Light chain part of A-band Cytoskeleton: TM38A Sodium-potassium ATPase MYL1 Cytoskeleton, actin Sodium-potassium ATPase AT1A3 M-band SEPT6 AT1A2 AT1B1 MYOM1 sept7 AT1B2 Extracellular matrix (ECM): OBSCN ACTG Oxygenation (muscle): NID2 Contraction ADDG MYG FINC PHKG1 ACTC ECM: CO1A1 MYLK2 ANK2 CO6A1 CO1A2 TNNC2 ARPC2 COEA1 CO6A3 TNNI2 ARPC4 ITB1 BGH3 TNNT3 CCDC6 NID1 HPLN1 TPM2 COF1 PEPD Neuronal: ACTN4 COF2 PGS2 AINX Cytoskeleton: DNJC7 Neurogenesis: VAMP3 Cytoskeleton, actin FERM2 NDKA ACTS PROF2 NDRG2 ML12B SLMAP Proteins on the × and y axis of Figure 6, indicating unique identification in MLRS TCPA the corresponding tissue, were listed based on biological process, molecular MLRV TLN2 function, and cellular localization. See Additional File 1 for expanded names of all abbreviated proteins. Actin capping-binding Actin capping-binding CAPZB CAZA2 RADI SPTB1 XIRP1 Cytoskeleton, microtubule promote AChR clustering [57]. In addition, dynamin supports clathrin-coated vesicles formed upon agrin- Cytoskeleton, microtubule DYN1 induced endocytosis of MuSK, which is translocated CLIP1 SIRT2 into lipid rafts for activation and signaling. Mate et al. Skeletal Muscle 2011, 1:20 Page 12 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Several studies support the ‘signaling endosome Lrp4/Src/Rapsyn network involved in AChR cluster for- hypothesis’ in which neurotrophic factors initiate ligand- mation and stabilization (Figure 5). The second is the mediated endocytosis of receptor tyrosine kinases into Agrin-MuSK/NRG-ErbB/MAPK/GABP network for clathrin-coated vesicles that contain activators such as G transcriptional activation of synaptic genes (Figure 5). proteins and downstream effector molecules involved in Absent molecules involved in these pathways include Ras-mitogen-activated protein kinase (MAPK) signaling downstream of tyrosine kinase 7 (Dok7), dishevelled [58-61]. In the electric organ, we identified guanine (Dvl), PAK, RAF-1, and extracellular signal-regulated nucleotide binding proteins and inhibitors that act on kinase (ERK). In addition, several Torpedo proteins with Rho family of Ras-related G proteins that may be UniProtKB/TrEMBL annotation that are expressed at involved in signaling endosomes. These include G subu- the NMJ were not detected by tandem mass spectrome- nit b-1 (GNB1), subunit b-2-like1 (RACK1), G(s) subu- try analysis of subcellular fractions. These include a and nit a (GNAS1), and Rho GDP-dissociation inhibitor 1 b dystroglycan homologs, the receptor tyrosine kinase (ARHGDIA). GNB1 composes part of the catalytic similar to MuSK, and protein tyrosine kinases Fyn and machinery of GTPases and provides docking regions for Fyk. However, we did identify dystrophin, dystrobrevin, interacting proteins. ARHGDIA prevents the release of and syntrophin that compose the dystroglycan complex GDP from Rho proteins (Rho, Rac, cdc42, TC10). and we did show several molecules that may be up and RACK1 is the receptor of protein kinase C (PKC), which downstream of receptor tyrosine-protein kinase ErbB is known to inactivate Rho; PKC also phosphorylates (neuroplastin (NPTN), Ras-related protein R-Ras2 serine residues of AChR δ subunit to promote receptor (RRAS2) or Ras-related protein Rap-1A (RAP1A), desensitization and disassembly [62-64]. Identification of HSP90b). several proteins involved in ligand-mediated endocytosis, We failed to identify relatively well characterized activators and inhibitors of GTPases, signaling, lysoso- mammalian NMJ proteins in our survey, including Lrp4, mal and proteasomal degradation support the mainte- MuSK, Dok7, Src and Fyn Kinase, Dvl, ErbB2, PKC nance of protein function across myogenic-derived cell (category 1), and agrin, laminin subunits a4, a5, PAK1, types. Rho, cyclin-dependent kinase 5 (cdk5), ephexin1, neure- Interestingly, Rac1 is involved in clathrin/dynamin- gulin, ETS transcription factor, Raf, MEK, MKK4, c-Jun independent endocytosis of AChR following binding N-terminal kinase (JNK), and c-Jun (category 2). This with bungarotoxin [65]. Rac1 functions in actin polymer may reflect technical issues with the sensitivity of our rearrangement to create compartments for AChR sur- proteomics methods and parameters (for example, false face sequestration and, most likely, polymerization of negative and low maximum mass range for glycosylated the cytoskeletal network involved in vesicle transport to peptides), challenges in mapping peptide spectral data to the lysosome for degradation. Rac is a key mediator of the partial cDNA sequence coverage or to cross-species receptor surface sequestration in addition to its role in transcript units, or significant differences in the struc- actin polymerization and rearrangement, which controls ture and function of the electric organ compared to the the number and arrangement of receptors at the synapse mammalian NMJ. Our study is based on non-targeted to modulate synaptic transmission. proteomics and it may be possible to identify these spe- Our proteome also includes an inhibitory protein of cific proteins in the electric organ using a more targeted synaptic gene expression. The 14-3-3 g (YWHAG), approach. The literature on the Torpedo electric organ extracted from Torpedo electric organ, reduced the supports the presence of agrin, a and b dystroglycan, expression of MuSK, AChR subunits ε and a,utrophin, MuSK, and Src kinases, which strengthens the organ’s and rapsyn and resulted in aberrant NMJ morphology use as a model NMJ. [43]. It is known that 14-3-3 g interacts with the N-ter- minus of Raf-1, HSP90 interacts with the C-terminus of Conclusions Raf-1, and Ras (RRAS2 (TC21)) binds to the Raf-1- The virtual electrocyte revealed that the Torpedo electric HSP90-p50 complex, causing the complex to translocate organ is a resource for several uncharacterized proteins to the plasma membrane and become an active kinase whose function may be clarified in future studies. for phosphorylating mitogen-activated protein kinase Knockout and reporter assays of C6orf130, C1orf123, kinase (MEK) [66-68]. PKC also is a target of 14-3-3 g. matrix-remodeling-associated protein 7, protein NipS- nap homolog 2, septin-6, prohibitin 2, GATS-like pro- T. californica proteome: limits as a NMJ model tein 2, SH3 domain-binding glutamic acid-rich protein, Several critical NMJ proteins are absent from our data. and 14-3-3 protein ζ/δ in mouse skeletal muscle may Most notable are proteins within the two major net- clarify their subcellular roles, which may reveal novel works responsible for postsynaptic stabilization and components involved in AChR expression and mainte- gene expression. The first network is the Agrin-MuSK- nance. The electric organ will continue to serve as a Mate et al. Skeletal Muscle 2011, 1:20 Page 13 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 model of membrane excitability and electrogenesis as it filtered through a 100 μm nylon filter (BD, Franklin + + is abundant in AChR and the Na /K -ATPase channels Lakes, NJ,USA)toremoveconnectivetissue. Thefil- and may be used as a model to design a prototype trate was centrifuged at 627 g for 15 min at 4°C. This biobattery. pellet was resuspended in 2 M STM buffer (2 M Based on our identification of electric organ proteins sucrose, 50 mM Tris-HCl pH 8.0, 5 mM MgCl ,Roche that match proteins in the three categories of our Mini Complete Protease Inhibitor and PhosStop Phos- defined ‘NMJ proteome’ and the persistent similarity to phatase Inhibitor) and placed in an ultracentrifuge at skeletal muscle, the electric organ can serve as a reposi- 80,000 g for 35 min at 4°C. The resultant pellet was tory of these NMJ molecules that are in low abundance resuspended in EBC buffer (50 mM Tris-HCl pH 8.0, in skeletal muscle. However, the absence of several NMJ 120 mM NaCl, 1% Triton-X 100, Roche Mini Complete components involved in synaptic gene expression and Protease Inhibitor and PhosStop Phosphatase Inhibitor). AChR clustering in our model limits our ability to con- After 15 min incubation at 4°C, the suspension was clude that it indeed represents the mammalian NMJ passed through a 20-gauge needle ten times to lyse any that is maintained similarly. This study offers a more cells. Soluble (S1) and insoluble fractions were separated detailed understanding of the electrocyte protein reper- by centrifugation at 9,000 g for 30 min at 4°C. The pel- toire with insight into the presence and absence of pro- let (P1.1) was resuspended in EBC buffer. teins between these two related tissues. It reflects their P2 was resuspended in 0.5 ml HDP buffer (10 mM 4- unique tissue-function specializations and insight into (2-hydroxyethyl)-1-piperazineethanesulfonic acid evolutionary conservation and divergence between (HEPES), 1 mM dithiothreitol (DTT), Roche Mini Com- synaptic gene expression, maintenance, and regulation. plete Protease Inhibitor and PhosStop Phosphatase Inhi- The data raises questions whether the pathways respon- bitor). After 30 min incubation on ice, the suspension sible for AChR clustering are required in the electrocyte was sonicated (Sonifier Cell Distributer 350, Branson given its dense innervations and high AChR expression Scientific Danbury, CT) on ice for five initial pulses, or whether electromotor neurons support the postsy- paused for 30 s, then a final ten pulses (50% Duty Cycle, napse with different neurotrophic or signaling molecules Pulsed-Hold, Output Control Limit 3). The lysate was than mammalian motor neurons such that the neuregu- centrifuged at 9,000 g for 30 min at 4°C. The superna- lin-ErbB pathway is unnecessary. tant was saved as S2. P2.1 was resuspended in ME buf- fer (20 mM Tris-HCl, 0.4 M NaCl, 15% glycerol, 1 mM Methods DTT, 1.5% TritonX-100, Roche Mini Complete Protease Sequencing and mass spectral database indexing of T. Inhibitor and PhosStop Phosphatase Inhibitor), incu- bated for 30 min at 4°C with rocking, then centrifuged californica cDNA library The 10,326 cDNA sequences utilized for proteomics at 9,000 g for 30 min at 4°C. The supernatant of solu- mass spectral mapping database have been previously blized P2.1 was saved as P2.1. described [40]. All T. californica sequences were saved S1 and S3 fractions were concentrated in a speed as a Fasta database and indexed in BioWorks 3.3.1 SP1 vacuum. P3 was suspended in EBC buffer. Each fraction (Thermo Fisher Scientific, Waltham, MA) as trypsin except P2.1 and S2 was desalted by passing the sample digested protein sequences from the translation across through a BioSpin6 column before protein quantitation all six reading frames. using the DC Protein Assay (BioRad, Hercules, CA, USA). Protein extracts were stored at 80°C until Fractionation of the electric organ electrophoresis. T. californica electric organ was fractionated by grinding and homogenizing electroplax in lysis buffer (0.25 M Protein isolation and identification sucrose, 20 mM Tris pH 8.0, 25 mM KCl, 5 mM Protein separation MgCl , Roche Mini Complete Protease Inhibitor and Proteins within each fraction were resolved using one- PhosStop Phosphatase Inhibitor (Roche, Branchburg, NJ, dimensional SDS-PAGE on Novex NuPage 3% to 8% USA) [69]. Tissue homogenate was centrifuged at 627 g Tris-Acetate MidiGel and 4% to 12% Bis-Tris MiniGel (2,500 rpm) for 15 min at 4°C. The pellet (P1) was Systems (Invitrogen, Carlsbad, CA, USA) according to saved for further purification and the supernatant was manufacturer’s directions such that 2.5-400 kDa pro- centrifuged at 10,000 g for 20 min at 4°C. The pellet teins may be isolated and prepared for ESI-MS/MS ana- (P2) was saved for further purification and the superna- lysis. In addition, 250 μg of cytosolic proteins were tant was ultracentrifuged at 100,000 g for 60 min at 4°C resolved by two-dimensional electrophoresis as pre- resulting in pellet P3 and supernatant S3. viously described with minor differences [70]. The P1 was processed further by homogenizing the iso- immobilized pH gradient (IPG) strip was rehydrated for lated pellet in 2-3 ml lysis buffer. The homogenate was 12 h at 20°C and was focused at 20°C using the Mate et al. Skeletal Muscle 2011, 1:20 Page 14 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 following conditions: 250 V for 15 min, 8,000 V for 2.5 15.0) by MASCOT using the following parameters: MS h, 500 V hold. Following isoelectric focusing (IEF), the peak filtering mass range 800-4,000 Da, minimum S/N IPG strip was incubated in equilibration buffer (6 M 10, peak density filter 50 per 200 Da, maximum num- urea, 50 mM Trizma preset crystals (pH 8.8), 2% SDS ber peaks 65; MS/MS peak filtering: mass 60 Da to 20 (w/v), 30% glycerol (w/v), 0.002% bromophenol blue) Da below precursor mass, lowest precursor 707.46 Da, with 1% DTT for 20 min followed by a 20 min incuba- peak density filter 50 per 200 Da, maximum number tion in equilibration buffer with 2% iodoacetamide. Each peaks 65, fixed modification carbamidomethyl (C), gel was fixed for 30 min in 5% acetic acid, 45% metha- variable modification oxidation (M), fragment ion tol- nol solution, stained with Bio-Safe Coomassie (Bio-Rad) erance 0.3, precursor tolerance 0.5. Proteins identified for 60 min, and destained in distilled water overnight. by the MASCOT algorithm were filtered based on pro- Protein digestion teins identified with MS/MS spectra, protein score CI Multiple molecular weight bands and spots were manu- >95%, protein score >69, proteins with PI and MW ally excised from the gel (Additional file 5) and pro- that match the gel spot. All .dat files of spectral data cessed for in-gel digestion with 12.5 ng/μl Trypsin Gold were also uploaded to the PRIDE database under the (reconstituted according to manufacturer’sdirections, same project title as ESI-LTQ-MS/MS data stated Promega, Madison, WI, USA) in 50 mM NH HCO as above. 4 3 previously described [71]. Lipid raft assay of fraction Protein identification: ESI-LTQ-MS/MS To isolate membrane proteins localized to lipid rafts, Recovered peptides from SDS-PAGE were analyzed using membrane was isolated from 3 g of electric organ nanospray ESI-LTQ MS/MS as previously described, according to the above procedure (protein separation) with minor differences [72]. Peptides were loaded onto a with minor modifications. Tissue homogenate was cen- C18 reverse-phase column for 10 min at a flow rate of 5 trifuged twice at 627 g for 15 min at 4°C and the super- nl/min then separated at a flow rate of 250 nl/min. A 65 natant passed through a 40 μm filter to clear cellular min linear gradient eluted peptides. The LTQ operated debris. The supernatant was ultracentrifuged at 100,000 in data-dependent mode to perform one full MS scan g for 60 min at 4°C to collect an insoluble pellet rich in (300-2,000 m/z) to select the five most intense peaks membrane proteins. Lipid rafts were isolated from elec- through dynamic exclusion for MS/MS analysis via colli- tric organ membrane fraction following previously pub- sion-induced dissociation (CID) with helium at 35% nor- lished guidelines with the following modifications: the malized energy. Raw spectra were analyzed by the gradient was ultracentrifuged at 100,000 g [77]. Visible SEQUEST algorithm in BioWorks 3.3.1 software, crossre- bands were collected and centrifuged at 14,000 g for 30 ferencing our T. californica cDNA library translated into min. The resultant pellets were resuspended in EBC buf- six reading frames and The Universal Protein Resource fer. Intermediate solutions were also collected and con- (UniProtKB/Swiss-Prot) release 14.0 [73]. Peptide accep- centrated by vacuum centrifugation. All collected tance criteria was set at ΔCn >0.1, a variable threshold of fractions were subjected to one-dimensional SDS-PAGE Xcorr versus charge state: Xcorr = 1.9 for z = 1, Xcorr = on Novex NuPAGE 4% to 12% Bis-Tris MiniGel using 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr NuPAGE MES SDS Running Buffer (Invitrogen, Carls- >40, and a peptide probability based score with a P value bad, CA) according to the manufacturer’sinstructions. <0.01. Spectral data (.raw files) were first converted into Protein bands were manually excised and processed for MS2 file format (.ms2 files) using pXtract, default set- ESI-LTQ-MS/MS analysis as described earlier. tings, and then into PRIDE XML format using PRIDE Converter for upload onto the PRIDE database [74-76]. Additional material Data can be found under the project name ‘Torpedo cali- fornica Electric Organ Proteome’, accession numbers: Additional file 1: Torpedo californica electric organ proteome. All 435 proteins, identified across different sample processing and mass 16,474-16,476. spectral data acquisition techniques, representing the T. californica Protein identification: MALDI-TOF/TOF MS proteome are listed in separated tabs based on the method of Two-dimensional IEF SDS-PAGE separated cytosolic identification. Electric organ fractions were separated by SDS-PAGE and analyzed by nanospray electrospray ionization quadrupole linear ion-trap peptides were processed and analyzed for protein iden- tandem mass spectrometry (ESI-LTQ MS/MS) or matrix-assisted laser tification as previously described with the following desorption/ionization - time of flight/time of flight mass spectrometry additional details. Data was acquired using the follow- (MALDI-TOF/TOF MS). For ESI-LTQ MS/MS, mass spectral matching of raw spectra against UniProtKB and Torpedo cDNA library was performed in ing parameters: mass range 500-4,000 Da, minimum S/ BioWorks 3.3.1 in which the peptide acceptance criteria was set at ΔCn N 20, mass tolerance ± 2 m/z, minimum peak match >0.1, a variable threshold of Xcorr versus charge state: Xcorr = 1.9 for z = 4, maximum outlier error 10 ppm, monoisotopic mass 1, Xcorr = 2.2 for z = 2, and Xcorr = 2.5 for z = 3, protein Xcorr >40, and a peptide probability based score with a P value <0.01. All cDNA [70]. MS and MS/MS spectra of peptides were sequences were queried in blastx (standard genetic code, Swiss-Prot, searched against the UniProtKB/Swiss-Prot (release Mate et al. Skeletal Muscle 2011, 1:20 Page 15 of 17 http://www.skeletalmusclejournal.com/content/1/1/20 Acknowledgements default algorithm parameters except for BLOSSUM80 scoring matrix) for We would like to acknowledge Aaron Lorsong for his work in generating identification via sequence similarity with a known protein, first across all the mouse skeletal muscle proteome and providing the proteomic data for species and then against Homo sapiens selected database (column C: all comparison with the Torpedo electric organ. In addition, we thank the PRIDE species/Homo sapiens). Score of blastx alignments: black ≥200, pink = 80- support team for their overwhelming assistance and technical support for 200, green = 50-80. For MALDI-TOF/TOF MS of cytosolic proteins the conversion and submission of spectral data. Supported by the National resolved two dimensionally, acquired data were searched against Center for Medical Rehabilitation Research (NIH 5R24HD050846-06), the UniProtKB/Swiss-Prot (release 15.0) by MASCOT using the following Intellectual and Developmental Disabilities Research Center (IDDRC) (NIH parameters: MS peak filtering mass range 800-4,000 Da, minimum S/N 10, P30HD40677), the WM Keck Foundation, and the Erynn Godla Family via the peak density filter 50 per 200 Da, maximum number peaks 65; MS/MS Juvenile ALS Foundation http://www.juvenileals.org/. peak filtering: mass 60 Da to 20 Da below precursor mass, lowest precursor 707.46 Da, peak density filter 50 per 200 Da, maximum number Author details peaks 65, fixed modification carbamidomethyl (C), variable modification 1 Department of Biochemistry and Molecular Genetics, IBS, George oxidation (M), fragment ion tolerance 0.3, precursor tolerance 0.5. 2 Washington University, Washington DC, USA. Department of Pediatrics, Proteins identified by the MASCOT algorithm were filtered based on 3 George Washington School of Medicine, Washington DC, USA. Research proteins identified with MS/MS spectra, protein score CI >95%, protein Center for Genetic Medicine, Children’s National Medical Center, Washington score >69, proteins with isoelectric points (PI) and molecular weights 4 DC, USA. Department of Integrative Systems Biology, George Washington (MW) that match the gel spot. School of Medicine, Washington DC, USA. Additional file 2: Validation of identified Torpedo proteins in spectral data using Public Access Databases. Torpedo protein Authors’ contributions sequences listed in GenBank were collected in a single database to SEM carried out the proteomic profiling of the Torpedo californica electric directly search Torpedo electric organ fractions with a Torpedo-specific organ, data comparison with mouse skeletal muscle proteome, the protein database. Results include Torpedo proteins also identified by a development of images projecting the electrocyte proteome as well as the search against UniProtKB, all species. UniProtKB accessions are used for proteome within the context of the NMJ, and preparation of the manuscript. consistency. The chart categorizes proteins positively identified, not KJB helped design and execute proteomic profiling and data analysis as well identified that are expected to be found with a possible explanation, and as assisting in the write-up of methodology and manuscript editing. EPH proteins not identified that are not expected to be identified. Reviewed provided the conceptual design of the project and insight into data analysis sequences are from the UniProtKB/Swiss-Prot database and unreviewed as well as drafting of the manuscript. All authors have read and approved sequences from UniProtKB/TrEMBL. this manuscript. SEM is a predoctoral student in the Biochemistry and Molecular Genetics Program of the Institute for Biomedical Sciences at the Additional file 3: Torpedo californica electric organ proteome George Washington University. This work is from a dissertation to be classified according to tissue expression or associated function. All presented to the above program in partial fulfillment of the requirements proteins identified by mass spectral mapping (listed in Additional file 1) for the PhD degree. were queried in UniProtKB for annotation of tissue expression and or function then categorized as electric organ (EO) specific, neuromuscular Competing interests junction (NMJ) specific, muscle specific or highly expressed in muscle, The authors declare that they have no competing interests. likely expressed in muscle based on function, expressed in neurons, or functions in metabolism and energy production. Torpedo cDNA Received: 20 December 2010 Accepted: 4 May 2011 sequences were queried by blastx (standard genetic code, Swiss-Prot, Published: 4 May 2011 default algorithm parameters except for BLOSSUM80 scoring matrix) to obtain a protein identification with high sequence similarity. References Additional file 4: Neuromuscular junction (NMJ) proteins from the 1. Bennett MVL: Comparative physiology: electric organs. Annu Rev Physiol literature categorized by the degree of influence on synapse 1970, 32:471-528. architecture. The NMJ proteome was defined by searching the current 2. Connolly JA, St John PA, Fischbach GD: Extracts of electric lobe and literature and categorizing the influence of proteins on the synaptic electric organ from Torpedo californica increase the total number as well structure and function. 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