Inventory of the Proteins in Neisseria meningitidis ... - ACS Publications

Inventory of the Proteins in Neisseria meningitidis Serogroup B Strain MC58 Giuseppina Mignogna,†,⊥ Alessandra Giorgi,†⊥ Paola Stefanelli,‡ Arianna Neri,‡ Gianni Colotti,§ Bruno Maras,† and M. Eugenia Schinina` *,†,| Dipartimento di Scienze Biochimiche, Universita` “La Sapienza”, Rome, Italy; Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanita`, Rome, Italy; Istituto di Biologia e Patologia Molecolari, C.N.R., Rome, Italy; and Laboratorio di Genomica Funzionale e Proteomica in Organismi Modello, Universita` “La Sapienza”, Rome, Italy Received March 4, 2005

A protein inventory of Neisseria meningitidis strain MC58, a meningococcal strain belonging to the serogroup B, was performed by proteomics. A differential extraction procedure was employed and 238 protein species were identified by 2D mini-maps and MALDI-ToF analyses. In this catalog, we detected protein products from 33 genes, which were not yet annotated in previous N. meningitidis proteomic studies. This approach is suitable for high-throughput studies on differential expression of N. meningitidis genomes. Keywords: Neisseria meningitidis • functional genomics • proteome • MALDI-TOF • two-dimensional electrophoresis • 2D-minigel • peptide mass fingerprint

Introduction The Gram-negative bacterial pathogen Neisseria meningitidis is a major cause of morbidity and mortality worldwide, with an estimated 500 000 to 1 000 000 cases of septicemia and meningitis reported every year. Invasive meningococcal infections represent a major childhood disease with a mortality of 10% and high morbidity.1 The post-genomic era of N. meningitidis began with the publication of the genome sequences of the serogroup A (menA) Z2491 strain and of the serogroup B (menB) MC58 strain.2,3 Nevertheless which of the 2158 N. meningitidis genes are involved in the disease process is not definitely known, and a substantial proportion of the proposed open reading frames (ORFs) have no assigned function. Therefore, methods for highthroughput analysis of the gene products are trying to exploit the information from the genome projects. Oligonucleotide-based DNA microarrays studies on the menB MC58 strain provided for the first time the technological platform to analyze the transcriptional changes occurring in the bacterial cell during the infection of human host cells.4 This experimental approach was able to reveal that several genes are submitted to a transcriptional regulation upon adherence * To whom correspondence should be addressed. Dipartimento di Scienze Biochimiche, Universita` “La Sapienza”, Piazzale A. Moro 5, I-00185 Rome, Italy. Tel: +39 06 49910605. Fax: +39 06 4440062. E-mail: eugenia.schinina@ uniroma1.it. † Dipartimento di Scienze Biochimiche, Universita` “La Sapienza”. ‡ Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanita`. § Istituto di Biologia e Patologia Molecolari, C.N.R. | Laboratorio di Genomica Funzionale e Proteomica in Organismi Modello, Universita` “La Sapienza”. ⊥ These authors contributed equally to this work. 10.1021/pr0500511 CCC: $30.25

 2005 American Chemical Society

of menB strain MC58 strain to human epithelial or endothelial cells, two key events in the N. meningitidis pathogenicity. Microarray analyses demostrated that 13 menB genes are differentially transcripted in bacteria adhering to both epithelial or endothelial cells, whereas several other genes were found to be differentially expressed in only one of the two adhesion systems. These trascriptional differences encompass genes known to be involved in transcription, translation and also in metabolism, but genes of unknown function are also included in this list. Collection of protein lists is an essential starting point to investigate global gene expression in all studies that may follow in the N. meningitidis post-genomic era. Two-dimensional electrophoresis (2-DE) showed its potential to separate the N. menigitidis protein complement and has been successfully applied to spotlight transcriptional and translational changes in rifampin resistant strains.5 An alternative application of the 2-DE separation, following gene amplification and in vitro transcription and translation of a specific subset of proteins of the N. meningitidis proteome, has been proposed to identify membrane proteins of the menB MC58 strain, candidates for vaccination against this bacterial serogroup.6 A 2-DE map of the serogroup C F5 strain is also available at the Aberdeen University website (http://www.abdn.ac.uk/∼mmb023/neismen/neisf5_f.htm) and, more recently, two-dimensional diagonal SDS-PAGE was successfully employed to identify N. meningitidis and N. lactamica membrane complex proteins with temperature-dependent mobility.7 Nowadays, the advances in mass spectrometry have allowed both the accomplishment of a partial catalog of 47 soluble proteins in a N. meningitidis L3 immunotype strain by a 2-DEindependent approach, and the systematic annotation of the Journal of Proteome Research 2005, 4, 1361-1370

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Published on Web 06/10/2005

research articles proteomic 2D-maps (protein products from 235 genes) of the menA strain Z4970, a clinical isolate responsible for most of the African and Asian epidemics.8,9 The aim of our work was to implement N. meningitidis proteomics, generating a faster and reliable reference catalog of proteins by the N. meningitidis strain MC58, a meningococcal strain belonging to the serogroup B, responsible for endemic infections in Europe and America. We mapped 238 protein species, relating to 116 different genes covering a wide range of cellular functions. Therefore, this work provides the first global proteome analysis of a meningococcal strain belonging to the B serogroup. Data from this study are available on-line at the web site http://schubert.bio.uniroma1.it/neisseria/index.html, where a 2-DE databank of Neisseria meningitidis strain MC58 is under construction.

Materials and Methods Bacterial Growth Conditions and Sample Preparation. N. meningitidis serogroup B strain MC58 was grown to confluence on GC agar plates (Oxoid, Basingstoke, UK) plus supplements at 37 °C in a humidified atmosphere containing 5% CO2. Bacteria were harvested from a single plate (1011 cells), resuspended in 2 mL of 10 mM Tris (pH 7.4), centrifuged (5000 × g, 4 °C, 5 min) and washed twice with the same buffer. The cell pellet was resuspended in 1.0 mL of 40 mM Tris (pH 7.4) containing 0.3 mg/mL protease inhibitor mixture (CompleteMini, Roche Diagnostic, Mannheim, Germany) and stored at -80 °C prior to fractionation. All chemicals were obtained from Sigma (St. Louis, MO), unless indicated otherwise. Protein Prefractionation. Cellular protein fractionation procedure was largely performed according to Molloy, with the following modification.10 Cells were broken using a sonic dismembranor (Ultrasons) for six 10 s bursts at 0 °C. After 15 min incubation at 37 °C in the presence of DNase I (150 U; Roche Diagnostic), a first protein fraction was obtained by centrifugation at 14 000 × g for 10 min at 4 °C. The supernatant (fraction A) was desalted using the PlusOne 2D Clean-Up kit (Amersham Biosciences, San Francisco, CA) and resuspended in 10 mM Tris (pH 7.4) buffer containing 5 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 0.2% v/v carrier ampholytes pH range 3-10 (Bio-Lytes, Bio-Rad, Hercules, CA), and 0.001% w/v bromophenol. The pellet from the previous centrifugation was resuspended in 40 mM Tris (pH 7.4), containing 8 M urea, 2% w/v CHAPS, 50 mM DTT, 0.2% v/v Bio-Lytes pH range 3-10, 1 mM EDTA, and 0.3 mg/mL protease inhibitor mixture. After centrifugation (14 000 × g, 4 °C, 10 min) the supernatant (fraction B) was collected and stored at -80 °C prior to electrophoresis. Finally, the pellet separated from fraction B was redissolved in 1.0 mL 40 mM Tris (pH 7.4), containing 2 M thiourea, 5 M urea, 2% w/v CHAPS, 50 mM DTT and 0.2% v/v Bio-Lytes pH range 3-10. After gentle mixing for 5 min at 20 °C, the supernatant (fraction C) obtained by centrifugation at 14 000 × g at 4 °C for 10 min was stored at -80 °C prior to electrophoresis. First-Dimension Electrophoresis: Aliquots (approximately 200 µg of protein) from each fraction were loaded by passive rehydration on an 11-cm precast Immobiline strip with a linear pH 4-7 gradient. All IEFs were carried out on the Protean IEF Cell at 20 °C using the following program: 250 V for 15 min, 250-8000 V for 2.5 h and 8000 V for 5 h. After the isoelectrofocusing separation was completed, the strips were first soaked for 30 min in 130 mM DTT and then treated for 1362

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30 min with 135 mM iodoacetamide, both in the equilibration buffer (50 mM Tris, pH 8.8, 6 M urea, 20% v/v glycerol, 2% w/v SDS). All electrophoresis chemicals and apparatus were obtained from BioRad (Hercules, CA), unless indicated otherwise. Second-Dimension Electrophoresis: The second separation by molecular weight was carried out on precast 12% Bis-Tris gels (13 × 8.5 cm) by using a Criterion apparatus. A prestained standard peptide mixture (10 µL, broad range) was applied on a corner of the gel to determine the relative molecular masses of proteins. Electrophoretic analyses were delevoped in NuPAGE MOPS SDS running buffer (Invitrogen, Paisley, UK) at constant voltage (200 V). Gels were stained using Coomassie Blue G-250 (Bio-Safe; fractions A and B) or the staining kit SilverQuest (Invitrogen, Paisley, UK; fractions C). Image Analysis. After destaining, gels were digitalized using a computing densitometer (GS-710 Imaging Densitometer, BioRad) with a pixel size of 36.3 × 36.3 µm and were analyzed with PDQuest image analysis system (version 7.2.0, Bio-Rad). Using PDQuest tools, total numbers of spots were determined according to manufacturer’s procedures. Briefly, automated spot detection was carried out following selection of spots 122, 167, and 172 (Figure 1) as the largest, faintest and smallest spot, respectively. According to this selection, detection was automatically set to: sensitivity 20.18, size scale 9 and minimum peak 6691. Molecular mass and isoelectric point of the major spots were automatically determined by bilinear interpolation between landmark features on each image. Mass Spectrometric Analysis of Protein Spots. Selected spots were manually excised from gels and a large number of samples were simultaneously digested with trypsin using the In-gel Digest96 Kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Briefly, gel pieces were placed in the upper 96-well plate provided by the manufacturer and submitted to destaining steps at room temperature. Destaining solutions were removed under vacuum at the end of the incubation. Destaining was carried out in 5% v/v (30 min) and 50% v/v (2 times, 30 min each) acetonitrile in 25 mM ammonium bicarbonate, and 100% v/v (10 min) acetonitrile, respectively. After the final drying step, each gel piece was imbibed with 15 µL of the trypsin provided in the kit (approximately 5 µg/mL in 25 mM ammonium bicarbonate) and incubated for 3 h at 37 °C. Enzymatic digestion was stopped by adding 8 µL of pure acetonitrile. Tryptic peptides were extracted by incubating each gel piece in 130 µL of a 0.2% v/v TFA aqueous solution for 30 min at room temperature. After the ZipPlate component was assembled, loading of the tryptic mixtures on its C18 beads was directly achieved on the bottom plate by applying low level vacuum. Finally, peptides were eluted from the microcolumns to the microtiter plate by extraction with 15 µL of 50% v/v acetonitrile containing 0.1% v/v TFA, under vacuum. A minimal aliquot of the volume collected from each gel spot was mixed with an equal volume of a solution of R-cyano-4-hydroxy-trans-cinnamic acid matrix saturated in 50% v/v acetonitrile containing 0.1% v/v TFA. The obtained mixture was spotted onto a MALDI target plate and allowed to air-dry at room temperature. MALDI-TOF-MS analyses were performed in a Voyager-DE STR instrument (Applied Biosystems, Framingham, MA) equipped with a 337 nm nitrogen laser and operating in reflector mode. Mass data were obtained by accumulating several spectra from laser shots with an accelerating voltage of 20 000 V. All mass spectra were externally calibrated using a standard peptide mixture

N. meningitidis Serogroup B Strain MC58 Proteome

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Figure 1. Reference maps of N. meningitidis serogroup B strain MC58 proteins. In the 2D-maps on minigel (13 × 8.5 cm), protein spots whose tryptic peptide mixture provided protein identification, are numbered according to their relative location in the gel (from the upper right corner to the lower left corner). The panel B shows a Coomassie stained map of the protein fraction soluble in a buffer containing 8 M urea and 2% w/v CHAPS (fraction B). Lower panels show the most representative minimap achieved for the two protein fractions extracted in 40 mM Tris-HCl (fraction A, panel A, Coomassie stained) and with a buffer containing 2 M thiourea, 5 M urea and 2% w/v CHAPS (fraction C, panel B, Silver stained), respectively. Positive protein identifications are listed in Table 1.

containing des-Arg-bradykinin (904.4681), angiotensin I (1296.6853), 1-17 (2093.0867), and 18-39 (2465.1989) adrenocorticotropic hormone fragments. Two tryptic autolytic peptides were also used for the internal calibration (m/z 842.5100 and 2807.3145). Database Searches. A monoisotopic mass list from each protein spot was obtained from MALDI-TOF data after exclusion of expected contaminant mass values (autolytic tryptic peptides and tryptic human keratin fragments), automatically achieved by the PeakErazor program (http:// www.protein.sdu.dk/gpmaw/Help/PeakErazor/peakerazor.html). These peptide mass fingerprints (PMF) were used to search for protein candidates in the NCBInr database using the Mascot

search engine11 at the site http://www.matrixscience.com, with the following parameters: one missed cleavage permission, 50 ppm measurement tolerance, and at least five matching peptide masses. Oxidation at methionine (variable modification) and S-carboxyamidomethylation at cysteine residues (fixed modification) were also considered. No post-translational modifications were allowed. Positive identifications were accepted with P values (the probability that the observed match is a random event) higher than 0.05. The computational similarity search of protein spots in the menA genome was performed using the BLAST network service, whereas the theoretical molecular mass and pI values of the identified proteins were calculated from their predicted amino acid Journal of Proteome Research • Vol. 4, No. 4, 2005 1363

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Figure 2. MALDI-TOF mass spectrum of the in-gel digested spot n. 95 (fraction B). The mass signals providing identification of the spot as EF-Tu isoform A are indicated in the spectrum by their m/z values and peptide position in the sequence. Protein sequence is also reported, with the region covered by the peptide mass fingerprint shadowed. In the inset, the database search result using MASCOT is reported.

sequences by using the ProtParam tool, both via the Expasy website (http://au.expasy.org/cgi-bin/blast.pl and http:// www.expasy.ch/tools/protparam.html, respectively).12 In the cases in which a proteolytic process was annotated into the relative SwissProt/TrEMBL entry (http://us.expasy.org/sprot), values refer to the expected mature polypeptide chain.

Results With the aim to setup a proteomic platform to collect the most possible information on the global protein content in a menB strain on a largest number of growing conditions, we approached the meningococcal proteomics by 2-DE separation on minigels, which requires less time and reagents than larger gels. To avoid the intrinsic risk of the mini 2D gel reduced resolving power, an up-front protein fractionation was carried out, limiting the total number of protein species loaded on the same gel. Using the 4-7 range IPG strip, highly reproducible 2D mini-maps (13 × 8.5 cm) over approximately 10 replicates were obtained for proteins extracted without chaotropic agent (fraction A, Coomassie-stained gels, 221 spots automatically detected), in the presence of urea and detergents (fraction B, Coomassie-stained gels, 653 spots automatically detected), or in the presence of a strong chaotropic agent (fraction C, Silverstained gels, 83 spots automatically detected). In Figure 1, the most representative gel of each fraction is shown; those protein spots from which menB gene products have been successfully identified by MALDI-TOF analyses, as described in Materials and Methods section, are highlighted and numbered according to their relative position on the maps (from the upper right to the lower left corners). An example of the mass peptide fingerprint quality, the spectrum of one of the protein spot, together with details of its identification, is shown in Figure 2. Protein identification was considered positive when peptide mass fingerprint yielded a statistically significant score value. 1364


All these identifications are listed in Table 1, with the corresponding accession number in the SwissProt/TrEMBL database. Spots no. 185-188 were only detected in some of the 2-DE replicates of fraction B. In the case of the protein EF-Tu, the N. meningitidis strain MC58 genome codes for two isoforms, differing only by residue 37 (alanine in isoform A coded by the gene tufA and serine in isoform B by tufB). In the N. meningitidis immutype L3 proteome both isoforms were detected.8 We were able to unequivocally detect the isoform A by the presence in the peptide mass fingerprint of spots no. 94c, 99, 100, and 101 of a signal at m/z 1317.8, corresponding to the discriminating peptide (Figure 2). Peptide signals encompassing residue 37, even taking into account a possible phosphorylation of the serine residue, were missing in the MALDI-ToF spectra of spot nos. 95, 96a, 97a, and 98 from the same 2D-map, making it impossible to unequivocally identify the relative EF-Tu isoform. In the menB protein list shown in Table 1, the theoretical pI and Mr values were also reported for each spot and compared with the experimental values. In the cases in which a proteolytic processing is annotated in the corresponding SwissProt/ TrEMBL entry (e.g., for some outer membrane proteins), theoretical values refer to the expected mature polypeptide chain. For the majority of the proteins identified these values are in agreement with the corresponding experimental values. For a more immediate comparison with data from the literature, proteome identifications are related in these Tables with the OrderedLocusNames of the N. meningitidis MC58 genome. In two cases, two ORF codes are reported. In the first (spot nos. 94-101), the two different isoforms of the elongation factor Tu are annotated, while in the other (spot no. 154), the two identical noncontiguous menB genes (NMB1127 and NMB1165) coding for a unique primary structure of a putative oxidoreductase are reported.

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N. meningitidis Serogroup B Strain MC58 Proteome Table 1. Protein Components Identified in 2D-Minimaps pI

spot no. a

1 2 3 4 5 6 7 8a 9 10a 8b 10b 11 19 20 12a 13 12b 13b 14 15 16 17 18 21 22 23 24 25 26 27 27b 28 29 30 31a 31b 32 33a 34 35 33b 36 37 38 39 40a 41a 42a 40b 41b 42b 43 44 45 46 47 48 49 50 51a 52 189 190 191 51b 53a 53b

protein nameb

mass (Da)

accession number (Swiss-Prot) theoreticalc measuredd theoreticalc measuredd

Phosphoribosylformyl-glycinamidine synthase Carbamoyl-phosphate synthase, large subunit

Q9JXK5

5.3

Q9JXW8

5.1

Preprotein translocase SecA subunit Aconitate hydratase 2

Q9JYK8 Q9JYI4

5.0 5.4

ClpB protein Pyruvate dehydrogenase, E1 component Phosphoenolpyruvate synthase

Q9JYQ8 Q9JZ12 Q9K0I2

5.4 5.6 5.0

Outer membrane protein (Omp85)

Q9K1H0

8.6

PilQ protein (pilus secretin)

Q70M91

9.4

Isocitrate dehydrogenase

Q9JZS1

5.6

Elongation factor G (EF-G)

Q9K1I8

5.1

Polyribonucleotide nucleotidyltransferase Glycyl-tRNA synthetase, β chain Succinate dehydrogenase, flavoprotein subunit Transketolase

Q9K062

5.3

Q9JXQ5 Q9JZP8

5.3 5.9

Q9JYS0

5.4

Aspartyl-tRNA synthetase Pyruvate dehydrogenase, E3 component

Q9K0U5 Q9JZ09

5.3 5.1

GTP-binding protein TypA Chaperone protein dnaK

Q9JZB7 Q9K0N4

5.0 4.8

Formate-tetrahydrofolate ligase

Q9JXY2

5.8

Aminopeptidase

Q9JYU4

5.3

ABC transporter, ATP-binding protein

Q9K112

5.2

Na+ - t ranslocating NADH quinone reductase, subunit A Bifunctional purine biosynthesis protein purH 30S ribosomal protein S1

Q9K0M3

6.2

Q9JZM7

5.9

Q9JZ44

5.0

2-oxoglutarate dehydrogenase, E3 component

Q9JZP5

5.9

Acetyl-CoA carboxylase, biotin carboxylase Pyruvate kinase II Lysyl-tRNA synthetase, heat inducible

Q9JXW3

5.9

5.6 5.5 5.6 5.5 5.3 5.3 5.7 5.6 5.5 5.5 5.6 5.5 5.3 5.0 4.9 or or or or 5.7 5.7 5.6 5.5 5.5 5.3 5.3 5.2 5.1 5.1 5.6 5.5 5.5 6.0 5.9 5.5 5.5 5.5 5.0 5.0 4.9 4.9 5.0 4.8 4.8 6.2 6.1 5.5 5.4 5.3 5.5 5.4 5.3 6.6 6.6 6.3 6.1 5.0 4.9 4.9 6.3 6.2 6.1 6.3 6.2 6.1 6.2

Q9K1M1 Q9JYU6

5.3 5.3

5.5 5.5

143853

49599

or or or or or or 96800 95600 94900 94000 95600 94000 87200 86500 86200 90900 91800 90900 91800 85600 85200 84000 82600 83100 82100 81600 83000 82200 82100 79400 76500 76500 73000 72800 74400 73600 73600 70900 70400 70100 70400 70400 72900 72600 70200 69300 67900 67700 67300 67900 67700 67300 65400 65300 65900 65200 64400 63500 64600 61300 60300 60600 51200 51200 51300 60300

52435 57312

61400 61400

117376 103294 92715

95195 99562 87170 88437 81668 80163 77244

76421 74573 64502 71658 68124 61829

67259 68792 59062 64852 62072 48636 56802 61177 50093

gene namee

NMB

NMA

menA proteomef

NMB1996 NMA0445

+

NMB1855 NMA0602

+

NMB1536 NMA1735 NMB1572 NMA1761

+

NMB1472 NMA1683 NMB1341 NMA1554 NMB0618 NMA0826

+ + +

NMB0182 NMA0085

+

NMB1812 NMA0650

-

NMB0920 NMA1116

+

NMB0138 NMA0135

+

NMB0758 NMA0969

+

NMB1930 NMA0523 NMB0950 NMA1145

+ +

NMB1457 NMA1669

+

NMB0466 NMA2019 NMB1344 NMA1556

+ +

NMB1199 NMA1370 NMB0554 NMA0736

+ +

NMB1839 NMA0617

+

NMB1428 NMA1640

+

NMB0387 NMA2101

+

NMB0569 NMA0752

+

NMB0983 NMA1182

+

NMB1301 NMA1515

+

NMB0957 NMA1151

+

NMB1861 NMA0596

+

NMB0089 NMA0177 NMB1425 NMA1638

+ +

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Table 1. (Continued) pI

spot no. a

54 55 56 57 58 63 64 59 60 61 62 65 66 67 68a 68b 69 70 71 72 192 193 194 195 73 74 75a 77 78 79 80 81 75b 76 82 83 84 85 86 87 196 88 89 90 91 92 93 94a 94b 94c 95 96a 97a 98 99 100 101 96b 97b 102 103a 103b 104 105 106a 106b 107 108 109 1366

protein nameb

mass (Da)


Glutamate-ammonia ligase 60 kDa chaperonin

Q9K134 P42385

5.2 4.9

Malate:quinone oxidoreductase

Q9JXD7

5.5

ATP synthase F1, R subunit

Q9JXQ0

5.4

Seryl-tRNA synthetase Aldehyde dehydrogenase A

Q9JY95 Q9JXM8

5.6 5.2

Twitching motility protein Serine hydroxy methyltransferase Glutamate dehydrogenase, NADPspecific

Q9K1N4 P56990 Q9JY71

6.5 6.3 6.0

Argininosuccinate synthase

Q9JXC1

5.2

Phosphate acetyltransferase Pta

Q9K0H1

4.8

Hypothetical protein Histidinol dehydrogenase Malate oxidoreductase ATP synthase F1, β subunit

Q9K1K7 Q9JYH8 Q9K0D8 Q9JXQ2

9.1 5.1 5.1 5.0

Trigger factor Major outer membrane protein P. IA, class 1

Q9JZ37 Q51240

4.7 8.7

Twitching motility protein PilT

Q9K053

6.1

Acetate kinase 1

Q9JYM1

5.8

3-oxoacyl-(acyl-carrier-protein) synthase II Cell division protein FtsA Elongation factor Tu (EF-Tu)

Q9K1D8

5.4

Q9K0X8 P64027

5.3 5.1 5.1

Enolase

Q9JZ53

4.8

Aminomethyltransferase Carbamoyl-phosphate synthase, small subunit ATP phosphoribosyl-transferase, regulatory subunit Alcohol dehydrogenase, zinc-containing

Q9K0L8 Q9JXX4

5.6 5.5

5.3 4.9 4.8 4.8 5.8 5.8 5.6 5.6 5.5 5.4 5.9 5.3 5.2 5.1 6.6 6.6 6.4 6.1 5.9 5.7 6.4 6.2 6.1 5.9 5.3 5.2 5.1 4.7 4.6 or 5.1 5.1 5.1 5.0 5.0 4.9 4.8 4.6 or or or 6.3 6.2 6.0 5.9 5.8 5.6 5.5 5.5 5.5 5.3 4.8 4.7 4.6 4.5 4.4 4.3 4.8 4.7 5.9 5.8

Q9K013

5.4

Q9K0J3

5.5

Polysialic acid capsule biosynthesis protein SiaC Cell division protein ftsZ

Q7DDU0

5.3

Q51130

4.9

Succinyl-CoA synthetase, β subunit

Q9JZP4

5.1


52137 57423

gene namee

NMB

NMA

menA proteomef

NMB0359 NMA2128 NMB1972 NMA0473

+ +

NMB2096 NMA0333

+

NMB1936 NMA0517

+

NMB1684 NMA1943 NMB1968 NMA0480

+ +


+ +

NMB2129 NMA0303

+

NMB0631 NMA0841

+

NMB0109 NMB1581 NMB0671 NMB1934

NMA0165 NMA1770 NMA0870 NMA0519

+ + +

NMB1313 NMA1526 NMB1429 NMA1642

+ +

NMB0768 NMA0979

+

NMB1518 NMA1718

+

NMB0219 NMA0044

+


+

NMB1285 NMA1495

+

NMB0574 NMA0758 NMB1849 NMA0608

+ +

39740 40587

59600 59400 59600 60800 58700 56300 58500 58800 58000 58100 56700 56700 56500 56100 56800 56800 55300 54100 53200 52900 51400 50200 50000 49900 53500 53300 53100 53300 53400 51900 49600 51100 53100 52500 51200 50800 50800 51100 47400 47400 42100 48000 47600 49500 48600 48400 49300 48500 48500 48500 46200 46100 46200 46900 45500 45700 45500 46100 46200 44700 46100

5.8

41746

46100

NMB0814 NMA1023

+

5.7 5.6 5.6 5.6

37921

43500 43400 43000 43000

NMB0604 NMA0808

+

NMB0068 -

-

4.9 4.8 5.0

41487

43000 42900 41700

NMB0427 NMA2057

+

NMB0959 NMA1153

-

53968 55291 47883 52256 45674 44916 48490

49664 52200 50120 46323 46025 50391

48325 40129 41508 42409 43221 44060 42909 42925

46134

38348

41336

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N. meningitidis Serogroup B Strain MC58 Proteome Table 1. (Continued) pI

spot no. a

110 111 112 113 114a 115a 116 117a 118 119 120a 121a 197 114b 115b 115c 117b 120b 121b 122 123 124 125 198 199 200 208 126 133 127 128 129 130 131 132 134 135 136 137 138 139 140 201 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156a 156b 157 159 162 158 160 161a 161b

protein nameb

mass (Da)


Alcohol dehydrogenase, propanolpreferring

Q9K0P0

5.6

Ketol-acid reductoisomerase

Q9JYI2

5.6

Fructose-bisphosphate aldolase

Q9JXV5

5.5

ADP-heptose synthase Holliday junction DNA helicase RuvB Major outer membrane protein P. IB, class3

Q9K004 Q9JZ86 P30690

5.3 5.2 6.5

Ribose-phosphate pyrophosphokinase

P65235

5.4

Phosphoribosylformyl glycinamidine cyclo-ligase Glutathione synthetase Succinyl-CoA synthetase, R subunit

Q9JZ80

4.7

Q9JYJ3 Q9JZP3

6.1 6.0

Elongation factor Ts (EF-Ts)

P64051

5.3

Outer membrane protein, class 4

P38367

6.0

UTP-glucose-1 phosphate uridylyltransferase Phosphoribosylaminoimidazole succinocarboxamide synthase Electron-transfer flavoprotein, R subunit Septum site-determining protein MinD

Q9K0G7

5.7

4.9 4.8 4.8 6.0 5.9 5.9 5.8 5.8 5.7 5.6 5.5 5.5 5.9 5.9 5.9 5.9 5.8 5.5 5.5 6.6 6.5 6.3 6.1 6.6 6.3 6.0 6.2 5.4 5.6 4.6 4.6 6.3 6.3 6.1 5.9 5.4 5.3 5.1 6.6 6.4 6.2 6.1 6.6 6.1

Q9K063

5.3

Q9JXA0

5.0

Q7DDS7

5.7

2,3,4,5-tetrahydropyridine-2carboxylate N-succinyltransferase Hypothetical protein 3-hydroxyacid dehydrogenase

Q9K152

5.4

Q9JYT8 Q9JYH6

5.3 5.3

Oxidoreductase, short-chain dehydrogenase/reductase family Peroxiredoxin 2 family protein/glutaredoxin Oxidoreductase, short chain dehydrogenase/reductase family Iron-starvation protein PigA 3-oxoacyl-(acyl-carrier-protein) reductase Stringent starvation protein A 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase

Q9JZR8

5.0

Q7DDK4

4.8

Q9JRT0

gene namee

NMB

NMA

menA proteomef

32030

43000 42900 41500 42100 42500 41900 41400 42200 40000 40100 40000 40200 40100 42500 41900 41900 42200 40000 40200 37300 38700 39400 39000 39200 39200 39200 35100 37300 37300 36500 36600 38400 37100 36400 35700 35300 35000 34400 35400 34300 32800 33400 33200 35200

5.4

32283

32600

NMB0757 NMA0968

+

5.0 4.9 6.1 5.8 5.7

32579

32600 32500 31000 29900 29500

NMB2154 NMA0241

+

NMB0171 NMA0100

+

NMB0335 NMA2153

+

5.5 5.5 5.2 5.1

28253 30378

28100 25000 27700 30000

NMB1436 NMA1648 NMB1584 NMA1773

-

NMB0924 NMA1120

+

NMB0946 NMA1141

+

NMB1127 NMA1336 NMB1165 NMB1669 NMA1927 NMB1921 NMA0533

-

NMB1953 NMA0498 NMB1604 NMA1801

+ +

36548

36438 38337 34998 37660 33786

35598 36974 35145 30548 30330 23925

29559 29409

30119

NMB0546 NMA0725

+

NMB1574 NMA1763

+

NMB1869 NMA0587

+


+

NMB0875 NMA1093

+

NMB1252 NMA1421

-

NMB1559 NMA1747 NMB0960 NMA1154

+

NMB2102 NMA0327

+

NMB0382 NMA2105

+

NMB0638 NMA0848

-

26912

6.0

4.8 4.7 6.5

25916

26400 26400 27500

Q9JYA7 Q9JXR1

5.9 5.9

6.2 6.4

23433 26067

26900 26200

Q9JXN8 Q9JYF7

6.2 5.6

23164 25958

DNA-binding response regulator

Q7DDM6

5.4

+

Q9JZI6

5.5

24264

26200 26300 26200 27500 25600 25000 24800

NMB0595 NMA0798

3-isopropylmalate dehydratase, small subunit Hypothetical protein

6.4 5.9 5.7 5.5 5.8 5.5 5.7

NMB1034 NMA1452

-

Q9JXV3

5.6

5.7

23762

24800

NMB1871 NMA0585

-

24780

+ +

Journal of Proteome Research • Vol. 4, No. 4, 2005 1367

research articles

Mignogna et al.

Table 1. (Continued) pI

spot no. a

163

164 166 165 167 168 169 170 171 172 173 174 175 176 177 204 178 179 180 181 182 183 205 184 185 186 187 188 202 203 206 207 209 210

protein nameb

mass (Da)


gene namee

NMB

NMA

menA proteomef

1-(5-phosphoribosyl)-5-[(5phosphoribosylamino) methylideneamino] imidazole-4carboxamide isomerase Hypothetical protein

Q9K0H3

5.2

5.2

25924

24900

NMB0629 NMA0839

-

Q9JYT7

5.1

25898

-

P49980 P63337 Q9JZQ8 Q9JZW3 P65592

5.0 5.1 4.8 6.6 6.0

24700 24600 25400 24100 24300 24200 23500

NMB1437 NMA1649

Adenylate kinase 6-phosphogluconolactonase Elongation factor P 50S ribosomal protein L25 Transcription antitermination protein nusG Superoxide dismutase

5.2 5.0 5.1 5.1 4.8 6.6 6.4

NMB0823 NMB1391 NMB0937 NMB0876 NMB0126

NMA1032 NMA1608 NMA1133 NMA1094 NMA0147

+ + +

Q9JZV6

5.8

NMB0884 NMA1104

-

Hypothetical protein

Q9JY11

5.7

NMB1796 NMA0666

+

Peptidyl-prolyl cis-trans isomerase B ATP synthase F1, delta subunit Hypothetical protein

Q7DDL3 Q9JXP9 Q9JYN4

5.0 5.0 5.1


+ + +

50S ribosomal protein L9 Nucleoside diphosphate kinase Lactoylglutathione lyase

Q9JZ31 P65533 O33393

6.6 5.4 5.2


-

Bacterioferritin A Protein-export protein SecB

P72080 Q9JY16

4.7 4.2

NMB1207 NMA1377 NMB1789 NMA0674

-

50S ribosomal protein L7/L12 β-phosphoglucomutase

P80716 Q9K108

-

Cysteine synthase Electron-transfer flavoprotein, β subunit 2-oxoglutarate dehydrogenase, E1 component Single-strand binding protein Inorganic pyrophosphatase Oligopeptidase A Citrate synthase Outer membrane protein, class 5 50S ribosomal protein L1

23217 24980 20880 20956 20550

4.6 5.1

6.0 6.0 6.1 5.7 5.0 5.1 5.2 5.3 6.4 5.7 5.3 5.1 4.6 4.2 4.2 4.5 5.1

21892

12491 23702

23400 23000 22400 21900 20400 19200 18000 17200 17400 15500 15900 15900 16000 16300 16200 14800 24100

Q7DDL5 Q9JX99

6.1 6.1

6.1 6.1

32821 26947

34000 30100

NMB0131 NMA0143 NMB0391 NMA2093 NMA2097 NMB0763 NMA0974 NMB2155 NMA0242

Q7DDJ9

6.2

6.3

105082

110000

NMB0955 NMA1149

+

P66849 Q9K0G4 Q9K1E2 Q7DDK0 Q7DDI3 P66088

5.8 4.7 5.2 6.3 9.7 9.6

6.0 4.8 5.3 6.2 or or

19453 19811 76054 48121 29991 24102

20200 22000 78200 43400 29200 26700

NMB1460 NMB0641 NMB0214 NMB0954 NMB1053 NMB0128

+ + + -

20931 18852 19525 16524 15747 15427 15669 17961 16319

NMA1672 NMA0851 NMA0054 NMA1148 NMA1251 NMA0145

+ -

a Numbering according to Figure 1. Spots no. 185-188 were only detected in some of the fraction B 2D replicates. Spots in the numbering range 189-205 and 206-210 relate to A and C fractions, respectively. Different numbers for the same protein entry indicate that the protein was identified in several spots. Different proteins identified in the same spot are distinguished by an alphabetic index. b Protein name according to the SwissProt/TrEMBL entry (http:// au.expasy.org/sprot/). c Theoretical pI and molecular weight are according to the SwissProt/TrEMBL entry. In the cases in which a proteolytic process is annotated, values refer to the expected mature polypeptide chain. d Experimental pI and molecular weight were automatically determined by bilinear interpolation between landmark features on each image. e Gene name according to the OrderedLocusNames of the N. meningitidis MC58 (NMB) and Z2491 (NMA) genomes, respectively. Similarity searches between menB and menA genes were carried out using the BLAST network service via the Expasy website (http://au.expasy.org/ cgi-bin/blast.pl). f Positive sign (+) marks a protein detection achieved also in the menA proteome; negative sign (-) marks a protein identification achieved only in the menB proteome. or Out of experimental ranges.

For an easier comparison with functional genomics data, for each protein identification the counterpart menA strain Z2491 was assessed by BLAST analysis and the relative gene code is included in Table 1. This was possible in all cases except for spot no.106, in which the SiaC protein fingerprint was determined by mass spectrometry. As known, this enzymatic protein is involved in the biosynthesis of the polysialic acid capsule that characterizes the menB phenotype. Finally, in the Table 1 the menB β-phosphoglucomutase gene (spot no.185) is associated to the two high homologous menA genes, since it shares the same degree of identity with both genes (approximately 95%). Our 2D-minigel patterns and the protein index have been made publicly available via the Internet on the web page http:// schubert.bio.uniroma1.it/neisseria/index.html. At the same web 1368


site, a Neisseria proteome databank, in which mass spectra, peptide mass lists and Mascot protein identification will be stored, is also under construction.

Discussion Traditionally, global studies of cellular protein content have relied on two-dimensional electrophoresis. Standard-size twodimensional gels (typically about 18 × 20 cm) are expected theoretically resolve up to 5000 spots.13 Proteomics routine has been demonstrated that these gels are capable of separating about 1500 to 2000 protein spots and as many as 3000 spots under favorable conditions.14 Therefore, 2DE-based proteomics provides the suitable technique platform allowing resolving the protein complexity challenging in microbial research (typically

Inventory of the Proteins in Neisseria meningitidis ... - ACS Publications

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