in vivo 24: 443-456 (2010)

A Two-dimensional Proteomic Profile of Tetrahymena thermophila Whole Cell Lysate

ALEXANDRA G. XANTHOPOULOU1,2, DIMITRIOS ANAGNOSTOPOULOS2, KONSTANTINOS VOUGAS1, ATHANASIOS K. ANAGNOSTOPOULOS1, ANASTASIA ALEXANDRIDOU3, GEORGE SPYROU3, ATHANASSIA SIAFAKA-KAPADAI2 and GEORGE TH. TSANGARIS1

1Proteomics Research Unit, Centre of Basic Research II, and 3Biomedical Informatics Unit, Biomedical Research Foundation of the Academy of Athens, Athens, Greece; 2Department of Biochemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Athens; Greece

Abstract. Tetrahymena thermophila is a unicellular eukaryotic considered to be a valuable model organism for genetic and model organism used for a variety of biochemical, molecular biological studies (2, 3). Its use has led to the discovery of and biological studies. According to its macronucleus genome important biomolecules such as catalytic RNA, telomere, sequence, it is expected to contain more than 27,000 protein- telomerase and the cell motor dynein, as well as elucidation coding genes, although only a small proportion of them have of the role of histone acetylation in gene expression (4). information published specifically about them. Here, we present Lately, T. thermophila has been widely used for enzyme a reference map for whole cell lysate of T. thermophila obtained purification and in bioreactors (5-8). using two-dimensional gel electrophoresis (2-DE) combined Like many ciliates, T. thermophila possesses two types of with mass spectrometry. Although (2-DE) is one of the most nuclei, distinct in their function (9). The micronucleus (MIC) efficient techniques for resolving complex protein mixtures and contains five pairs of mostly transcriptionally silent revealing the relative high-abundance proteins, it has not yet chromosomes, while the macronucleus (MAC) determines been applied generally to ciliates. In order to obtain qualitative the phenotype of the cell. The genome of the latter has only protein samples for analysis, an appropriate homogenization recently been annotated and found to consist of 225 method is required. Optimization of the homogenization method transcriptionally active chromosomes with a total size of 104 led to the analysis of nearly 4500 protein spots, the final Mb; 27,000 protein-encoding sequences have been identified identification of 375 different proteins using Mascot software (10). The mitochondrial proteome of T. thermophila was and an additional 258 gene products using a newly developed recently published (11), while from previous studies, data web service, called Peptide Finder, resulting in a total of 631 about its ciliome (12), its phagosomic proteins (13), basal different gene products that are considered to constitute the body proteins (14) and four novel β/γ crystalline domain proteomic profile of the whole cell lysate of T. thermophila. proteins from a subcellular fraction, enriched in granules (15) were made available. Out of the 27,000 predicted Tetrahymena thermophila is a unicellular eukaryotic protein-encoding sequences, approximately 15,000 have organism that belongs to the division of protists. It is a non- strong BLAST matches to known or predicted genes from pathogenic, aerobian that possesses characteristic structures, other organisms. However, only 53 proteins are annotated in such as cilia. Due to its structural and functional complexity, the protein database of UniProtKB/Swiss-Prot and 17,715 in as well as the fact that its genome preserves many primary UniProtKB/TrEMBL. In the present work, we applied biochemical procedures of eukaryotes (1), T. thermophila is proteomic technologies coupled with mass spectrometry and bioinformatics approaches in order to characterize the proteome of T. thermophila and construct its reference map.

Correspondence to: Dr. George Th. Tsangaris, Proteomics Research Materials and Methods Unit, Centre of Basic Research II, Biomedical Research Foundation of the Academy of Athens, Soranou Ephessius 4, 11527 Athens, Cell cultures. T. thermophila was cultured at 25˚C under aerobic Greece. Tel: +30 2106597075, Fax: +30 2106597545, e-mail: conditions in a medium consisting of 2% (w/v) proteose-peptone, [email protected] 0.5% (w/v) sucrose, 0.2% (w/v) yeast extract and 1% (v/v) Fe2+ - 9 mM EDTA (pH 5.5) (16). At the end of the logarithmic phase Key Words: MALDI-TOF, proteome, proteomics, Tetrahymena (~72 h), cells were harvested, centrifuged and the cell pellet was thermophila, TRI reagent, 2-D electrophoresis, Peptide Finder. washed using 0.9% NaCl.

0258-851X/2010 $2.00+.40 443 in vivo 24: 443-456 (2010)

Figure 1. Peptide map of total protein extract from T. thermophila. The first dimensional separation was conducted in a 3-10 immobilized pI gradient strip. The identified proteins are annotated by their accession numbers as listed in Table I.

Homogenization. Protein extraction was achieved using TRI- The solution was centrifuged at 12000×g for 5 min to remove Reagent (17), an organic solution of phenol and guanidine undiluted material and 200 μl chloroform were added to the thiocyanate that facilitates protein extraction with the supernatant. After incubation for 15 min and centrifugation at simultaneous removal of DNA and RNA content. DNA and RNA 12000×g for 10 min, the RNA phase was removed and 300 μl are endogenous charged particles that enclose proteins and inhibit ethanol were added to the residual phenol phase and interface to them from being separated during two-dimensional gel sediment the DNA. After 2-3 min, the solution was then electrophoresis (2-DE). The use of TRI Reagent not only centrifuged at 2000×g for 5 min. Three volumes of acetone were facilitates cell homogenization due to its organic compounds, but added to the supernatant, incubated for 10 min at room enriches the sample’s protein content as it removes inhibiting temperature and centrifuged at 12000×g for 10 min. molecules, giving clear, highly reproducible gel images and The protein pellet was washed with 1 ml of protein Wash 1 buffer identifications. (300 mM guanidine hydrochloride in 95% ethanol, 2.5% glycerol Cells from cell cultures were homogenized in TRI Reagent as (v/v)) and incubated for 10 min at room temperature, centrifuged at recommended by the manufacturer (Ambion/Applied Biosystems, 8000×g for 5 min, washed twice more with 1 ml of Wash 1 buffer Austin, TX, USA). A total of 10×106 cells were homogenized in and then washed with Protein Wash 2 (ethanol containing 2.5% 1 ml TRI reagent and incubated for 5 min at room temperature. glycerol (v/v)) for 10 min.

444 Xanthopoulou et al: Proteomic Profile of Tetrahymena thermophila

Two-dimensional electrophoresis (2-DE). Protein pellets were pooled and resuspended in urea lysis buffer (20 mM Tris, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS), 10 mM 1,4-dithioerythritol, 1 mM EDTA) and a mixture of protease inhibitors [1 mM PMSF and 1 tablet complete™ (Roche Diagnostics, Basel, Switzerland), per 50 ml of suspension buffer] and phosphatase inhibitors (0.2 mM Na2VO3 and 1 mM NaF). The protein content in the supernatant was determined by applying the Bradford method (18) using BIO- RAD protein assay (BIO-RAD Laboratories, Hercules, CA, USA). 2-D Gel electrophoresis was performed as previously reported (19). Samples of 1.0 mg total protein were applied on immobilized 3-10 pI, 4-7 pI and 6.3-8.3 pI non-linear gradient strips (17 cm) at their basic and acidic ends. Focusing for the former two strips started at 250 V for 30 min and the voltage was gradually increased to 5000 V at 3 V/min and remained constant for an additional 16 h. For pI 6.3-8.3, strip rehydration lasted only 6 hours and focusing started at 250 V for 30 min and the voltage was gradually increased to 8000 V at 3 V/min and remained constant for an additional 15 h. The second-dimensional separation was performed in 12% SDS- polyacrylamide gels (180×200×1.5 mm), running at 40 mA per gel in a PROTEAN apparatus (BIO-RAD). After fixation with 50% Figure 2. Functional classification of identified proteins from T. methanol containing 10% acetic acid for 2 h, the gels were stained thermophila. Proteins from Table I and II were classified into functional overnight with colloidal Coomassie blue (Novex, San Diego, CA, groups according to the UniProtKB/TrEMBL ontologies. USA), washed twice with water and scanned in a densitometer (GS- 800 Calibrated Densitometer; BIO-RAD). Samples not identified by PMF (probability significance of p<0.05) Peptide mass fingerprint and post source decay. Peptide analysis and were automatically selected for post-source decay (PSD) MS-MS protein identification were performed as previously described (20). analysis with MALDI-MS-MS in the same spectrometer. The Spots were detected using Melanie 4.02 software (GeneBio, Geneva peptide masses chosen for PSD-MS-MS analysis had a signal Swiss) on the Coomassie blue-stained gel and the spots were then intensity of >600 counts and were excluded from the trypsin excised by Proteineer SPII (Bruker Daltonics, Bremen, Germany), autodigest, matrix and keratin peaks. The resulting PSD spectra destained with 30% acetonitrile in 50 mM ammonium bicarbonate were also interpreted by the Mascot Software and Mascot and dried in a speed vacuum concentrator (MaxiDry Plus; Heto, probability-based scores of p<0.02 were considered significant. Allered, Denmark). Each dried gel piece was rehydrated with 5 μl of The identified proteins were automatically annotated on the gel 1 mM ammonium bicarbonate, containing 50 ng trypsin (Roche image by ProteinScape software (Bruker Daltonics). Obtained Diagnostics) and left for 16 h at room temperature. A total of 10 μl of spectra did not result in reliable significance in protein identification 50% acetonitrile, containing 0.3% trifluoroacetic acid, were added to through the Mascot software, re-evaluated with a newly developed each gel piece and incubated for 20 min with constant shaking. A web service called Peptide Finder (21). The search was carried out peptide mixture (1.5 μl) was simultaneously applied with 1 μl of through a database of completely digested T. thermophila proteins matrix solution, consisting of 0.025% α-cyano-4-hydroxycinnamic (http://bioserver-1.bioacademy.gr/Bioserver/ PeptideFinder/). For the acid, CHCA (Sigma-Aldrich, St. Louis, MO, USA) and the internal identification, complete peak lists with measured molecular weights standard peptides [Des-Arg]-bradykinin (904.4681 Da; Sigma) and obtained by the MALDI-MS analysis were uploaded to the software adrenocorticotropic hormone fragment 18-39 (2465.1989 Da; Sigma) and mapping to the proteins of the database requested. Additionally, in 65% ethanol, 35% acetonitrile and 0.03% trifluoroacetic acid. various filters are available in order to obtain a more refined list of Samples were analyzed for peptide mass fingerprint (PMF) with peptides and proteins with the requested molecular weights. Peptide matrix assisted laser desorption ionization – Mass Spectrometry Finder Score is an index used to comparatively rank the candidate (MALDI-MS) in a time-of-flight mass spectrometer (Ultraflex II proteins based on the statistics of the molecular weight distributions. MALDI-TOF-TOF MS/MS; Bruker Daltonics, Bremen, Gernamy). It suggests that a good identification should be based on the number Peptide matching and protein searches were performed of molecular mass matches, the frequency they present inside the automatically, as described by Berndt et al. (20). Each spectrum was Swiss-Prot database for the species (i.e. their randomness index) and interpreted by the Mascot Software (Matrix Sciences Ltd., London, the molecular mass (indicating the size) of the suggested protein. UK) and Peptide Finder. For peptide identification, the monoisotopic masses were used and a mass tolerance of 0.0025% Results was allowed. Unmatched peptides or peptides with up to one miscleavage site were not considered. The peptide masses were The objective of the present study was to characterize the compared with the theoretical peptide masses of all available proteins from all species using Swiss-Prot and TrEMBL databases. relevant proteins of high and medium abundance of T. The probability score identified by the software was used as the thermophila homogenate. For this reason, we employed 2-D criterion of the identification (http:/www.matrixscience.com). gel electrophoresis technique. The homogenates were analysed

445 in vivo 24: 443-456 (2010)

Table I. Proteins from T. thermophila were extracted and separated by 2-D gel electrophoresis as described in the Materials and Methods. Proteins were identified by MALDI-MS and MS/SM, following in-gel digestion with trypsin. The proteins identified by Mascot Software are designated by their Swiss-Prot accession numbers and their full names. The theoretical MW as well as the protein amino acid sequence coverage by the matching peptides are given as an indication of the confidence of the identification. Mascot Score is –10 log(P), where P is the probability that the observed match is a random event.

Protein name Protein description MW Mascot Cover- score age

A0BH21_PARTE Chromosome undetermined scaffold_107, whole genome shotgun sequence - Paramecium tetraurelia 33190.70 66 44 A0BNX8_PARTE Chromosome undetermined scaffold_119, whole genome shotgun sequence - Paramecium tetraurelia 27994.69 111 66 A0BNX9_PARTE Chromosome undetermined scaffold_119, whole genome shotgun sequence - Paramecium tetraurelia 20873.41 74 34 A0C5P1_PARTE Chromosome undetermined scaffold_150, whole genome shotgun sequence - Paramecium tetraurelia 24292.62 86 46 A0DJD3_PARTE Chromosome undetermined scaffold_53, whole genome shotgun sequence - Paramecium tetraurelia 44219.08 68 22 A0E0C7_PARTE Chromosome undetermined scaffold_71, whole genome shotgun sequence - Paramecium tetraurelia 45466.54 128 41 A0EB19_PARTE Chromosome undetermined scaffold_87, whole genome shotgun sequence - Paramecium tetraurelia 42336.25 63 19 A4VCW8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 26399.32 82 46 A4VD76_TETTH Hypothetical protein - Tetrahymena thermophila SB210 32806.01 75 27 gi|113305 Actin, cytoplasmic (Actin, micronuclear) - Tetrahymena thermophila SB210 42523.17 67 26 gi|115809 25 kDa calcium-binding protein (TCBP-25) [Contains: 10 kDa calcium-binding protein (TCBP-10)] 24801.51 73 22 gi|116475 Citrate synthase, mitochondrial precursor (14 NM filament-forming protein) - Tetrahymena thermophila SB210 52940.97 74 17 gi|118345684 Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 33775.26 68 30 gi|118347591 Hypothetical protein TTHERM_00418110 - Tetrahymena thermophila SB210 19193.41 68 47 gi|118348110 Enoyl-CoA hydratase/isomerase family protein - Tetrahymena thermophila SB210 31520.35 105 33 gi|118348344 Hypothetical protein TTHERM_00059150 - Tetrahymena thermophila SB210 31978.26 62 30 gi|118348384 Copper/zinc superoxide dismutase family protein - Tetrahymena thermophila SB210 21341.75 63 28 gi|118350268 Regulator of chromosome condensation - Tetrahymena thermophila SB210 53433.28 104 17 gi|118350492 Lysozyme - Tetrahymena thermophila SB210 23862.57 61 26 gi|118352740 Carboxyvinyl-carboxyphosphonate phosphorylmutase-related - Tetrahymena thermophila SB210 32042.91 62 32 gi|118353774 Hypothetical protein TTHERM_00559980 - Tetrahymena thermophila SB210 15660.56 85 50 gi|118354074 Hypothetical protein TTHERM_01018540 - Tetrahymena thermophila SB210 42359.19 63 3 gi|118355860 Hypothetical protein TTHERM_00145490 - Tetrahymena thermophila SB210 14707.62 75 23 gi|118356235 Hypothetical protein TTHERM_00433490 - Tetrahymena thermophila SB210 15074.74 133 41 gi|118358966 Co-chaperone GrpE family protein - Tetrahymena thermophila SB210 40667.41 73 20 gi|118362498 Hypothetical protein TTHERM_00522980 - Tetrahymena thermophila SB210 186847.78 115 6 gi|118362866 Hypothetical protein TTHERM_00046970 - Tetrahymena thermophila SB210 24096.53 125 33 gi|118363006 V-type ATPase, G subunit family protein - Tetrahymena thermophila SB210 14269.39 76 39 gi|118363098 Dihydrolipoamide dehydrogenase family protein - Tetrahymena thermophila SB210 57575.95 64 11 gi|118363256 Ribosomal S3Ae family protein - Tetrahymena thermophila SB210 24864.23 73 30 gi|118363404 Mov34/MPN/PAD-1 family protein - Tetrahymena thermophila SB210 35199.68 80 37 gi|118364445 Hypothetical protein TTHERM_00378630 - Tetrahymena thermophila SB210 61905.68 63 12 gi|118364497 Granule lattice protein 5 precursor, putative - Tetrahymena thermophila SB210 41540.68 130 6 gi|118364519 Viral A-type inclusion protein repeat containing protein - Tetrahymena thermophila SB210 149982.71 125 9 gi|118365441 Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 35241.61 76 28 gi|118366717 Hypothetical protein TTHERM_00188980 - Tetrahymena thermophila SB210 30371.22 73 21 gi|118367835 Ribosomal protein S5 containing protein - Tetrahymena thermophila SB210 46734.52 87 26 gi|118368405 Hypothetical protein TTHERM_00477020 - Tetrahymena thermophila SB210 13811.59 119 48 gi|118368469 Hypothetical protein TTHERM_00481300 - Tetrahymena thermophila SB210 32768.96 79 18 gi|118368471 Hypothetical protein TTHERM_00481310 - Tetrahymena thermophila SB210 42766.79 106 20 gi|118370059 Hypothetical protein TTHERM_00283760 - Tetrahymena thermophila SB210 18505.74 73 29 gi|118370910 Hypothetical protein TTHERM_00289380 - Tetrahymena thermophila SB210 14464.14 69 61 gi|118372704 Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 42060.61 94 14 gi|118373447 Hypothetical protein TTHERM_00590090 - Tetrahymena thermophila SB210 86611.74 71 16 gi|118374567 14-3-3 protein - Tetrahymena thermophila SB210 28228.01 76 32 gi|118374637 Hypothetical protein TTHERM_00218340 - Tetrahymena thermophila SB210 17712.73 61 15 gi|118376155 Hypothetical protein TTHERM_00777250 - Tetrahymena thermophila SB210 35423.16 216 78 gi|118376768 Ribosomal family S4e containing protein - Tetrahymena thermophila SB210 43053.42 81 39 gi|118376862 Ribosomal protein S6e containing protein - Tetrahymena thermophila SB210 112254.14 63 4 gi|118378644 Pyridine nucleotide-disulphide oxidoreductase family protein - Tetrahymena thermophila SB210 70081.99 65 24 gi|118378850 Granule lattice protein 3 precursor, putative - Tetrahymena thermophila SB210 61634.26 94 9 gi|118379843 Adenosylhomocysteinase family protein - Tetrahymena thermophila SB210 54143.22 79 16

Table I. continued

446 Xanthopoulou et al: Proteomic Profile of Tetrahymena thermophila

Table I. continued

Protein name Protein description MW Mascot Cover- score age gi|118380689 Hypothetical protein TTHERM_00535990 - Tetrahymena thermophila SB210 13211.45 67 37 gi|118381449 Hypothetical protein TTHERM_00473020 - Tetrahymena thermophila SB210 47766.66 74 28 gi|118381726 Hypothetical protein TTHERM_00657230 - Tetrahymena thermophila SB210 13018.32 63 41 gi|118383479 Succinate dehydrogenase and fumarate reductase iron-sulfur protein - Tetrahymena thermophila SB210 28274.93 83 25 gi|118384233 60s Acidic ribosomal protein - Tetrahymena thermophila SB210 11701.87 86 55 gi|118386523 Ubiquitin-conjugating enzyme family protein - Tetrahymena thermophila SB210 17954.99 67 44 gi|118388862 Hypothetical protein TTHERM_01055600 - Tetrahymena thermophila SB210 44483.58 74 18 gi|118389110 Hypothetical protein TTHERM_00569430 - Tetrahymena thermophila SB210 15045.74 61 28 gi|118389519 Ribosomal protein L7/L12 C-terminal domain containing protein - Tetrahymena thermophila SB210 34276.05 77 25 gi|118389764 Oxidoreductase, zinc-binding dehydrogenase family protein - Tetrahymena thermophila SB210 37771.60 61 23 gi|118395199 Hypothetical protein TTHERM_01205250 - Tetrahymena thermophila SB210 20742.39 103 32 gi|118395488 Predicted protein - Tetrahymena thermophila SB210 24292.31 99 25 gi|118397556 Hypothetical protein TTHERM_00927010 - Tetrahymena thermophila SB210 17377.15 88 17 gi|118399603 CAP-Gly domain containing protein - Tetrahymena thermophila SB210 28943.46 79 36 gi|118399953 Hypothetical protein TTHERM_00649060 - Tetrahymena thermophila SB210 39192.12 79 30 gi|118401044 Hypothetical protein TTHERM_00486310 - Tetrahymena thermophila SB210 37574.06 76 30 gi|118401200 Fructose-bisphosphate aldolase class-I family protein - Tetrahymena thermophila SB210 38859.07 74 22 gi|118401253 Hypothetical protein TTHERM_00470570 - Tetrahymena thermophila SB210 28640.46 63 39 gi|118401433 SNF7 family protein - Tetrahymena thermophila SB210 27485.98 64 35 gi|118573853 40S ribosomal protein S4 (40S ribosomal protein S7) 29557.96 107 57 gi|135483 Tubulin beta chain (Beta-tubulin) 50349.13 67 25 gi|136675 Ubiquitin 8519.59 66 56 gi|161744 Calcium-binding 25 kDa protein 24818.50 70 29 gi|3366651 Granule lattice protein 3 precursor; Grl3p - Tetrahymena thermophila SB210 41636.52 62 9 gi|860923 Alpha-tubulin - Euplotes aediculatus 42622.15 61 24 O00945_9HYMN Ubiquitin - Tetrahymena vorax 17091.23 154 42 O43956_TETTH Phosphoglucomutase - Tetrahymena thermophila 65720.41 92 24 O61174_9CILI Alpha-tubulin - Frontonia sp. 42465.39 143 52 O76219_TETTH Granule lattice protein 4 - Tetrahymena thermophila 43236.37 90 18 O76849_9CILI Beta-tubulin - Colpoda sp. 42109.64 84 16 O96774_TETTH Ribosomal protein L3 - Tetrahymena thermophila 44562.81 114 21 P90618_TETTH Granule lattice protein 1 - Tetrahymena thermophila 45417.20 86 25 Q1HFX5_TETTH Dynein light chain 3-likeB - Tetrahymena thermophila 12943.58 71 65 Q229S0_TETTH ADP-ribosylation factor, arf - Tetrahymena thermophila SB210 19971.51 109 66 Q22A30_TETTH Ribosomal protein L15 - Tetrahymena thermophila SB210 24175.16 62 35 Q22A35_TETTH Hypothetical protein - Tetrahymena thermophila SB210 8179.05 63 36 Q22A75_TETTH Hypothetical protein - Tetrahymena thermophila SB210 63050.92 192 47 Q22AI5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 22354.30 95 56 Q22AJ2_TETTH AhpC/TSA family protein - Tetrahymena thermophila SB210 23139.58 117 65 Q22AS4_TETTH Hypothetical protein - Tetrahymena thermophila SB210 171353.75 100 11 Q22AV9_TETTH KH domain containing protein - Tetrahymena thermophila SB210 28892.44 66 35 Q22AX5_TETTH Ribosomal protein L6 containing protein - Tetrahymena thermophila SB210 21436.66 119 61 Q22AZ2_TETTH Isocitrate lyase family protein - Tetrahymena thermophila SB210 64768.67 87 27 Q22B31_TETTH Hypothetical protein - Tetrahymena thermophila SB210 36222.14 90 61 Q22B43_TETTH Ubiquitin carrier protein - Tetrahymena thermophila SB210 17194.86 68 26 Q22B85_TETTH Glucose-6-phosphate isomerase family protein - Tetrahymena thermophila SB210 64135.83 106 28 Q22BB4_TETTH Proliferating cell nuclear antigen - Tetrahymena thermophila SB210 29030.53 137 62 Q22BK0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 29100.33 143 64 Q22BP5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 15919.04 108 80 Q22BQ2_TETTH Oxidoreductase, short chain dehydrogenase/reductase family protein - Tetrahymena thermophila SB210 31171.00 66 37 Q22BV6_TETTH Glutamine synthetase, catalytic domain containing protein - Tetrahymena thermophila SB210 45136.95 93 40 Q22BV7_TETTH Glutamine synthetase, catalytic domain containing protein - Tetrahymena thermophila SB210 45220.10 170 59 Q22BW3_TETTH Phospholipase/Carboxylesterase family protein - Tetrahymena thermophila SB210 33302.00 76 36 Q22CQ5_TETTH Phytanoyl-CoA dioxygenase - Tetrahymena thermophila SB210 41292.78 82 37 Q22CW1_TETTH DNAK protein - Tetrahymena thermophila SB210 70962.23 249 54 Q22CX0_TETTH 26S Proteasome subunit P45 family protein - Tetrahymena thermophila SB210 143564.74 89 13 Q22D31_TETTH Hypothetical protein - Tetrahymena thermophila SB210 48661.23 92 25

Table I. continued

447 in vivo 24: 443-456 (2010)

Table I. continued

Protein name Protein description MW Mascot Cover- score age

Q22D32_TETTH Hypothetical protein - Tetrahymena thermophila SB210 48407.97 94 31 Q22D58_TETTH Oxidoreductase, aldo/keto reductase family protein - Tetrahymena thermophila SB210 39813.41 244 78 Q22D83_TETTH Hypothetical protein - Tetrahymena thermophila SB210 24256.29 107 42 Q22DB4_TETTH Hypothetical protein - Tetrahymena thermophila SB210 36626.99 63 26 Q22DV8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 18956.00 129 17 Q22DW0_TETTH Endoribonuclease L-PSP, putative family protein - Tetrahymena thermophila SB210 16769.62 63 44 Q22DW1_TETTH Acetyl-CoA acyltransferases family protein - Tetrahymena thermophila SB210 41520.60 128 56 Q22G04_TETTH AhpC/TSA family protein - Tetrahymena thermophila SB210 26073.80 79 49 Q22GF6_TETTH UTP-glucose-1-phosphate uridylyltransferase family protein - Tetrahymena thermophila SB210 68645.44 78 28 Q22GI5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 120324.27 171 5 Q22GX4_TETTH Elongation factor 1-alpha - Tetrahymena thermophila SB210 48729.36 175 34 Q22H00_TETTH Copper/zinc superoxide dismutase family protein - Tetrahymena thermophila SB210 18059.87 69 36 Q22HE6_TETTH Bis(5'-adenosyl)-triphosphatase - Tetrahymena thermophila SB210 20072.20 72 41 Q22HK2_TETTH Viral A-type inclusion protein repeat containing protein - Tetrahymena thermophila SB210 145351.97 98 12 Q22KC0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 18469.26 71 62 Q22KJ9_TETTH Hypothetical protein - Tetrahymena thermophila SB210 35071.38 142 49 Q22KQ8_TETTH Nucleotide binding protein 2, putative - Tetrahymena thermophila SB210 63303.75 177 46 Q22KR4_TETTH Hypothetical protein - Tetrahymena thermophila SB210 36112.33 106 39 Q22LX1_TETTH Non-canonical purine NTP pyrophosphatase, rdgB/HAM1 family protein - Tetrahymena thermophila SB210 25980.00 88 54 Q22LY6_TETTH Hypothetical protein - Tetrahymena thermophila SB210 20638.18 62 41 Q22M72_TETTH Aconitate hydratase 1 family protein - Tetrahymena thermophila SB210 110144.77 163 25 Q22MB3_TETTH TCP-1/cpn60 chaperonin family protein - Tetrahymena thermophila SB210 59472.66 164 53 Q22MS9_TETTH Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 25287.45 66 23 Q22MV3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 44967.20 87 47 Q22MV4_TETTH Malate dehydrogenase family protein - Tetrahymena thermophila SB210 38108.69 110 37 Q22N23_TETTH Hypothetical protein - Tetrahymena thermophila SB210 9065.41 62 40 Q22NS8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 35651.99 73 35 Q22PA7_TETTH AAA family ATPase, CDC48 subfamily protein - Tetrahymena thermophila SB210 93840.61 227 38 Q22R57_TETTH Bm44, putative - Tetrahymena thermophila SB210 24409.41 72 45 Q22R83_TETTH Ras family protein - Tetrahymena thermophila SB210 25690.16 126 47 Q22RA1_TETTH Phosphoenolpyruvate phosphomutase - Tetrahymena thermophila SB210 34059.46 182 71 Q22RF2_TETTH Regulator of chromosome condensation - Tetrahymena thermophila SB210 51046.95 184 55 Q22RI9_TETTH Cytidyltransferase-related domain containing protein - Tetrahymena thermophila SB210 52337.36 72 29 Q22RN3_TETTH TPR Domain containing protein - Tetrahymena thermophila SB210 68679.59 195 50 Q22RQ9_TETTH UBA/TS-N domain containing protein - Tetrahymena thermophila SB210 41080.48 90 29 Q22SZ9_TETTH Hypothetical protein - Tetrahymena thermophila SB210 53038.16 70 15 Q22T77_TETTH Ubiquitin-activating enzyme - Tetrahymena thermophila SB210 49544.17 70 29 Q22T89_TETTH Triosephosphate isomerase - Tetrahymena thermophila SB210 28209.71 222 70 Q22TE0_TETTH DNAK protein BiP - Tetrahymena thermophila SB210 72174.43 304 55 Q22TF3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 68091.91 63 14 Q22U45_TETTH Cytosol aminopeptidase family, catalytic domain containing protein - Tetrahymena thermophila SB210 61410.47 122 34 Q22UE7_TETTH Glutaredoxin-related protein - Tetrahymena thermophila SB210 48152.78 114 28 Q22UL2_TETTH Glyceraldehyde-3-phosphate dehydrogenase, type I family protein - Tetrahymena thermophila SB210 37116.23 139 53 Q22UP3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 121020.38 103 20 Q22V96_TETTH TPR Domain containing protein - Tetrahymena thermophila SB210 37404.01 117 64 Q22W11_TETTH 2Fe-2S iron-sulfur cluster binding domain containing protein - Tetrahymena thermophila SB210 19813.77 115 36 Q22W19_TETTH Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 37360.59 120 42 Q22W26_TETTH Ribosomal protein L7Ae containing protein - Tetrahymena thermophila SB210 14086.39 70 34 Q22W31_TETTH Nucleoside diphosphate kinase family protein - Tetrahymena thermophila SB210 17241.77 130 58 Q22W51_TETTH Oxidoreductase, FAD/FMN-binding family protein - Tetrahymena thermophila SB210 63219.62 77 19 Q22W55_TETTH 14-3-3 protein - Tetrahymena thermophila SB210 28730.26 170 64 Q22W82_TETTH Hsp90 protein - Tetrahymena thermophila SB210 81996.84 196 34 Q22WB3_TETTH Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 30020.29 106 49 Q22WD0_TETTH RanBP1 domain containing protein - Tetrahymena thermophila SB210 22080.03 82 56 Q22WE4_TETTH TPR Domain containing protein - Tetrahymena thermophila SB210 56291.01 119 29 Q22WQ4_TETTH Actin family protein - Tetrahymena thermophila SB210 51279.58 203 50

Table I. continued

448 Xanthopoulou et al: Proteomic Profile of Tetrahymena thermophila

Table I. continued

Protein name Protein description MW Mascot Cover- score age

Q22WU7_TETTH Ribosomal protein S7 containing protein - Tetrahymena thermophila SB210 22746.98 116 61 Q22WZ2_TETTH Hypothetical protein - Tetrahymena thermophila SB210 37586.05 109 34 Q22X00_TETTH Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 31403.78 70 24 Q22XI7_TETTH Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 27557.40 96 49 Q22XM3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 42359.19 81 9 Q22XU5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 31446.02 94 43 Q22XV3_TETTH Metallopeptidase family M24 containing protein - Tetrahymena thermophila SB210 41340.66 220 62 Q22XW3_TETTH Superoxide dismutase , putative - Tetrahymena thermophila SB210 25363.04 83 59 Q22YJ5_TETTH SerH3 immobilization antigen, putative - Tetrahymena thermophila SB210 43223.47 72 28 Q22YL0_TETTH Putative uncharacterized protein - Tetrahymena thermophila SB210 24031.29 102 31 Q22YL1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 86837.45 81 25 Q22YU1_TETTH TCP-1/cpn60 chaperonin family protein - Tetrahymena thermophila SB210 60095.66 94 11 Q22Z06_TETTH Pyruvate kinase family protein - Tetrahymena thermophila SB210 56395.63 138 41 Q22Z08_TETTH Hypothetical protein - Tetrahymena thermophila SB210 34636.90 116 31 Q22Z34_TETTH Seryl-tRNA synthetase family protein - Tetrahymena thermophila SB210 54438.43 243 68 Q22Z89_TETTH Hypothetical protein - Tetrahymena thermophila SB210 48359.78 169 41 Q22ZB6_TETTH Transketolase, pyridine binding domain containing protein - Tetrahymena thermophila SB210 72129.57 190 42 Q22ZH1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 26386.84 95 21 Q230V2_TETTH Ribosomal protein S4 containing protein - Tetrahymena thermophila SB210 21131.59 94 45 Q230X4_TETTH Enoyl-CoA hydratase/isomerase family protein - Tetrahymena thermophila SB210 41424.65 121 45 Q230X6_TETTH Hypothetical protein - Tetrahymena thermophila SB210 19844.39 158 78 Q230Y9_TETTH Hypothetical protein - Tetrahymena thermophila SB210 22232.23 67 52 Q231A8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 10646.13 101 62 Q231M1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 72883.84 87 16 Q231P6_TETTH F-actin capping protein, beta subunit containing protein - Tetrahymena thermophila SB210 32002.16 157 55 Q232G7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 85853.87 128 23 Q232U3_TETTH Adenosine/AMP deaminase family protein - Tetrahymena thermophila SB210 82243.66 69 20 Q233B2_TETTH Hypothetical protein - Tetrahymena thermophila SB210 74975.50 113 26 Q233T1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 278294.42 63 7 Q233X7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 22714.03 110 46 Q234A8_TETTH Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 27828.11 123 47 Q234B0_TETTH Glycyl-tRNA synthetase family protein - Tetrahymena thermophila SB210 72503.40 67 10 Q234F5_TETTH Profilin family protein - Tetrahymena thermophila SB210 16869.49 63 50 Q234I5_TETTH DNAK protein - Tetrahymena thermophila SB210 71681.94 374 59 Q234R8_TETTH Thioredoxin family protein - Tetrahymena thermophila SB210 42949.77 117 37 Q236H9_TETTH Ser/Thr protein phosphatase family protein - Tetrahymena thermophila SB210 39354.88 182 55 Q236K0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 46986.42 95 24 Q236L9_TETTH Hypothetical protein - Tetrahymena thermophila SB210 71762.39 149 46 Q236M1_TETTH Co-chaperone GrpE family protein - Tetrahymena thermophila SB210 38259.08 151 46 Q236M7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 85374.28 129 34 Q236V1_TETTH FHA domain containing protein - Tetrahymena thermophila SB210 52468.31 66 18 Q238Z7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 45212.53 165 54 Q239D8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 14023.83 64 42 Q239E3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 39156.71 86 33 Q239L8_TETTH Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 36335.85 94 48 Q239R1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 18302.15 82 39 Q23AD3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 70118.30 102 24 Q23AI8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 84373.23 107 26 Q23AY2_TETTH Putative uncharacterized protein - Tetrahymena thermophila SB210 32227.91 77 23 Q23AY3_TETTH Mov34/MPN/PAD-1 family protein - Tetrahymena thermophila SB210 32798.61 141 51 Q23B10_TETTH Hypothetical protein - Tetrahymena thermophila SB210 19121.44 115 63 Q23BN3_TETTH Glycosyl hydrolases family 38 protein - Tetrahymena thermophila SB210 122254.58 109 23 Q23BQ5_TETTH Translation initiation factor eIF-5A family protein - Tetrahymena thermophila SB210 24023.07 99 47 Q23BZ5_TETTH XYPPX repeat family protein - Tetrahymena thermophila SB210 27154.14 77 21 Q23CV0_TETTH Cystathionine beta-lyase - Tetrahymena thermophila SB210 45975.90 156 55 Q23D94_TETTH Hypothetical protein - Tetrahymena thermophila SB210 64200.06 218 41 Q23DC8_TETTH Ser/Thr protein phosphatase family protein - Tetrahymena thermophila SB210 22284.40 109 55

Table I. continued

449 in vivo 24: 443-456 (2010)

Table I. continued

Protein name Protein description MW Mascot Cover- score age

Q23DE2_TETTH Hypothetical protein - Tetrahymena thermophila SB210 57826.29 203 44 Q23DE3_TETTH Ribosomal S3Ae family protein - Tetrahymena thermophila SB210 29805.98 184 56 Q23DG8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 41275.67 78 34 Q23DI3_TETTH Succinate dehydrogenase, flavoprotein subunit containing protein - Tetrahymena thermophila SB210 70562.71 101 28 Q23DJ4_TETTH Hypothetical protein - Tetrahymena thermophila SB210 20516.60 65 34 Q23DJ9_TETTH TATA box-binding protein - Tetrahymena thermophila SB210 51796.65 78 29 Q23DP3_TETTH Enolase family protein - Tetrahymena thermophila SB210 50329.06 142 56 Q23DU3_TETTH Fumarylacetoacetase family protein - Tetrahymena thermophila SB210 48682.46 135 42 Q23DV2_TETTH Transaldolase family protein - Tetrahymena thermophila SB210 43755.56 73 25 Q23DV5_TETTH Proteasome A-type and B-type family protein - Tetrahymena thermophila SB210 27760.91 133 68 Q23DV6_TETTH Dihydrolipoamide dehydrogenase family protein - Tetrahymena thermophila SB210 52758.39 272 65 Q23F08_TETTH Hypothetical protein - Tetrahymena thermophila SB210 21801.20 105 56 Q23F09_TETTH Phosphoglycerate mutase 1 family protein - Tetrahymena thermophila SB210 36061.76 120 49 Q23F17_TETTH Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 37871.32 72 31 Q23F81_TETTH Hypothetical protein - Tetrahymena thermophila SB210 39169.99 156 41 Q23F83_TETTH Protein phosphatase 2C containing protein - Tetrahymena thermophila SB210 32579.11 203 78 Q23F99_TETTH Squalene and phytoene synthases family protein - Tetrahymena thermophila SB210 37593.80 117 31 Q23FB2_TETTH Proteasome, putative - Tetrahymena thermophila SB210 32954.27 106 38 Q23FB7_TETTH Viral A-type inclusion protein repeat containing protein - Tetrahymena thermophila SB210 132972.50 132 17 Q23FE8_TETTH Peptidase family M20/M25/M40 containing protein - Tetrahymena thermophila SB210 52932.01 160 54 Q23FF5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 69166.04 222 40 Q23FF7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 26618.63 201 72 Q23FG0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 21750.06 89 41 Q23FL2_TETTH Hsp90 protein - Tetrahymena thermophila SB210 82105.55 97 26 Q23FR9_TETTH Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 34782.35 68 23 Q23FT7_TETTH Electron transfer flavoprotein, alpha subunit containing protein - Tetrahymena thermophila SB210 36874.57 67 45 Q23FU1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 77549.45 106 27 Q23FY6_TETTH KE2 family protein - Tetrahymena thermophila SB210 15464.91 97 53 Q23G19_TETTH Mitochondrial glycoprotein - Tetrahymena thermophila SB210 28825.21 103 40 Q23H37_TETTH Peptidyl-prolyl cis-trans isomerase, cyclophilin-type family protein - Tetrahymena thermophila SB21 21527.86 80 42 Q23JH0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 85569.82 84 15 Q23JH9_TETTH Transketolase, pyridine binding domain containing protein - Tetrahymena thermophila SB210 47389.56 95 35 Q23JZ7_TETTH TCP-1/cpn60 chaperonin family protein - Tetrahymena thermophila SB210 62213.12 271 63 Q23KA3_TETTH WD domain, G-beta repeat - Tetrahymena thermophila SB210 39089.85 71 24 Q23KA9_TETTH NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial, putative - Tetrahymena thermophila SB 87854.83 111 24 Q23KG1_TETTH Ribosomal protein S5 containing protein - Tetrahymena thermophila SB210 35314.73 80 41 Q23KL2_TETTH Pyruvate dehydrogenase E1 component - Tetrahymena thermophila SB210 49638.75 146 55 Q23MB8_TETTH Alpha-soluble NSF attachment protein - Tetrahymena thermophila SB210 35192.20 136 49 Q23MN3_TETTH TPR Domain containing protein - Tetrahymena thermophila SB210 117085.70 90 15 Q23ND5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 24223.58 96 59 Q23PZ8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 38822.96 66 30 Q23QV9_TETTH Putative uncharacterized protein - Tetrahymena thermophila SB210 15226.78 75 21 Q23QZ4_TETTH Inorganic pyrophosphatase family protein - Tetrahymena thermophila SB210 21903.91 124 48 Q23R09_TETTH Biopterin-dependent aromatic amino acid hydroxylase family protein - Tetrahymena thermophila SB210 52651.61 177 44 Q23RC7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 33046.28 95 42 Q23RE9_TETTH Glucose kinase - Tetrahymena thermophila SB210 46450.68 158 50 Q23RK6_TETTH La domain containing protein - Tetrahymena thermophila SB210 39269.26 118 55 Q23RS7_TETTH Nucleotidyl transferase family protein - Tetrahymena thermophila SB210 81796.88 81 23 Q23RT7_TETTH Papain family cysteine protease containing protein - Tetrahymena thermophila SB210 43537.61 114 21 Q23RZ9_TETTH Hypothetical protein - Tetrahymena thermophila SB210 87770.08 83 15 Q23TB8_TETTH 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain - Tetrahymena thermophila SB210 32673.06 126 52 Q23TC6_TETTH Proteasome subunit alpha type 7-like, putative - Tetrahymena thermophila SB210 27506.92 80 26 Q23U16_TETTH DEAD/DEAH box helicase family protein - Tetrahymena thermophila SB210 54434.96 88 29 Q23VX8_TETTH Purine nucleoside phosphorylase I, inosine and guanosine-specific family protein - T. thermophila 30125.28 111 60 Q23W16_TETTH Cofilin/tropomyosin-type actin-binding protein - Tetrahymena thermophila SB210 16024.87 95 60

Table I. continued

450 Xanthopoulou et al: Proteomic Profile of Tetrahymena thermophila

Table I. continued

Protein name Protein description MW Mascot Cover- score age

Q23W20_TETTH Dehydrogenase, isocitrate/isopropylmalate family protein - Tetrahymena thermophila SB210 53026.95 190 54 Q23WS8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 35987.42 165 71 Q23WS9_TETTH Hypothetical protein - Tetrahymena thermophila SB210 35829.21 176 56 Q23WT0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 36928.99 191 69 Q23YB7_TETTH Acetyl-CoA acyltransferases family protein - Tetrahymena thermophila SB210 44538.05 163 73 Q23YQ0_TETTH Hypothetical protein - Tetrahymena thermophila SB210 80567.22 93 25 Q23YT4_TETTH EF-1 guanine nucleotide exchange domain containing protein - Tetrahymena thermophila SB210 25122.95 84 37 Q23ZD8_TETTH Glutamate/leucine/phenylalanine/valine dehydrogenase family protein - Tetrahymena thermophila SB210 56163.42 118 35 Q23ZH0_TETTH Nucleosome assembly protein - Tetrahymena thermophila SB210 39672.67 110 49 Q240Q4_TETTH Metallopeptidase family M24 containing protein - Tetrahymena thermophila SB210 67237.54 199 42 Q240R0_TETTH Granule lattice protein 3, putative - Tetrahymena thermophila SB210 41666.53 68 12 Q240U0_TETTH Methionine aminopeptidase, type II - Tetrahymena thermophila SB210 56847.53 130 29 Q241P0_TETTH EF hand family protein - Tetrahymena thermophila SB210 16796.93 64 33 Q244X0_TETTH Oxidoreductase, FAD/FMN-binding family protein - Tetrahymena thermophila SB210 42740.55 163 40 Q245F2_TETTH Translation initiation factor eIF-6, putative family protein - Tetrahymena thermophila SB210 27415.62 101 38 Q245Z0_TETTH Ubiquitin carboxyl-terminal hydrolase, family 1 protein - Tetrahymena thermophila SB210 28541.71 77 34 Q248A7_TETTH Peptidyl-prolyl cis-trans isomerase, FKBP-type family protein - Tetrahymena thermophila SB210 15672.96 92 64 Q24BB1_TETTH SCP-2 sterol transfer family protein - Tetrahymena thermophila SB210 13010.93 101 69 Q24BD4_TETTH Hypothetical protein - Tetrahymena thermophila SB210 46828.85 108 47 Q24BX6_TETTH Glutamate/leucine/phenylalanine/valine dehydrogenase family protein - Tetrahymena thermophila SB210 55010.12 85 32 Q24C97_TETTH Hypothetical protein - Tetrahymena thermophila SB210 14767.57 84 56 Q24CA7_TETTH AMP-binding enzyme family protein - Tetrahymena thermophila SB210 73695.80 95 27 Q24CI4_TETTH Hypothetical protein - Tetrahymena thermophila SB210 205585.27 77 10 Q24CJ0_TETTH UDP-N-acetylglucosamine pyrophosphorylase-like - Tetrahymena thermophila SB210 61902.80 76 14 Q24CJ7_TETTH Phosphoglycerate kinase family protein - Tetrahymena thermophila SB210 45999.43 120 33 Q24CY7_TETTH Translationally controlled tumor protein - Tetrahymena thermophila SB210 19237.37 69 37 Q24D42_TETTH Hypothetical protein - Tetrahymena thermophila SB210 38738.49 239 77 Q24D68_TETTH Pyridine nucleotide-disulphide oxidoreductase family protein - Tetrahymena thermophila SB210 79755.42 147 31 Q24DJ3_TETTH Hypothetical protein - Tetrahymena thermophila SB210 47516.52 64 27 Q24DP2_TETTH Hypothetical protein - Tetrahymena thermophila SB210 81011.25 160 28 Q24F10_TETTH Catalase family protein - Tetrahymena thermophila SB210 55754.61 103 27 Q24F70_TETTH Plectin/S10 domain containing protein - Tetrahymena thermophila SB210 18685.48 84 47 Q24FH6_TETTH Hypothetical protein - Tetrahymena thermophila SB210 60217.56 186 52 Q24FH8_TETTH Hypothetical protein - Tetrahymena thermophila SB210 16940.24 66 53 Q24FS1_TETTH Indigoidine synthase A like protein - Tetrahymena thermophila SB210 34061.70 104 44 Q24FT7_TETTH DJ-1/PfpI family protein - Tetrahymena thermophila SB210 23358.81 67 38 Q24FV5_TETTH Hypothetical protein - Tetrahymena thermophila SB210 31402.29 130 56 Q24HM7_TETTH Hypothetical protein - Tetrahymena thermophila SB210 46278.61 63 18 Q24HT1_TETTH Hypothetical protein - Tetrahymena thermophila SB210 16609.86 70 47 Q24HX2_TETTH Glutathione S-transferase, N-terminal domain containing protein - Tetrahymena thermophila SB210 30578.81 82 40 Q24HX6_TETTH Phosphoglycerate mutase - Tetrahymena thermophila SB210 29446.16 128 57 Q24HY8_TETTH ATP synthase F1, alpha subunit family protein - Tetrahymena thermophila SB210 60031.79 66 13 Q24I09_TETTH Hypothetical protein - Tetrahymena thermophila SB210 36361.91 121 50 Q24I43_TETTH Hypothetical protein - Tetrahymena thermophila SB210 30887.73 79 33 Q24I54_TETTH Glutathione S-transferase, N-terminal domain containing protein - Tetrahymena thermophila SB210 23121.05 172 79 Q24I95_TETTH EF hand family protein - Tetrahymena thermophila SB210 23932.93 139 60 Q24IM4_TETTH Ribosomal protein L7/L12 C-terminal domain containing protein - Tetrahymena thermophila SB210 31336.41 110 18 Q2I2L2_TETTH C-terminal crystallin fold containing protein 9p - Tetrahymena thermophila 42240.87 90 47 Q2I2L4_TETTH C-terminal crystallin fold containing protein 12p - Tetrahymena thermophila 71762.39 131 46 Q2I2L5_TETTH C-terminal crystallin fold containing protein 3p - Tetrahymena thermophila 48407.97 148 45 Q2I2L6_TETTH C-terminal crystallin fold containing protein 4p - Tetrahymena thermophila 48661.23 152 54 Q2I2L9_TETTH C-terminal crystallin fold containing protein 11p - Tetrahymena thermophila 63162.69 89 23 Q2VA70_THELE Hypothetical protein - Theileria lestoquardi 17935.63 85 36 Q38J86_TETTH Radial spoke head 2 - Tetrahymena thermophila 57139.37 222 49 Q3LS02_9CILI Beta-tubulin - Pseudocohnilembus persalinus 49958.91 63 22

Table I. continued

451 in vivo 24: 443-456 (2010)

Table I. continued

Protein name Protein description MW Mascot Cover- score age

Q3S4V5_TETTH Granule tip protein 1 - Tetrahymena thermophila 85812.86 160 32 Q3SEG0_PARTE Alpha tubulin, putative - Paramecium tetraurelia 50114.77 79 27 Q3SEG8_PARTE Alpha_tubulin, putative - Paramecium tetraurelia 50070.74 96 25 Q4UFV5_THEAN Tpr-related protein family member, putative - Theileria annulata 95473.78 69 13 Q4UIB3_THEAN Hypothetical protein - Theileria annulata 57567.35 65 20 Q4XA93_PLACH Hypothetical protein (Fragment) - Plasmodium chabaudi 3134.45 63 100 Q4XDU1_PLACH Hypothetical protein (Fragment) - Plasmodium chabaudi 33460.14 66 27 Q52H29_TETTH Acidic ribosomal P0 protein - Tetrahymena thermophila 35611.64 65 35 Q546B0_PARTE Beta-tubulin - Paramecium tetraurelia 49937.02 211 63 Q5CLC4_CRYHO Translation initiation factor if-2, 73082-68138 - Cryptosporidium hominis 74798.51 62 14 Q6LDR3_TETPY Ubiquitin - Tetrahymena pyriformis 10608.73 75 45 Q6TP40_9HYMN Heat shock protein 90 - Tetrahymena bergeri 65493.73 90 14 Q6U682_TETTH Putative catalase - Tetrahymena thermophila 55452.39 103 16 Q76LX4_PARCA Beta-tubulin - Paramecium caudatum 49937.02 244 61 Q7RIL1_PLAYO Hypothetical protein PY03608 (Fragment) - Plasmodium yoelii yoelii 86631.24 65 25 Q7Z001_TETTH Actin - Tetrahymena thermophila 42528.22 156 60 Q7Z2D1_TETPY Beta-tubulin - Tetrahymena pyriformis 50005.10 153 58 Q86N78_TOXGO Putative vacuolar H+ ATPase subunit B - Toxoplasma gondii 45380.90 70 10 Q8IEV7_TETTH P28 protein - Tetrahymena thermophila 19250.43 88 52 Q8IEV8_TETTH Phospholipase A1 - Tetrahymena thermophila 35562.53 111 30 Q8IS25_TETTH Actin-related protein - Tetrahymena thermophila 44031.24 112 32 Q8IS26_TETTH Actin-related protein 3 - Tetrahymena thermophila 48131.80 141 46 Q8ITP9_9SPIT Alpha-tubulin (Fragment) - Favella ehrenbergii 40638.32 69 30 Q8ITQ0_9SPIT Alpha-tubulin - Favella ehrenbergii 40617.43 119 34 Q8ITR3_9SPIT Alpha-tubulin - Strombidinopsis sp. 40040.21 99 29 Q8ITR6_9SPIT Alpha-tubulin - Strombidinopsis sp. 39805.94 70 16 Q8ITS1_9SPIT Alpha-tubulin (Fragment) - Strombidinopsis sp. 41334.81 83 18 Q8MM93_9STIC Beta-tubulin - Gastrostyla steinii 49924.95 68 9 Q8MU24_9CILI Alpha-tubulin (Fragment) - Chilodonella uncinata 38295.26 81 27 Q8MU83_TETTH Elongation factor 2 - Tetrahymena thermophila 94292.56 202 35 Q8T925_TETTH Igr2p - Tetrahymena thermophila 27154.14 70 25 Q8WQI1_TETTH Lycopene biosynthesis-enhancing protein - Tetrahymena thermophila 43577.90 82 29 Q95WA9_TETTH Enolase - Tetrahymena thermophila 42083.44 198 61 Q95WT3_TETPY CCTepsilon subunit - Tetrahymena pyriformis 59398.47 93 24 Q9BK15_TETTH Glyceraldehyde-3-phosphate dehydrogenase - Tetrahymena thermophila 33055.13 157 41 Q9BME9_TETTH Ndc1 protein - Tetrahymena thermophila 44468.60 68 29 Q9GRF9_TETTH Tetrin C protein - Tetrahymena thermophila 89734.85 113 25 Q9NJH9_TETTH Centrin - Tetrahymena thermophila 19367.86 65 38 Q9U5Y4_TETTH P85 - Tetrahymena thermophila 86611.74 133 27 Q9U697_TETTH SerH3 immobilization antigen - Tetrahymena thermophila 43223.47 154 29 Q9U6C5_STYMT Beta tubulin - Stylonychia mytilus 49959.95 71 17 Q9UAH0_TETPY 14-3-3 protein - Tetrahymena pyriformis 28284.14 116 53 Q9Y0T0_TETTH Hsp82 heat shock protein - Tetrahymena thermophila 81185.52 223 42

in three different pH ranges (3-10, 4-7, 6.3-8.3) and 4,457 were able to use very narrow windows of mass tolerance spots were totally detected by the Melanie 4.02 software. (0.0025%) and hence, increase the confidence of These spots were excised from the gels and analyzed for identification, as well as the total identification rate up to 85%. protein identification following in-gel digestion with trypsin. This procedure resulted in the identification of 375 different Each spot was analyzed for PMF with MALDI-MS in a time- gene products (Figure 1). The abbreviated and full names of of-flight mass spectrometer and proteins were identified the proteins, the theoretical MW as well as data from the MS automatically by the peptide mass matching. Proteins not analysis, such as the probability that the identification is a identified by PMF were subsequently selected for PSD-MS- random event (Score), and the coverage of the protein by the MS and analyzed with MALDI-MS-MS. Using an internal identified peptides are listed in Table I. The protein peptide standard to correct the measured peptide masses, we identification data are available at the PRIDE database

452 Xanthopoulou et al: Proteomic Profile of Tetrahymena thermophila

Table II. Proteins from T. thermophila were extracted and separated by 2-D gel electrophoresis as described in the Materials and Methods. Proteins were identified by MALDI-MS and MS/SM, following in-gel digestion with trypsin. The proteins identified by the PeptideFinder software are designated by their SWISS-PROT accession numbers and their full names. The theoretical MW as well as the protein amino acid sequence coverage by the matching peptides are given as an indication of the confidence of the identification. PeptideFinder score is an index used to comparatively rank the candidate proteins based on the statistics of the molecular weight distribution.

Accesion No MW IP Score No of MWs matched Accesion No MW IP Score No of MWs matched

Q24BP6 47663.1 9.16 10.3 2 Q23R69 88541 5.84 8.3 3 Q22G27 90815.8 8.65 14.7 7 Q23MC0 80534 10.49 26.1 12 A4VD69 68914.8 8.34 15.2 6 Q236S3 96952.8 10.03 25.8 14 Q22WX1 90513.9 10.5 19.9 11 Q23TE4 88668.1 8.61 20.3 9 Q23GZ0 70969.7 8.96 13.7 4 Q24FA1 81908.4 7.5 14.9 6 Q23GA4 84762.2 6.98 14 5 Q23RD8 94831 9.85 11.5 5 Q2I2L4 69520.5 7.6 17.3 6 Q228A8 96973.2 5.11 18.6 8 Q240Y6 80245.6 9.97 10.5 4 Q239J6 49693.2 8.1 22.1 5 Q22AN0 97542.2 9.9 19.4 12 Q22FY8 70473.4 9.33 16.7 5 Q23CR5 67697.2 7.64 24.3 9 Q22UT1 99278.4 9.01 16.5 7 Q23A42 81586.1 7.12 22 9 Q24CE3 83245.1 9.88 14.8 7 Q23JX3 99314.9 9.43 3.8 2 Q229T8 38302.1 9.56 38.3 7 Q23TD5 71771.3 6.79 13.8 7 Q22LX7 95632.3 10.16 5.7 3 Q23E77 79829.3 7.79 14.6 8 Q23FK3 99546.1 9.19 18.6 8 Q231U7 68704.7 10.71 29.2 12 Q22EI7 86646.4 9.2 15.3 8 Q23NL2 86837.7 11.61 11.7 7 Q22BK0 28740.2 5.42 33.4 4 Q23RV4 86293.9 10 4.1 2 Q23Q69 85544.1 9.51 16.5 7 Q22EZ2 81073.3 10.09 10.7 4 Q23RB1 72307.3 10.07 15 6 Q22Z30 80339.2 8.65 8.1 3 Q23M81 76144.3 6.24 10.6 4 Q22S62 43745.7 8.57 14.3 4 Q22HI7 90916.8 4.83 20.6 12 Q238V2 95109 5.48 11.1 7 Q22MK2 62091 6.83 11.8 3 Q235X2 83110.9 10.4 13 5 Q22LQ1 87176.6 7.11 14.8 7 Q23YX0 92895.9 10.51 18.1 10 Q24GH1 85660.8 8.78 18.8 10 Q22R68 76027.6 10.26 21.4 10 Q23UG7 49694.9 6.36 13.4 5 Q23CZ9 94457.7 9.14 38.9 20 Q23A24 49007.5 8.55 13.3 4 Q22M96 95211.9 8.43 17.2 8 Q22BA2 59319.5 6.72 12.7 4 Q22LR7 63478.2 8.64 14.6 5 Q24GL3 91006.2 7.31 15 6 Q22G16 63180.6 5.36 10.5 3 Q23D71 38872.3 7.6 18.1 3 A0MNQ4 87056.1 6.15 21 8 Q229S1 89333.6 5.05 26.1 15 Q231F8 34126.3 4.77 32.5 5 Q228L0 73265 5.32 13.2 7 Q23ZD6 41357.7 7.33 24.4 5 Q22SS6 59225.3 8.2 10.9 4 Q22AC1 51607.1 6.69 40.1 9 Q22CG0 62424.6 9.1 19.3 7 Q23VX6 69460.8 9.28 19.1 7 Q230T8 37219.5 9.24 10 2 Q234F3 62686.2 10.46 18.2 5 Q23EV8 33845 5.53 19.7 5 Q24GL8 92469.4 9.65 15.7 7 Q24BT1 61948.1 8.54 10.8 4 Q237P3 95969.7 10.1 18.6 8 Q24HK0 23728 8.79 21.2 3 Q22U30 92853 9.98 10.6 6 Q227A0 27661.9 6.93 19.3 4 Q22NV7 63799.1 7.91 20 7 Q224Z4 35839.5 5.96 13.7 2 Q248D6 88078.6 9.63 18.6 10 Q240I6 99169.1 9.45 18.5 10 Q22AN4 96049 4.93 9.6 4 Q23R68 95751.6 10.02 10.9 6 Q22AT1 99812 9.82 12 5 Q23D37 93168.2 9.96 11.7 5 Q22Z98 78625.3 8.56 19.5 11 Q24GE7 77885.7 7 11.3 4 Q22HF5 83512 9.51 13.2 6 Q245B8 84032.3 6.81 17.1 9 Q22GG3 87630.6 10.3 7.3 3 Q23R24 72359.6 9.28 42.4 19 Q241B3 69607.8 9.33 17 7 Q24DD7 80656.2 10.2 15.6 7 Q9GRG0 87126.6 5.86 24.2 12 Q23YB8 64126 9.48 7.8 3 Q22HJ1 41720.4 5.72 36.2 7 Q233G4 63010.8 8.16 11.1 4 Q237F4 71633.9 7.99 20.1 6 Q23G03 61286.5 9.67 13.2 4 Q22TH7 32515.8 5.02 11.3 2 Q23TF3 86068.7 8.28 8.7 3 Q22V19 35161.9 6.38 20.3 3 Q23BV8 49974 4.98 24.4 6 Q22CT8 61889.9 5.11 24.7 9 Q23K92 99673.3 6.66 10.4 6 Q22L22 67802.5 10.21 7.2 2 Q22X70 89444.6 5.73 10.3 5 Q23BT8 91385.4 5.64 16.9 9 Q22BQ9 75534.6 10.18 10.1 5 Q23E39 62592.4 7.25 23.4 8 Q23CR8 95759.9 10.37 7.8 5

Table II. continued

453 in vivo 24: 443-456 (2010)

Table II. continued

Accesion No MW IP Score No of MWs matched Accesion No MW IP Score No of MWs matched

Q236D9 49823.4 10.04 16.9 4 A4VD69 68914.8 8.34 31.8 12 Q24C00 86566 9.79 8.2 4 Q233G3 66228.3 4.73 20.2 7 Q22BV0 44955.9 7.07 15 3 Q238S7 90378.3 7.65 6.8 3 Q22AK7 53504.1 7.75 14.6 3 Q24BZ4 91664.4 6.92 11 6 Q23KA9 86867.5 8.27 10.4 6 Q235L3 90589.2 6.91 10.9 7 Q22WB5 43570.4 8.23 15.2 3 Q23H78 61941.8 9.38 24.9 8 Q23FU3 87417.3 10.4 11.7 6 Q24I48 99791.3 9.78 13.5 7 Q23FQ2 37824.5 6.88 19.7 3 Q23G97 91057.5 4.9 14 7 Q24CJ8 80970.4 5.38 16.7 7 Q24GJ2 73251.1 5.64 12.1 4 Q24CI3 21611.9 10.71 33.9 3 Q231B6 60875 8.82 16.8 7 Q236M5 59982.2 8.92 10.3 4 Q9GRF9 89374.7 6.08 15.1 8 Q22V38 76830 4.86 8 4 Q23AF6 87994.2 6.14 12.5 5 Q24HH5 48894.4 10.79 17.8 6 Q2I2L7 85836.3 4.94 10.9 6 Q23YC3 95020.7 9.88 5.5 4 Q23YC0 87467.5 8.43 10.6 6 Q240U4 78458.6 8.92 14.7 8 Q22KR4 35809.2 4.84 21.1 5 Q22KA8 43475.8 9.61 21.4 6 Q23AZ7 69208.9 5.23 11.8 3 Q23QI1 14822.5 5.95 36.4 2 Q22PA5 80302.7 5.51 10 4 Q23RG5 95432.7 7.01 14 6 Q24GK5 79567.2 8.8 10 5 Q23D31 51309.8 9.3 14.9 3 Q238V9 81385.2 10.34 15.4 8 Q227B7 39306.5 7.47 20.7 4 Q23J97 90879.8 10.47 8.7 6 A3F819 39061.1 10.11 22.3 4 Q22US8 95628.1 9.94 12.2 7 Q22ZH8 92831.8 4.9 16.4 10 Q23A68 78037.7 7.18 17.1 6 Q234A4 96913.5 6.36 15.4 9 Q22AR9 95015.9 9.98 7.9 5 A0MNQ4 87056.1 6.15 13.3 8 Q239P0 83766.2 5.97 11.2 4 Q238T3 94178.3 9.38 9.7 6 Q23A57 96555.6 7.25 7.7 4 Q24HN3 68570.4 11.12 24.5 11 Q23DC6 99141.5 6.07 15 8 Q23RC0 73022.4 10.34 18.9 7 Q22TT1 73439.6 9.03 15.1 6 Q24CK5 87975.5 9.44 19.1 10 Q22AE1 50382 6.78 16.9 5 A4VDG5 80519.7 9.37 17.2 9 Q23ZG0 83499.5 7.02 13.1 7 Q22GH1 99859.2 10.32 12.3 8 Q22SJ4 73487.1 8.06 10 5 Q23D86 44219.7 10.2 9.6 3 Q239Q0 64755.7 5.1 7.7 3 Q22S53 93171 9.94 12.8 8 Q248I1 46317.9 9.97 20.2 6 Q22GF7 68549.9 9.48 21 9 Q22EF5 83490.2 7.57 12.1 7 Q232W5 95160.4 9.3 9.7 6 Q23CZ6 89695.4 10.33 19.1 10 Q233N9 94098.6 8.42 8.3 5 Q22G40 92946.3 5.29 8 4 Q23PY5 88257.8 5.17 11.1 5 Q22F28 92650.1 10.14 24.1 11 Q245D7 97643.1 9.84 11.7 7 Q951C0 50660 11.53 7.5 2 Q22A01 63128.2 6.34 14.9 6 A4VDC6 53673.1 9.86 15.4 5 Q22T71 84786.8 8.93 10.1 5 Q23T79 58801.1 6.44 9.1 3 Q24F47 92864.9 9.79 7.3 4 A4VD76 32502.9 10.94 27.6 4 Q22T70 66160.2 8.75 10.7 4 Q22MT3 15569.5 10.95 24.3 2 Q239G8 86458.8 5.96 7.6 4 Q23MP7 52582.2 4.19 18.5 6 Q23D79 79775.2 4.66 11.7 6 Q23EA3 83107.8 9.11 18.5 10 Q229W2 69696.4 9.85 7.4 4 A4VD72 39226.8 5.79 18.9 5 Q241S7 82783.8 9.71 9.3 5 Q23F42 67403.8 7.21 14.5 5 Q23QS3 52969.4 8.53 9.3 3 Q22DC5 64198.4 6.85 22.8 7 Q248A0 90437.8 7.76 9.7 7 A4VDC8 82698.4 4.58 21.1 12 Q24E37 98164.7 8.26 8.5 6 Q226U5 29840.8 10.4 12.7 2 Q22Y56 90841.1 8.92 8 5 Q231D3 77371.7 10.25 11.2 5 Q22GH2 51620.6 10.03 10.9 4 Q23CN5 77333.5 4.44 17.9 8 Q22RM6 93944.2 6.55 5.9 3 Q23JQ1 66375.9 10.07 45.4 16 Q22C94 25002.6 10.6 29.6 5 Q22Y20 49010.3 10.36 13.2 5 Q23MH1 88212.9 9.93 10.4 6 Q22WR4 71152.1 9.84 17.5 7 Q237Q9 94262.7 9.05 5.5 3 Q241A6 82931.8 10.16 19 8 Q24DG7 75789.4 6.3 9.4 4 Q24FW3 99364.6 9.09 20.7 13 Q22VV4 87963.5 9.38 6.8 4 Q23FY0 84089.6 9.36 11.7 6 Q24F51 66370.6 7.77 9.6 4 Q227M6 93083.6 7.56 16.7 8 Q23PS0 97884.2 7.82 33.9 19 Q229R9 75163.5 9.7 52.4 24 Q23TF4 94327.3 8.84 29.3 16 Q234H0 61138.7 10.02 16.4 5

Table II. continued

454 Xanthopoulou et al: Proteomic Profile of Tetrahymena thermophila

Table II. continued analysed all at once, with a single technique. Each technique has its limitations and requires compatibility of the Accesion No MW IP Score No of MWs matched homogenization and identification methods. In this study, we Q234T9 84223.1 8.27 20.4 10 were able to identify a significant number of proteins, but Q23F08 21669.1 9.68 21.1 2 only after proper optimization of the homogenization Q236K7 47898 9.93 11.5 4 method, namely, by the removal of nucleic acids with the use Q23QL9 70138.4 5.92 13.9 7 of Tri-Reagent for the protein isolation instead of the Q23FZ1 99190.3 9.83 15.6 9 conventional method (urea-thiourea) (data not shown). Q23J80 66036 5.14 17.3 7 Q236L3 99735.7 9.3 17 8 Out of the 631 identified proteins, 8 have already been Q23CZ5 53682.6 8.1 20 5 annotated in the Swiss Prot database, indicating that the Q22V90 76013.3 8.57 17.2 7 remaining 623 proteins are as yet considered to be Q23JF4 80168.4 8.59 21.9 9 hypothetical. This is because Swiss Prot is a protein database Q23MM8 69701.8 5.94 16.9 7 that contains only proteins actually identified and studied, Q23K07 37809.5 8.03 31.4 6 Q24FC4 89767.7 5.46 10 5 while TrEMBL is generated by computer translation of the Q23TX1 49580.1 10.04 9.9 2 genetic information from the EMBL Nucleotide Sequence Q224W0 33809.8 10.02 19.7 5 Database. As expected, the latter procedure is subject to Q22AQ9 90312.1 9.36 13.8 7 errors, so proteins predicted by the TrEMBL database have a Q23FV0 99024.8 5.11 9.5 6 risk of being hypothetical. The identification of these Q23PX2 75078.3 6.74 11.2 5 Q22L39 65443.6 10.28 19.6 8 proteins and thus, the confirmation of a significant number Q22UN2 25748.8 8.4 19 2 of proteins found in the TrEMBL database all at once, was a Q22Y32 50081.7 5.24 4.9 2 major achievement. Q22EC1 71738.5 5.94 11.1 4 A total of 92% of the identified proteins are correlated to Q232Z1 91334.3 8.53 14 5 the genome of T. thermophila, while the remaining 8% show Q22BI0 88730.3 9.24 2.8 1 Q23D18 70910.9 6.71 13 4 high homology with proteins of other protozoa such as Q22HC3 85645.9 8.72 7.9 4 Stylonychia, Gastrostyla, Frontonia, Strombidinopsis, Q227U6 63869.6 5.57 7.9 3 Toxoplasma, Plasmodium, Paramecium, Cryptosporidium, Q22C27 84472.1 8.43 15.7 9 Theileria, Favella, and Chilodonella. Q22AG7 85464.4 6.53 14.4 7 A brief comparison between our work and other studies, Q247W2 56313.1 6.86 21.1 6 Q23KB1 68526 9.09 13.8 5 revealed only few similarities. Out of the 631 identified Q23K26 49071.8 6 32.2 8 proteins, only 11 are common to those published for its ciliome (12) and 34 proteins are found in both our results and those for the mitochondrial proteome (11). As to be expected, this is a result of the different preparation and (http://www.ebi.ac.uk/pride/, experiment #3700). It should be identification techniques used and mainly due to the fact that noted that many of the identified proteins in these gels were we chose to analyze the entire homogenate of the organism. represented by more than one spot with the same apparent Nearly half of these identified proteins have unknown molecular mass, but with different pI values, possibly due to functions, while of the rest 47.5% are enzymes (lyases, post-translational modifications or isoenzyme variations. ligases, isomerases, proteases, hydrolases, etc.) participating Mass spectra that did not result in protein identification in various metabolic reactions, 22.4% are proteins that bind by the above procedure were processed for identification by other molecules and ions for their action, such as ATP-, GTP- the Peptide Finder software (21). By that procedure, we were RNA and calcium-binding proteins (Figure 2). A total of able to identify 258 additional gene products (Table II), 13.8% correspond to structural proteins, such as actin, resulting in a total of 631 proteins that are considered to tropomyosin, centrin, dynein and Granule lattice proteins, make up the proteome of T. thermophila. 4.4% were regulatory proteins, such as 14-3-3, AhpC/TSA and N-ethylmaleimide-sensitive factor and 3.8% are ribosomal Discussion proteins involved in protein synthesis. Another 3.8% are proteins chaperones, such as heat-shock proteins 90, 82 and T. thermophila is one of the most studied model organisms in DNAK, and 3.2% are translational proteins, such as translation biology. Although numerous studies concerning specific elongation factor ef-1. Finally, 1.2% of the identified proteins proteins or subsets of proteins after fractionation exist, to are involved in signal transduction pathways, as the date nothing has been published regarding its whole Mov34/MPN/PAD-1 and Ras protein families. proteome. This is mainly due to the fact that T. thermophila The identified proteins are considered to be the most has a very large number of protein-encoding sequences to be abundant (22), but this number may easily seem small for the

455 in vivo 24: 443-456 (2010) entire homogenate of such a complicated organism. However, Wang Y, Cai H, Collins K, Stewart BA, Lee SR, Wilamowska K, since no further steps have been taken in proteomics for the Weinberg Z, Ruzzo WL, Wloga D, Gaertig J, Frankel J, Tsao analysis of total cell extract of T. thermophila and since CC, Gorovsky MA, Keeling PJ, Waller RF, Patron NJ, Cherry JM, Stover NA, Krieger CJ, del Toro C, Ryder HF, Williamson protein databases are continuously altered, as they are based SC, Barbeau RA, Hamilton EP and Orias E: Macronuclear on the translation of the mRNA without taking into account genome sequence of the ciliate Tetrahymena thermophila, a the alterations that may occur before the final product of model eukaryote. PLoS Biol 4: e286, 2006. protein is reached, their addition to the list of proteins found 11 Smith DG, Gawryluk RM, Spencer DF, Pearlman RE, Siu KW, to be expressed from T. thermophila is a notable contribution Gray MW: Exploring the mitochondrial proteome of the ciliate to the study of this organism. protozoon Tetrahymena thermophila: direct analysis by tandem The obtained results indicated that even though most of mass spectrometry. J Mol Biol 374: 837-863, 2007. 12 Smith JC, Northey JG, Garg J, Pearlman RE and Siu KW: the identified proteins are essential for several functions of Robust method for proteome analysis by MS/MS using an entire this protozoon and participate in various metabolic reactions, translated genome: demonstration on the ciliome of Tetrahymena there is still a significant percentage corresponding to thermophila. J Proteome Res 4: 909-919, 2005. proteins of unknown function. The future aim is to elucidate 13 Jacobs ME, DeSouza LV, Samaranayake H, Pearlman RE, Siu their role or even suggest a possible connection with the KW and Klobutcher L A: The Tetrahymena thermophila existing functions. This indicates that Tetrahymena is an phagosome proteome. Eukaryotic cell 5: 1990-2000, 2006. organism to be studied for many years and should still be a 14 Kilburn CL, Pearson CG, Romijn EP, Meehl JB, Giddings TH Jr, Culver BP, Yates JR 3rd and Winey M: New Tetrahymena reliable model organism for biochemical and biological basal body protein components identify basal body domain studies. structure. J Cell Biol 178(6): 905-912, 2007. 15 Bowman GR, Smith DG, Michael Siu KW, Pearlman RE and References Turkewitz AP: Genomic and proteomic evidence for a second family of dense core granule cargo proteins in Tetrahymena 1 Orias E: Mapping the germ-line and somatic genomes of a thermophila. J Eukaryot Microbiol 52: 291-297, 2005. ciliated protozoan, Tetrahymena thermophila. 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A homolog to yeast Gcn5p linking histone acetylation to gene 18 Bradford MM: A rapid and sensitive method for the quantitation activation. Cell 84: 843-851, 1996. of microgram quantities of protein utilizing the principle of 5 Noseda DG, Gentili HG, Nani ML, Nusblat A, Tiedtke A, Florin- protein-dye binding. Anal Biochem 72: 248-254, 1976. Christensen J and Nudel CB: A bioreactor model system 19 Fountoulakis M, Tsangaris G, Oh JE, Maris A and Lubec G: specifically designed for Tetrahymena growth and cholesterol Protein profile of the HeLa cell line. J Chromatogr A 1038: 247- removal from milk. Appl Microbiol Biotechnol 75: 515-520, 265, 2004. 2007. 20 Berndt P, Hobohm U and Langen H: Reliable automatic protein 6 Pauli W and Berger S: Toxicological comparisons of Tetrahymena identification from matrix-assisted laser desorption/ionization species, end points and growth media: supplementary mass spectrometric peptide fingerprints. Electrophoresis 20: investigations to the pilot ring test. Chemosphere 35: 1043-1052, 3521-3526, 1999. 1997. 21 Alexandridou A, Tsangaris GT, Vougas K, Nikita K and Spyrou 7 Valcarce G, Munoz L, Nusblat A, Nudel C and Florin- G: Peptide Finder: mapping measured molecular masses to Christensen J: The improvement of milk by cultivation with peptides and proteins. Bioinformatics 24: 2267-2269, 2008. ciliates. J Dairy Sci 84: 2136-2143s, 2001. 22 Maillet I, Bernt P, Malo C, Rodriguez S, Brunisholz RA, Pragai Z, 8 Weide T, Herrmann L, Bockau U, Niebur N, Aldag I, Laroy W, Arnold S, Langen H and Wyss M: From the genome sequence to Contreras R, Tiedtke A and Hartmann MW: Secretion of the proteome and back: evaluation of E. coli genome annotation functional human enzymes by Tetrahymena thermophila. BMC with a 2-D gel-based proteomics approach. Proteomics 7: 1097- Biotechnol 6: 19, 2006. 1106, 2007. 9 Hill DL: The Biochemistry and Physiology of Tetrahymena. Academic Press., New York and London, 1972. 10 Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, Wortman JR, Badger JH, Ren Q, Amedeo P, Jones KM, Tallon LJ, Delcher AL, Salzberg SL, Silva JC, Haas BJ, Majoros WH, Farzad M, Received October 14, 2009 Carlton JM, Smith RK Jr, Garg J, Pearlman RE, Karrer KM, Sun Revised April 14, 2010 L, Manning G, Elde NC, Turkewitz AP, Asai DJ, Wilkes DE, Accepted April 23, 2010

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