Electrophoresis 2000, 21, 3765±3780 Jörg T. Regula1* Barbara Ueberle1* Günther Boguth2 Angelika Görg2 Martina Schnölzer3 Richard Herrmann1 Rainer Frank4 1
Zentrum für molekulare Biologie (ZMBH), Universität Heidelberg, Heidelberg, Germany 2 Technische Universität München, Institut für Lebensmitteltechnologie und Analytische Chemie, Freising, Germany 3 Deutsches Krebsforschungszentrum Heidelberg, Zentrale Proteinanalytik, Heidelberg, Germany 4 GAG Bioscience AG, Bremen, Germany
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Towards a two-dimensional proteome map of Mycoplasma pneumoniae A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatographyelectrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions. Keywords: Proteomics / Genome annotation / Mass spectrometry / Two-dimensional gel electrophoresis
Approximately 30 bacterial genomes have been completely sequenced and published. For an updated list see www.tigr.org or www.ncbi.nlm.nih.gov/Entrez/Genome/ org.html. The annotation of these sequences permits 50± 70% of the proposed open reading frames (ORFs) to be assigned to a function. The assignment is based on significant sequence similarity to proteins with known function from other organisms. The functionally unassigned ORFs are the most interesting ones, since they are potential candidates for genes with new functions. Because the functional analysis is labor-intensive it is advantageous to preselect genes of interest. One possibility of preselection is to analyze genes that are expressed under one particular set of conditions. Expression analysis answers two important questions: Is a certain gene expressed at all and does the expression depend on certain conditions? Expression can be studied at the level of transcription or translation. Either analysis has its own limits. The methods for mRNA analysis that includes all genes, even of
Correspondence: Dr. Richard Herrmann, Zentrum für molekulare Biologie (ZMBH), Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany E-mail:
[email protected] Fax: +49-6221-545893
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000
the most complex genome (transcriptome), are well established [1, 2]. This is not the case in the identification of all proteins of a cell (proteome [3]), although a proteome analysis is more meaningful because the proteins are in most cases the functional elements and the mRNAs are only intermediates. mRNA measurements can not be used to predict protein concentrations since there is no strict correlation known [4]. For the identification of all expressed proteins of a cell, provided that the complete genome sequence is available, a combination of 2-D gel electrophoresis and MS is the method of choice [5]. Complex protein mixtures can be separated reproducibly into individual protein spots, resulting in 2-D maps of a proteome. Identification of individual proteins by peptide mass fingerprinting [6±8] and peptide fragmentation [9±11] proved to be efficient and reliable. The procedure matches the masses of in-gel generated proteolytical peptides with the theoretical peptide masses of all proteins in a database of the organism to be analyzed. Further evidence is obtained by matching the MS/MS fragmentation patterns of these peptides to the calculated fragment spectra.
* Both authors contributed equally
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Proteomics and 2-DE
1 Introduction
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The potential of the proteomic technology has been shown by a wide range of applications, particularly in connection with microorganisms [12]. For instance, expression profiles of several bacteria grown under standard and modified conditions have been established and led to the discovery of functional protein groups [13, 14], and the comparisons of protein maps of pathogenic and apathogenic isolates of the same species are being used to reveal pathogenicity-related proteins [15]. A further topic are the studies on gene expression of complete cells at the level of transcription and translation, which will provide insight into global mechanisms of regulation of expression [4]. For more examples see VanBogelen et al. [16], Cash [17], or the recent summary on proteomics [18]. Such a proteome analysis is limited, mainly in the first dimensions, by the resolution of the 2-D gel, the enormous differences in concentration of individual proteins in a complete bacterial protein extract, and the sensitivity of MS. Since proteome analyses are becoming increasingly more interesting [19±21], it is important to explore the limits of the applied technology with a simple cell, where the chance exists to identify almost all of the proposed proteins. For this purpose, we use Mycoplasma pneumoniae as a model organism for a proteome analysis. M. pneumoniae is one of the smallest bacteria known with a genome size of 816 kbp and a cell diameter of about 0.5 mm [22]. According to a recent reannotation, the number of ORFs increased from 677 to 688 [23]. Even in such a small genome only 67% of the ORFs could be assigned to a function. As a human pathogen, M. pneumoniae causes respiratory tract disease mainly in children and yound adults, fulfilling Koch©s postulats as shown by inoculation of volunteers [24]. In nature it depends strictly on a host cell, but it can be grown in the laboratory without a host cell in a rich medium complemented by 10±20% serum. M. pneumoniae coexists as a true parasite in close contact with its host, e.g., lung epithelia cells, by adhering to their surface and colonizing them. Although reports have been published indicating that M. pneumoniae can invade its host cell, growth and reproduction of M. pneumoniae inside the cell have not been proven [25]. The present parasitic lifestyle became necessary after loss of genetic information during degenerative evolution [26]. Many enzymes involved in the synthesis of essential compounds like amino acids, purine, and pyrimidine bases or fatty acids, as well as genes for the synthesis of a bacterial cell wall are absent. This was shown by biochemical studies [27] as well as the annotation [22] and recent reannotation [23] of the complete sequence of the 816 kbp long genome. So far, it has been difficult, with conventional genetic and biochemical approaches, to elu-
Electrophoresis 2000, 21, 3765±3780 cidate M. pneumoniae-specific functions, in particular considering that the standard tools of genetics that exist for Escherichia coli or Bacillus subtilis, such as efficient transformation, transduction or construction of mutants by homologous recombination, are either not at hand or not well developed for M. pneumoniae. Therefore, as an alternative approach, we combine protein expression analysis with functional analysis. The prerequisite for these studies is a 2-D proteome map of M. pneumoniae cells grown under standard conditions that serves as a reference map for all comparative analyses. This paper summarizes the present state of our efforts to establish a proteome map of M. pneumoniae.
2 Materials and methods 2.1 Materials and equipment Tissue culture flasks (150 cm2) were from Renner (Darmstadt, Germany). Horse serum, Pefabloc SC and Complete EDTA-free was from Roche Boehringer Mannheim (Germany). PPLO broth was from Difco (Detroit, MI, USA). The equipment for IEF and horizontal electrophoresis (Multiphor II electrophoresis unit, Macrodirve 5 power supply, IPGphorTM, Multitemp III, Immobiline DryStrip kit, reswelling tray), ready-made IPG DryStrips (pH 4±7, 3± 10 L, 6±11), 2-D Pharmalyte pH 3±10, the 2-D evaluation software (Labscan, Imagemaster 2D, Imagemaster 2D database) and the Sharp JX-330 scanning device were from Amersham Pharmacia Biotech (Uppsala, Sweden). The Protean 2D Xi cell was from Bio-Rad (Richmond, CA, USA). Acrylamide (Gel 30), PMSF, TEMED and SDS were from Roth (Karlsruhe, Germany). CHAPS, iodoacetamide, DTT and a-cyano-4-hydroxycinnamic acid were from Sigma-Aldrich (St. Louis, MO, USA). Modified trypsin (sequencing grade) was from Promega (Madison, WI, USA). HPLC water, acetonitrile (HPLC grade) and SDS were from Merck (Darmstadt, Germany). Zip Tips C-18 silica was from Millipore (Bedford, MA, USA). The HPLC Rheos 2000 was from Flux Instruments (Basel, Switzerland) equipped with an Acurate flow splitter and a Vydac C-18 reversed-phase column with 5 mm particles and 30 nm pore size (250 mm length and 300 mm inner diameter) from LC-Packings (Amsterdam, The Netherlands). A Finnigan LCQ ESI ion trap mass spectrometer (San Jose, CA, USA) and a Bruker-Daltonic Reflex-II MALDI-TOF mass spectrometer (Bremen, Germany) equipped with a SCOUT-26 multiprobe inlet and a 337 nm nitrogen laser was used.
2.2 Strains and growth conditions M. pneumoniae M129 cells (ATTC 29342, broth passage No. 23) were grown in modified Hayflick [28] medium at 37oC for 4 days (96 h). The cells were cultured in
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Table 1. Fragmented proteins and corresponding fragment positions No.a)
ORFNo.a)
Length Exp. in AA pI
Exp. Fragment Mr position
Theor. Theor. Number of pI Mr spots/fragments
102a 102b 350 384ab) 384b 384c 384d 396 431 439 461 548a 548b 552a 552b
D09_orf657 D09_orf657 P02_orf474 H08_orf1005 H08_orf1005 H08_orf1005 H08_orf1005 H08_orf1325 F11_orf760 F11_orf582 A19_orf1140 A65_orf787o A65_orf787o A65_orf794 A65_orf794
657 657 474 1005 1005 1005 1005 1396 760 582 1140 787 787 794 794
22 6 35 19 37 20 18 15 25 40 34 24 31 31 58
5.3 4.7 7.38 6.6 6.6 4.43 6 4.99 7.31 5.45 6.34 8.9 5.34 5.1 8.47
8.6 5 6 5 5 5.3 10 5.2 6.8 5.2 10 9.5 5.5 5.2 8.3
317± 450 598± 648 295± 440 167± 239 288± 506 657± 717 852± 926 1211±1323 589± 630 89± 470 80± 206 271± 469 563± 769 562± 757 202± 757
14.3 5.4 16.5 8.3 24.8 21.2 16.7 12.6 4.45 53.4 15.1 22.2 23 22.3 61.6
3 1 1 1 3 5 1 1 1 3 1 2 2 3 >6
a) Numbers according to Himmelreich et al. [22] b) Identified in a protein extract of cells grown at 25oC The fragment positions were derived from the first and last amino acid (AA) of the identified tryptic peptides. The experimental (exp.) pI and Mr give the relative position in a 2-D gel. Theoretical (theor.) pI and Mr are calculated from the sequence of the corresponding region as indicated by fragment position.
150 cm2 tissue culture flasks containing 100 mL medium. Fresh or frozen cultures were inoculated into medium at a ratio of 1:10. The color change of the phenol red indicator to yellow was used to monitor the growth of the culture. Cultures were harvested as soon as the medium turned orange (pH 7.1). One tissue culture flask with 100 mL medium contained 70±100 mg cells (wet weight), or approximately 6 mg protein. Cultures were stored at ±70oC in fresh medium.
2.3 Extract preparation M. pneumoniae cells from 100 mL of culture in a 150 cm2 tissue culture flask were washed twice with PBS, harvested, and immediately suspended in 500 mL lysis solution containing 9 M urea or 7 M urea and 2 M thiourea, 2% w/v CHAPS, 1% w/v DTT, 2% v/v Pharmalyte pH 3±10 and 2 mM Pefabloc SC or Complete EDTA-free [29, 30]. Cells were disrupted with a Branson sonifier for 6 ´ 15 s at 4oC and centrifuged (60 min, 75 000 ´ g) in a Beckman TL 100 ultracentrifuge (Palo Alto, CA, USA). The protein concentration of the supernatant was about 5±7 mg/mL. The protein samples were stored in aliquots at ±78oC until use.
2.4 Triton X-114 fractionation Triton X-114 fractionations according to Bordier [31] and Riethman et al. [32] were used to enrich membrane and membrane-associated proteins. M. pneumoniae cells from 100 mL of culture in a 150 cm2 tissue culture flask were washed twice with PBS, harvested in a 2 mL Eppendorf tube and extracted with 1% Triton X-114 in TBS (10 mM Tris, 150 mM NaCl, pH 7.5) with 1 mM PMSF for 30 min on ice in a total volume of 2 mL. The suspension was centrifuged for 20 min at 14 000 ´ g (4oC) and the sediment was re-extracted as described above. The supernatants were pooled, incubated at 37oC for 5 min and the detergent phase was separated from the aqueous phase by centrifugation for 5 min at 37oC (8000 ´ g). The detergent phase was washed twice with TBS and 1 mM PMSF by centrifugation at 37oC. Proteins were precipitated with ten volumes of methanol from the detergent phase and redissolved in lysis buffer for 2-D gel electrophoresis.
2.5 2-D gel electrophoresis 2-D electrophoresis with immobilized pH gradients (IPGDalt) was carried out according to Görg and colleagues [29, 33±35] with minor modifications. For details see
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Figure 1. Composite 2-D map of M. pneumoniae proteins. About 50 mg of a total protein extract were separated on a 13 cm IPG strip (3±10) followed by a vertical 12.5% polyacrylamide SDS gel (20 ´ 20 cm). The gel was stained with silver according to the method of Blum et al. [37]. The assignment of the protein spots to the ORF numbers [22] was based on results mostly derived from colloidal Coomassie blue-stained gels. Some proteins were identified from gels of different compositions (e.g., 5% polyacrylamide, 18% polyacrylamide, basic pH-gradients).
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Figure 2. Analytical 2-D gel of M. pneumoniae. Proteins were separated on an 18 cm IPG strip (4±12) in the first dimension and on a vertical 12.5% polyacrylamide SDS gel (20 ´ 20 cm) in the second dimension. The gel was stained with silver according to the method of Heukeshoven and Dernick [38]. The assignment of the protein spots to the ORF numbers [22] was based on results mostly from colloidal Coomassie blue-stained gels. http://www.weihenstephan.de/blm/deg. IPG strips were either run on the Multiphor or IPGphor system. The pH range of the IPG gradients mostly used was from 3±10, 4±12, 4±9 and 4±7. The length of the IPG strips was
18 cm unless otherwise stated. Equilibration of the focused IPG strips and 2-D SDS-PAGE was as described by Görg et al. [34]. The size of the gels was 30 ´ 20 cm. 1-D Tris/Tricine gels were run according to the instruc-
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Table 2. Examples for formation of multiple spots a)
a)
No.
Name
406
A05_orf595 DnaK K05_orf394 EF-Tu R02_orf648 C12_orf141 G12_orf184 P01_orf1033
177 73 217 314 366
Formation
Number of spots
Train of spots Fragments Train of spots Fragments Train of spots Two spots in a row Two spots in a row Monomer/dimer Several fragments
7 3 4 4 4 2 2 > 10 > 4
a) According to Himmelreich et al. [22] We distinguish between three categories: (i) train of spots: proteins are close together with the same Mr forming a horizontal line; (ii) row of spots: proteins are at the same Mr but separated by approximately 0.5 pH units; (iii) fragments: spots derived from one ORF which have a different pI and lower Mr than the proposed ORF.
tions of Schägger and von Jagow [36]. 2-D gels were either stained for 48 h with colloidal Coomassie blue from Novex (San Diego, CA, USA) according to the manufacturer©s instructions, with a prefixation step in 5% phosphoric acid, 40% methanol for 12 h, or with silver according to the method of Blum et al. [37] with slight modifications. Briefly, the gels were fixed overnight in 40% ethanol, 10% acetic acid in water. After the gels were washed three times with 30% ethanol in water, they were incubated for exactly 1 min in 0.05% w/v NaS2O3 in water. The gels were then washed twice with water for 20 s each and incubated for 20 min in 0.2% w/v AgNO3 and 0.025% v/v formaldehyde. The gels were washed 3 times for 20 s each in water and developed with 3% w/v Na2CO3 with 0.05% v/v formaldehyde in water until the desired contrast was reached (usually 2±5 min). The reaction was stopped by addition of 1% glycine in water and incubation for 10 min. Alternatively, when gels where not analyzed by MS, silver staining according to the method of Heukeshoven and Dernick [38] was used. Fixation was done with ethanol instead of methanol. Stained gels were scanned using a Sharp JX-330 scanning device in combination with the Labscan software from Amersham Pharmacia Biotech. Digitized 2-D images were evaluated using the Imagemaster 2D and Imagemaster 2D Database software from Amersham Pharmacia Biotech.
2.6 In-gel trypsin digestion The stained protein bands, precisely excised from the acrylamide gel, were cut into small cubes and rinsed several times with water. Then acetonitrile/water (1/1 v/v)
was added to remove the Coomassie dye and left for 15 min. To shrink the gel and to extract residual water, pure acetonitrile was added and left for 15 min. The acetonitrile was removed and 20 mL digestion buffer (50 mM N-methylmorpholine, pH 8.1, for LC-MS and 40 mM ammonium bicarbonate buffer for MALDI-MS) and 0.125 mg trypsin were added. Digestion was at 37oC for 6±15 h. For LC-MS the supernatants was removed and the gel pieces were extracted twice with 20 mL of 50% acetonitrile. The supernatants and extracts were combined and concentrated in a SpeedVac to approximately 5 mL. MALDI sample preparation was achieved by cocrystallization of matrix and ZipTiP C18 purified sample. Briefly, the peptides in the supernatant of the in-gel digestion were adsorbed to a prewashed (50% acetonitrile/ water) and equilibrated (0.1% trifluoroacetic acid/water) ZipTip C18 by repetitive pipetting steps. Following washing of the ZipTip by 0.1% trifluoroacetic acid/water, the sample was eluted from the ZipTip onto the MALDI target with 0.7 mL of matrix (a-cyano-4-hydroxycinnamic acid saturated in 50% acetonitrile/water).
2.7 LC-MS analysis Online LC-MS was done on a Finnigan LCQ quadrupole ion trap mass spectrometer equipped with an electrospray ionization (ESI) ion source and a Rheos 2000 micro-HPLC system. Online LC-separation of the peptides was done on a Vydac C18 reversed-phase column at a flow rate of 4 mL/min and by a 40 min gradient of acetonitrile (10±40%) in 0.05% TFA. The ionized peptides were analyzed in the quadrupole ion trap for peptide mass determination. Selected peptides were isolated in the ion trap in a signal-dependent mode and MS/MS fragment ion spectra were produced by collision-induced dissociation (CID). Full scan MS spectra (m/z 350 to m/z 1700) and MS/MS spectra were collected alternatingly at a scan cycle time of 3 s. The minimum signal for precursor ion selection and CID was set to 2.8 ´ 104 ions. Processing and analysis of MS data from the LCQ ion trap was done using the SEQUEST program [11]. The SEQUEST search mass tolerances were 3 Da for the peptide, 0 Da for the fragment ion and 1.4 Da for the precursor, the group scan was 1 or 2 and the minimum group count 2. The database searched was derived from the M. pneumoniae genome sequence and contained 677 at the beginning and now 688 predicted proteins as annotated by Himmelreich et al. [22] in FASTA format [39]. A second database containing all possible polypeptides was constructed by translating the DNA sequence in all six reading frames into protein sequence and defining a polypeptide by the sequence between two stop codons.
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Figure 3. Standardized spot volume of individual proteins. Most abundant proteins found on preparative colloidal Coomassie blue-stained gels with IPG 3±10 and 12.5% polyacrylamide. Blue, red and yellow bars indicate expression levels from three different gels. ORF numbers are according to Himmelreich et al. [22]. Protein A is most probably a serum component and could not be identified (see Section 3.2). ated by immunizing rabbits with proteins or fusion proteins as described by Proft [40] or with keyhole limpet hemocyanin-coupled synthetic polypeptides as described by Lerner et al. [42]. Figure 4. Structure of the proposed lipoprotein 384. The protein starts with a putative lipoprotein signal sequence. The protein is found in only four fragments as indicated by regions a, b, c, and d. A rabbit antiserum was generated against the synthetic polypeptide KNNNTSDNTQQNQTSSC coupled to keyhole limpet hemocyanin to identify a full-length product. In 1-D Western blots, the antibody recognized only a protein corresponding to the Mr of fragment c.
2.8 MALDI-MS analysis MALDI-mass spectra were recorded in the positive ion mode with delayed extraction and reflector on a Reflex-II time of flight instrument. Ion acceleration voltage was set to 20.0 kV. Mass spectra were obtained by averaging 50± 200 individual laser shots. Calibration of the spectra was done internally by a two-point linear fit, using the autolysis products of trypsin at m/z 842.50 and m/z 2211.10. Database searches were done with the peptide masses against the nonredundant NCBI database using the search program ProFound provided by the Rockefeller University pdtc.rockefeller-edu/pdtcmain/ms/msinfo.html. Mass tolerance for the monoisotopic peptide masses was set to 50 ppm.
2.9 Western blotting and preparation of antisera Western blotting of 1-D and 2-D gels was done as described by Proft and Herrmann [40]. Antisera were gener-
2.10 Nomenclature To avoid any further confusion with the nomenclature of genes, ORFs, and proteins from M. pneumoniae we use the following nomenclature. All ORFs and proteins were named according to the numbers given in the original publication of the genome sequence [22]. On the occasion of the reannotation of the genome sequence of M. pneumoniae the old nomenclature will be replaced by a new one consisting of the prefix ªMPNº and a new number [23]. This new nomenclature will be used together with the old one in Table 3. Additional information is available from the following websites: www.zmbh.de/M_pneumoniae and www.bork.EMBL-Heidelberg.DE/Annot/MP.
3 Results 3.1 2-D gels of complete cell extract The result of the separation of a complete cell extract from M. pneumoniae on a standard 2-D gel with an IPG gradient of 3±10 is shown in Fig. 1. About 450 protein spots could be visualized by silver staining, but only 225 spots with colloidal Coomassie blue. In our hands, the colloidal Coomassie blue stain was 5±10 times more sensitive than ordinary Coomassie blue staining, but still only 1/10 as sensitive as silver staining according to the method of Blum et al. [37]. Only recently it became possible to prepare immobilized pH gradients covering an extremely alkaline pH range [33, 43±45]. Using alkaline pH gradients, we were able to visualize about 60 additional spots
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Table 4. Functional classification of the identified proteins * Biosynthesis of cofactors, prosthetic groups and carrier * Cell envelope Membranes, lipoproteins and porines Surface structures and cytadherence Surface polysaccharides, lipopolysaccharides and antigens * Cellular processes Cell division Cell killing Chaperones Detoxification Protein and peptide secretion * Central intermediary metabolism * Energy metabolism Aerobic Amino acids and amines Anaerobic ATP-proton motive force interconvention Glycolysis Pentose phosphate pathway Pyruvate DHase Sugars * Fatty acid and phospholipid metabolism * Purines, pyrimidines, nucleosides and nucleotides 2¢Deoxyribonucleotide metabolism Nucleotide and nucleoside interconvention Purine ribonucleotide biosynthesis Salvage of nucleosides and nucleotides Sugar-nucleotide biosynthesis and conversions * Pyridine nucleotide metabolism * Regulatory function * Replication DNA replication, restriction, modification, recombination and repair * Transcription * Translation Amino acyl tRNA synthetases and tRNA modification Degradation of proteins, peptides and glycopeptides Protein modification and translation factors Ribosomal proteins: synthesis and modification * Transport and binding proteins ABC transporters PTS transporters Other transport systems * Other categoriesa) Adaptations and atypical conditions Other * No classification so farb) * Hypothetical ORFs derived from repetitive DNA elements
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5/8 21/54 15/42 6/8 0/4 10/20 1/2 0/1 4/7 0/1 5/9 5/6 27/39 3/3 1/5 0/1 4/9 9/10 2/2 4/4 4/4 2/9 15/18 3/3 1/2 3/3 6/8 2/2 1/1 1/8 14/46 14/46 6/13 47/99 10/24 7/8 11/15 19/52 9/44 6/34 3/7 0/3 48/191 2/3 46/188 9/86 0/46
Functional classes according to Himmelreich et al. [22]. The numbers indicate identified and annotated proteins from each category. Updated Tables 3 and 4 according to the genome reannotation by Dandekar et al. [23] will be available on our website. a) Proteins with significant similarities to proteins from other organisms, but without defined functions b) M. pneumoniae-specific proteins without significant similarities to proteins from other organisms
in the basic region from approximately pH 9±12 (Fig. 2). Combining the results from 2-D gel electrophoresis with the IPG gradients from 3±10 and 4±12, approximately 510 individual protein spots could be stained with silver. We consider these protein spots to be the minimal protein set for the proteome analysis of M. pneumoniae.
3.2 Identification of M. pneumoniae proteins The general strategy for constructing a proteome reference map was to identify proteins mainly from colloidal Coomassie blue-stained gels and to transfer this information via landmark proteins to silver-stained gels. To date, proteins from altogether about 350 individual separated protein spots could be successfully analyzed by MS and assigned to 224 genes of M. pneumoniae (Table 3)*. Most of these spots (225) came from 2-D gels with an IPG gradient of pH 3±10 stained with colloidal Coomassie blue. Additional protein spots were excised from 2-D gels with IPG gradients of pH 4±12 and 4±7 (data not shown) prepared from complete cell extracts or enriched subfractions (see Section 3.3) and in a few cases, samples from 1-D SDS-polyacrylamide gels were included. All colloidal Coomassie blue-stained spots could be identified by LCMS or MALDI-MS. However, the success rate with silverstained spots was only between 20 and 40% compared to the success rate of Coomassie-stained spots. The best results with silver-stained gels were obtained when the gels were processed immediately after staining. One intensive Coomassie blue-stained spot that could not be assigned by MS to a protein in the database (marked A in Fig. 1 and 2; pI ~ 5 and Mr ~ 25 000) seems to be a component from the medium, since it did not appear in autoradiograms of 2-D gels of cells grown in the presence of 14 C-labeled amino acids (B. Ueberle, unpublished). Among the most prominent spots (Fig. 1 and 2) were the heat shock proteins DnaK (406, hsp70), GroEL (269, hsp60), the elongation factor Tu (177), and two subunits (a b) of the pyruvate dehydrogenase complex (446 and 447, pdh). The proteins listed in Fig. 3 always gave the strongest signals in pH gradients from 3±10, independent of the staining procedure applied. All these strongly expressed proteins were found as a ªtrain of spotsº consisting of up to seven single spots which were all independently identified. These housekeeping proteins represent about 40% of the Coomassie blue-stainable protein as measured by spot volume. The 677 ORFs originally annotated by Himmelreich et al. [22] were classified into 16 different categories of functions (Table 4). From
* See 6 Addendum, pp. 3778±3780
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the following proteins were confirmed: protein 366, P65 (528), HMW3 (388), DnaK (406), GroEl (269), HMW1 (393), and subunit b (240) of the F0F1-type ATPase (also see Section 2.9).
3.3 Enrichment by fractionation with detergent Triton X-114
Figure 5. Newly identified and hitherto not annotated proteins. The proteins were identified by ESI ion trap-MS using the SEQUEST program and a protein database directly derived from the genome sequence. The identified peptides of these proteins are depicted in red. The corresponding protein starts with the first Met that is underlined. Preliminary results of this study have been included in a publication on the reannotation of the genome sequence of M. pneumoniae [23].
the categories covering essential functions like biosynthesis of cofactors, cellular processes, central intermediary metabolism, energy metabolism, and the synthesis of nucleic acid precursors, more than 50% of the listed proteins were identified. In addition, for 55 of the proteins with unknown function, expression could be verified. The methods used in this study were also effective for identifying four proteins that were excluded in the original genome annotation because of a cut-off point of 100 amino acids for ORFs without significant similarity to other proteins (Fig. 5). Three of them were smaller than 100 amino acids. Protein 460.1 and 346.1 were identified on 2-D gels using 18% polyacrylamide gels for the second dimension and protein 563.1 on a 1-D Tris-Tricine gel. Protein 346.1 (Fig. 5) had already been proposed by Reizer et al. [46] and protein 578.1 was suggested during the reannotation process [23]. The other two new proteins show no significant similarities to other sequences in the database [23]. As an independent control for protein identification by MS, N-terminal sequencing of enriched M. pneumoniae proteins from 1-D gels [41] (Table 3) as well as immunological identification of proteins were applied. By Western blotting of 2-D gels the positions for
After Triton X-114 fractionation of M. pneumoniae, the proteins were separated in pH gradients from 3 to 10. Approximately 250 protein spots could be seen in a silverstained gel (Fig. 6) but only about 150 in a colloidal Coomassie blue-stained gel. We identified 93 proteins by MALDI-MS, 37 of which had not been identified on a 2-D gel before. These proteins belonged to different functional categories and for 40 of them one or more transmembrane regions were predicted with the program TopPred II [47]. For example, we found adhesin P1 (protein 14), the first time as a complete protein (probably without signal peptide). This protein is predicted to have five transmembrane domains. So far, in 2-D gels of complete cell extracts, only fragments of this 176 kDa protein were identified. Other proteins only found after Triton X-114 fractionation were protein 123 with four predicted transmembrane domains and unknown function, seven putative lipoproteins belonging to the category cell envelope (proteins 72, 96, 384, 396, 431, 552, 632), and four transport and binding proteins; three of them were subunits of ABC transporters (protein 407, 424, 638) and protein 101 belongs to the PTS transport system.
3.4 Multiple spots and modifications Many proteins of M. pneumoniae identified on the 2-D gels we represented by multiple spots on several positions of the gel. Generally, there were three different configurations in which those multiple spots could be found on the gel: (i) they appeared at identical Mr with several spots close together in a row (ªtrains of spotsº), (ii) as two or more spots at the same Mr but separated by approximately 0.5 pH units (row of spots), or (iii) at different Mrs and pIs. Examples for each category are shown in Table 1 and 2. For the first two categories all the proteins of a ªtrain of spotsº were identified as the same polypeptide. For the third category the same ORF was identified but the peptides always belonged to either one of several subfragments, matching the experimentally obtained Mr and pI. These multiple spots derived from only one ORF were not restricted to a certain class. Heat shock protein DnaK (406) for example, is one of the most abundant proteins in Mycoplasma protein extracts. It is found on the gel as a long ªtrain of spotsº that fits into the prediction of pI and
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Figure 6. Analytical 2-D gel of the Triton X-114 soluble fraction of M. pneumoniae. Triton X-114 extracted proteins were separated on an 18 cm IPG strip (3±10) and a vertical 12.5% polyacrylamide SDS gel (20 ´ 20 cm). The gel was stained with silver according to the method of Blum et al. [37]. The assignment of the proteins to the ORF numbers [22] was based on MALDI-MS results from colloidal Coomassie blue- and silver-stained gels.
Electrophoresis 2000, 21, 3765±3780
2-D proteome map of M. pneumoniae
3775
Figure 7. Mock 2-D gel of M. pneumoniae. Molecular weights and pIs of the 677 predicted proteins [22] were calculated using the GCG program. The pIs ranged from 3.58 to 12.75 and more than 200 proteins had pIs between 10 and 12.75. Blue circles indicate proteins identified by the combination of 2-D electrophoresis and MS (proteins found only as fragments or no agreement with the prediction are not shown), yellow circles represent predicted proteins that have not yet been found on 2-D gels. Mr (Fig. 1 and Table 2). Each individual spot of the ªtrain of spotsº was identified as DnaK, but we can not explain the reasons for this spot formation with the data from the mass spectrometric analysis, e.g., by comparing molecular masses of tryptic peptides and their fragmentation patterns from proteins of different spots. This is also true for the other proteins appearing in several spots. In addition, DnaK was found in two spots with lower Mr, probably representing stable fragments (Table 2). The N-terminal sequence of the one with the Mr of about 25 000 was determined and starts at amino acid position 396 of the DnaK protein with N as the first amino acid [41]. Most of the proteins we identified as subfragments only were putative lipoproteins. For example, protein 384 appears on the gel in ten different spots corresponding to four fragments as discussed in Section 3.2 (Table 1, Fig. 4). Confirming published data from SDS-PAGE analyses we mapped the three proteins P65 (528) [48], HMW1 (393) [49], and HMW3 (388) [50] at positions in the 2-D gel that indicated a greater Mr than predicted from the DNA sequence-derived amino acid composition.
4 Discussion A proteome analysis pursues, as the name indicates, the aim to identify all the proteins of a cell that are synthesized under defined growth conditions and to display the results in a way that facilitates comparative proteome analyses of the same organism grown under modified conditions. Presently, the best way to show the results of the proteome analysis is the 2-D protein map. This technology improved considerably by extending the pH range of the separation in the first dimension according to charge [34] and by introducing new detergents for the solubilization of mainly hydrophobic proteins [51]. Nevertheless, the results of our proteome analysis showed clearly
that even for a simple cell like M. pneumoniae with less than 700 ORFs, 2-D gel electrophoresis was still not sufficient to visualize and analyze a complete proteome at one time. The limiting factors are the solubilization of membrane proteins, the still poor resolution of basic proteins, the identification of low copy number proteins, and, to a lesser extend, the analysis of proteins with an Mr below 10 000 or above 150 000. For instance, with the PRED-TMR2 program [52], and program TopPred II [47], 55 and 58 ORFs, respectively, with more than four transmembrane segments, were predicted for M. pneumoniae. With the possible exception of the P1 protein (14) none of these membrane proteins was on our list of identified proteins, indicating a strong bias against them although we enriched for membrane proteins by Triton X-114 fractionation. Our experience with the recovery of basic proteins was also not satisfactory. On 2-D gels with a 4±12 IPG, approximately 60 protein spots were visible in the pH range 9±12 after staining with silver. Only 16 proteins with a pI greater than 10 could be identified, whereas the annotation of the genome sequence predicts 244 ORFs. Note that most of the basic proteins including the ribosomal proteins with their low molecular weight were identified by 1-D SDS PAGE with different buffer systems. Even if not all basic proteins were expressed under our growth conditions, the analytical difficulties with basic proteins are obvious, as can be seen by comparing our experimental results with the predicted data (Fig. 7). Low copy number proteins are also a serious general problem, since the concentration of different proteins varies by orders of magnitudes. This problem could not be solved by simply increasing the amount of protein loaded onto the gel because this produced gels of lower quality
3776
J. T. Regula et al.
with overloaded spots forming long horizontal streaks. The best solution to this problem seems enrichment of low copy proteins by cell fractionations [21]; alternatively one could try to remove high copy proteins from the protein extracts before 2-D gel electrophoresis, e.g., by antibodies. Western blotting, as a very sensitive method, would also be a possibility to identify low copy proteins in total cell extracts, but this requires many specific antibodies and is presently only a realistic approach for a limited number of proteins. Western blotting is a good method to show heterogenicity in a single protein ([53, 54]; protein 366; Regula, unpublished). The high-molecular protein HMW2 (527, Mr 216 000), which has the tendency to form multimeres [55], could be identified only with 1-D polyacrylamide gels. We assume that this large protein does not migrate into the IPG strip during the separation in the first dimension and, therefore, only a small amount of this protein moves to its position in the 2-D gel. Both mass spectrometric protein identification methods used in this study were based on tryptic in-gel digestion of stained proteins and subsequent matching of the resulting peptides to the database. Peptide analysis was done by LC-MS or MALDI-MS. These methods exhibit very different features concerning speed of operation and mass accuracy. The duration of one LC-MS run was approximately 1 h, whereas in the same time ten MALDI analyses could be carried out. Protein identification with good statistical significance and by peptide mass fingerprint alone was only possible when a mass accuracy better than 50 ppm was obtained. Due to the internal calibration procedure the MALDI instrument routinely achieves this accuracy. To obtain good statistical evidence to identify a protein, a sequence coverage above 25% was required when using MALDI-MS. However, peptide mass determination with the LCQ ion trap when operated in the LC-MS mode was in the range of 500 ppm. Therefore, the additional information from the MS/MS fragmentation pattern, which was automatically produced by this instrument, was essential for confident identification. Our LC-MS analysis setup proved to be reliable. Even though this method is slower than MALDI-MS, the additionally obtained partial sequence information leads to highly confident protein identification. We could get away with even a lower peptide coverage than 10% if at least two peptides with partial sequences and high score values were available. To ensure that the entire nucleotide sequence of the genome was used for peptide matching without any restrictions, such as cut-off points of 100 amino acids, all six ORFs of the complete genome of M. pneumoniae were translated to amino acid sequence. A polypeptide was defined as the sequence between two stop codons and used as database entry. This database
Electrophoresis 2000, 21, 3765±3780 and the high statistical significance of the protein and peptide matches allowed us to identify four not yet annotated small proteins (Fig. 5) and to correct the N-termini from two proposed ORFs [23]. Generally, we considered an assignment only as proven when Mr and pI were in the predicted range. When we searched in a general protein database, with our mass spectrometric data we found that in competition with all possible protein data the highest scores were from M. pneumoniae. This gave additional support to our assignments. Except for the problems described in Section 3, the experimentally derived values for pIs agreed well with the predicted ones. We did not observe strong deviations (> 2 pH units) as it was recently reported for a number of proteins of Mycoplasma genitalium [20], in particular ribosomal proteins. A deviation of 2 pH units seems high; note, however, that even the programs available for calculation of PIs give different values. Many identified proteins were found in multiple spots on the gel. We do not think that the multiple spots are artifacts introduced during sample preparation because the very similar patterns with multiple spots were produced in different laboratories independently (A. Görg, P. Jungblut and U. Völcker, personal communication). In the case of train of spots and rows of spots of the same molecular weight, their appearance can be seen as a hint for post-translational modifications. The proteins DnaK (406) and EFTu (177) [56] were reported to be phosphorylated in E. coli, but the verification for M. pneumoniae by MS and the correlation to spot formation is still missing. Another explanation, at least for some of the spots, might be the binding of lipids to a protein, altering its pI, as was recently reported for the E. coli heat shock protein GroEL [57]. Most of the proteins found as fragments only belonged to the family of hypothetical lipoproteins. The best studied example is protein 384 (Fig. 4, Table 1). Four different nonoverlapping fragments of this protein were identified, raising the question whether this gene is translated as a larger precursor and then cleaved post-translationally, or whether these fragments are artifacts of our sample preparation. Since an antibody raised against a peptide from the 384c region recognized only this fragment and no additional larger precursor protein in a 1-D Western blot could be found, we exclude artificial protein disruption during the 2-D sample preparation procedure as a possible cause. In contrast to protein 384 and the other proteins listed in Table 1 subunit b (240) of the F0F1 ATPase protein is an experimentally confirmed lipoprotein [58] that could be identified as a complete protein in the Triton X114 soluble fraction after 2-D gel electrophoresis. A possible explanation might be the proposed membrane topology of protein 240, which does not predict any extended
Electrophoresis 2000, 21, 3765±3780 outside-exposed region for this lipoprotein but for all the others. The surface-exposed region might be a target for M. pneumoniae-specific proteases sticking on the surface of the bacterium or proteases from the medium which attack any exposed protein with an appropriate cleavage site. In conclusion, as the number of sequenced genomes is increasing rapidly, the need for further functional analyses becomes more and more evident. The transcriptome and proteome analysis, which show experimentally which genes are expressed, are the next steps towards the functional description of a complete cell. It has been shown that M. pneumoniae is an excellent model organism to explore the limits of the present proteomic technology. It is important to know these limits, because otherwise a realistic assessment of data from proteome research is impossible. Our aim to visualize and characterize the proteome of this minimal cell in one step, i.e., in one 2-D gel, is still to be expected, but might only be possible in the future by improving the technology. A combined quantitative analysis of transcriptome and proteome, to elucidate the mechanisms regulating gene expression at the cellular level, will be the focus of future research. Of particular interest will be the identification and functional analysis of M. pneumoniae genes showing induction or repression in response to the interaction of M. pneumoniae with its host cell. We thank E. Pirkl and A. Bosserhoff for skillful technical assistence, J. Weiss and co-workers the preparation of antibodies, and P. Jungblut and U. Völcker for running 2-D gels of M. pneumoniae protein extracts. The research was supporeted by grants from the Deutsche Forschungsgemeinschaft (He 780/10-1, 13-1, SFB 352), the Fonds der Chemischen Industrie, and by a short-term fellowship to R. F. from the ªH. and C. Schaller Stiftungº. Received June 22, 2000
5 References [1] Velculescu, V. E., Zhang, L., Vogelstein, B., Kinzler, K. W., Science 1995, 270, 484±487. [2] Schena, M., Shalon, D., Davis, R. W., Brown, P. O., Science 1995, 270, 467±470. [3] Wilkins, M. R., in: Wilkins, M. R., Williams, K. L., Appel, R. D., Hochstrasser, D. F. (Eds.), Proteome Research: New Frontiers in Functional Genomics, Springer, Heidelberg 1997, pp. 1±11. [4] Gygi, S. P., Rochon, Y., Franza, B. R., Aebersold, R., Mol. Cell Biol. 1999, 19, 1720±1730. [5] Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., Mann, M., Proc. Natl. Acad. Sci. USA 1996, 93, 14440±14445. [6] Cottrell, J. S., Pept. Res. 1994, 7, 115±124.
2-D proteome map of M. pneumoniae
3777
[7] James, P., Quadroni, M., Carafoli, E., Gonnet, G., Prot. Sci. 1994, 3, 1347±1350. [8] Mann, M., Wilm, M., Trends Biochem. Sci. 1995, 20, 219±224. [9] Biemann, K., Biomed. Environ. Mass Spectrom. 1988, 16, 99±111. [10] Katta, V., Chowdhury, S. K., Chait, B. T., Anal. Chem. 1991, 63, 174±178. [11] Eng, J. K., McCormack, A. L., Yates III, J. R., J. Am. Soc. Mass Spectrom. 1994, 5, 976±989. [12] Washburn, M. P., Yates III, J. R., Curr. Opin. Microbiol. 2000, 3, 292±297. [13] Bernhardt, J., Völker, U., Völker, A., Antelmann, H., Schmid, R., Mach, H., Hecker, M., Microbiology 1997, 143, 999±1017. [14] Dainese-Hatt, P., Fischer, H. M., Hennecke, H., James, P., Electrophoresis 1999, 20, 3514±3520. [15] Jungblut, P. R., Bumann, D., Haas, G., Zimny-Arndt, U., Holland, P., Lamer, S., Siejak, F., Aebischer, A., Meyer, T. F., Mol. Microbiol. 2000, 36, 710±725. [16] VanBogelen, R. A., Schiller, E. E., Thomas, J. D., Neidhardt, F. C., Electrophoresis 1999, 20, 2149±2159. [17] Cash, P., Electrophroesis 2000, 21, 1187±1201. [18] Blackstock, W., Mann, M. (Eds.), Proteomics: A Trends Guide, Vol. 8, Elsevier, London 2000. [19] Wasinger, V. C., Cordwell, S. J., Cerpa-Poljak, A., Yan, J. X., Gooley, A. A., Wilkins, M. R., Duncan, M. W., Harris, R., Williams, K. L., Humphery-Smith, I., Electrophoresis 1995, 16, 1090±1094. [20] Wasinger, V. C., Pollack, J. D., Humphery-Smith, I., Eur. J. Biochem. 2000, 267, 1571±1582. [21] Langen, H., Takacs, B., Evers, S., Berndt, P., Lahm, H. W., Wipf, B., Gray, C., Fountoulakis, M., Electrophoresis 2000, 21, 411±429. [22] Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C., Herrmann, R., Nucleic Acids Res. 1996, 24, 4420±4449. [23] Dandekar, T., Huynen, M., Regula, J. T., Ueberle, B., Andrade, M., Doerks, T., Sanchez, L., Snel, B., Suyama, M., Yuan, Y. P., Herrmann, R., Bork, P., Nucleic Acids Res. 2000, 28, 3278±3288. [24] Channok, R. M., N. Engl. J. Med. 1965, 273, 1199±1206; 1257±1264. [25] Baseman, J. B., Lange, M., Criscimagna, N. L., Giron, J. A., Thomas, C. A., Microb. Pathog. 1995, 19, 105±116. [26] Maniloff, J., Annu. Rev. Microbiol. 1983, 37, 477±499. [27] Pollack, J. D., Williams, M. V., McElhaney, R. N., Crit. Rev. Microbiol. 1997, 23, 269±354. [28] Hayflick, L., Tex. Rep. Biol. Med. 1965, 23, Suppl 1, 285± 303. [29] Görg, A., Postel, W., Günther, S., Electrophoresis 1988, 9, 531±546. [30] Rabilloud, T., Adessi, C., Giraudel, A., Lunardi, J., Electrophoresis 1997, 18, 307±316. [31] Bordier, C., J. Biol. Chem. 1981, 256, 1604±1607. [32] Riethman, H. C., Boyer, M. J., Wise, K. S., Infect. Immun. 1987, 55, 1094±1100. [33] Görg, A., Obermaier, C., Boguth, G., Csordas, A., Diaz, J. J., Madjar, J. J., Electrophoresis 1997, 18, 328±337. [34] Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., Weiss, W., Electrophoresis 2000, 21, 1037±1053. [35] Görg, A., Weiss, W., Methods Mol. Biol. 1999, 112, 189±195.
X
[50]
[51] [52]
[53]
[54]
Electrophoresis 2000, 21, 3765±3780
X
[55]
X
[56]
X X
[57]
X
J. T. Regula et al.
X
3778
X
N-utilization substance protein A homolog 130K protein (orf6; P1 operon) Adhesin P1 (orf5; P1 operon) sn-glycerol-3-phosphate transport system permease protein (ugpC) Putative lipoprotein, MGf)186 homolog MG207 homolog Heat shock protein grpE Ribosomal protein L35 ± Phenylalanyl-tRNA synthetase alpha-subunit (pheS) Putative lipoprotein, MG067 homolog Transketolase 1 (tklB) 1-phosphofructokinase (fruK) Phosphoribosylpyrophosphate synthetase (prs) Transcription antitermination factor (nusG) Phosphomannomutase (cpsG) Cytidine deaminase (cdd) Deoxyribose-phosphate aldolase (deoC) Purine-nucleoside phosphorylase (deoD) Signal recognition particle protein (ffh) O-sialoglycoprotein endopeptidase (gcp) Putative lipoprotein, MG045 homolog Phosphocarrier protein HPr (ptsH) Putative lipoprotein, MG040 homolog Aerobic glycerol-3-phosphate dehydrogenase (glpD) Glycerol kinase (glpK) Histidyl-tRNA synthetase (hisS) Thymidine kinase (tdk) Uracil phosphoribosyltransferase (upp) MG028 homolog Elongation factor P (efp) Fructose-bisphosphate aldolase (tsr) DNA-directed RNA polymerase delta subunit (rpoE) Methionyl-tRNA synthetase (metS) Proline iminopeptidase (pip) 5,10-methylene-tetrahydrofolate dehydrogenase (mtd1) DNA polymerase III subunit delta¢ (holB) Thymidylate kinase Seryl-tRNA synthetase (serS) DNA gyrase subunit A (gyrA) DNA gyrase subunit B (gyrB) DNA polymerase III beta subunit (dnaN) Protein (soj) homolog ± ± Glutamyl-tRNA synthetase (gltX) L-lactate dehydrogenase (ldh) Hypothetical protein (HI0671) homolog Osmotically inducible protein (osmC)
[58]
E07_orf540o E07_orf1218 E07_orf1627 E07_orf586 E07_orf301 C09_orf159 C09_orf217 C09_orf59 C09_orf165 C09_orf341 R02_orf533 R02_orf648 R02_orf300 D09_orf388 D09_orf320 D09_orf554 D09_orf133 D09_orf224 D09_orf238 D09_orf450 D09_orf319 D09_orf485 D09_orf88 D09_orf657 D09_orf384 D09_orf508 B01_orf414o B01_orf191 B01_orf178 B01_orf203 B01_orf190 B01_orf288 B01_orf146 B01_orf512 B01_orf309 D12_orf269 D12_orf253 D12_orf210 D12_orf420 K05_orf839o K05_orf650 K05_orf380 K05_orf270 K05_orf250 K05_orf1882 K05_orf484 K05_orf312 K05_orf169 K05_orf140
[59]
MPN154 MPN142 MPN141 MPN134 MPN133 MPN126 MPN120 MPN116 MPN109 MPN105 MPN083 MPN082 MPN079 MPN073 MPN067 MPN066 MPN065 MPN063 MPN062 MPN061 MPN059 MPN058 MPN053 MPN052 MPN051 MPN050 MPN045 MPN044 MPN033 MPN031 MPN029 MPN025 MPN024 MPN023 MPN022 MPN017 MPN007 MPN006 MPN005 MPN004 MPN003 MPN001 MPN688 MPN687 MPN684 MPN678 MPN674 MPN673 MPN668
Annotationa)
Stevens, M. K., Krause, D. C., J. Bacteriol. 1992, 174, 4265±4274. Rabilloud, T., Anal. Chem. 2000, 72, 48A±55A. Pasquier, C., Promponas, V. J., Palaios, G. A., Hamodrakas, J. S., Hamodrakas, S. J., Prot. Engineer. 1999, 12, 381±385. Zeindl-Eberhart, E., Jungblut, P. R., Rabes, H. M., Electrophoresis 1997, 18, 799±801. Janke, C., Holzer, M., Klose, J., Arendt, T., FEBS Lett. 1996, 379, 222±226. Stevens, M. K., Krause, D. C., Infect Immun. 1990, 58, 3430±3433. Lippmann, C., Lindschau, C., Vijgenboom, E., Schroder, W., Bosch, L., Erdmann, V. A., J. Biol. Chem. 1993, 268, 601±607. De Bruyn, J., Soetaert, K., Buyssens, P., Calonne, I., De Coene, J. L., Gallet, X., Brasseur, R., Wattiez, R., Falmagne, P., Montrozier, H., Laneelle, M. A., Daffe, M., Microbiology 2000, 146, 1513±1524. Pyrowolakis, G., Hofmann, D., Herrmann, R., J. Biol. Chem. 1998, 273, 24792±24796. Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A., Fleischmann, R. D., Bult, C. J., Kerlarage, A. R., Sutton, G., Kelley, J. M., et al., Science 1995, 270, 397±403.
1 13 14 21 22 29 35 39c) 46 50 72 73 76 82 87 88 89 91 92 93 95 96 101 102 103 104 109 110 121 123 125 129 130 131 132 137 147 148 149 150 151 153 154 155 158 164 168 169 174
N-term. Sequ.e)
[36] Schägger, H., von Jagow, G., Anal. Biochem. 1987, 166, 368±379. [37] Blum, H., Beiers, H., Gross, H. J., Electrophoresis 1987, 8, 93±99. [38] Heukeshoven, J., Dernick, R., Electrophoresis 1988, 9, 28±32. [39] Pearson, W. R., Lipman, D. J., Proc. Natl. Acad. Sci. USA 1988, 85, 2444±2448. [40] Proft, T., Herrmann, R., Mol. Microbiol. 1994, 13, 337±348. [41] Proft, T., PhD thesis, ZMBH, Universität Heidelberg 1995. [42] Lerner, R. A., Sutcliffe, J. G., Shinnick, T. M., Cell 1981, 23, 309±310. [43] Fountoulakis, M., Takµcs, B., Langen, H., Electrophoresis 1998, 19, 761±766. [44] Görg, A., Boguth, G., Obermaier, C., Weiss, W., Electrophoresis 1998, 19, 1516±1519. [45] Görg, A., Methods Mol. Biol. 1999, 112, 197±209. [46] Reizer, J., Paulsen, I. T., Reizer, A., Titgemeyer, F., Saier Jr., M. H., Microb. Comp. Genomics 1996, 1, 151±164. [47] Claros, M. G., von Heijne, G., Comput. Appl. Biosci. 1994, 10, 685±686. [48] Proft, T., Hilbert, H., Layh Schmitt, G., Herrmann, R., J. Bacteriol. 1995, 177, 3370±3378. [49] Stevens, M. K., Krause, D. C., J. Bacteriol. 1991, 173, 1041±1050.
Numbera) MPNORFa) Numberb)
6 Addendum
Table 3. Identified proteins
Number
MPNORF Numberb)
K05_orf291 K05_orf394 K05_orf151 E09_orf143V E30_orf375 E30_orf184 C12_orf235 C12_orf298 C12_orf244 C12_orf508 C12_orf141 C12_orf681 C12_orf157 C12_orf456 D02_orf105 D02_orf207 D02_orf518 D02_orf475 D02_orf569 D02_orf152 D02_orf353V D02_orf406 D02_orf116 D02_orf543 D02_orf445 H03_orf433 H03_orf248 H03_orf438 H03_orf191 H03_orf193o G12_orf57 G12_orf122 G12_orf161 G12_orf390 G12_orf715 G12_orf136 G12_orf184 G12_orf166b G12_orf166a G12_orf1391o P02_orf793 P02_orf163 P02_orf242 P02_orf159 P02_orf218 P02_orf474 P02_orf1300 P01_orf341 P01_orf197
Table 3. continued N-term. Sequ.e) X X
X X
X
X X
X X
X
Annotation
a)
UDP-glucose pyrophosphorylase (gtaB) Elongation factor TU (tuf) PilB homolog (fragment) PTS system mannitol-specific component IIA (EIIA-MTL)(mtlF) MG438 homolog Ribosome releasing factor (frr) Uridylate kinase (pyrH) Elongation factor Ts (tsf) Triosephosphate isomerase (tim) Phosphoglycerate mutase (pgm) MG427 homolog DNA polymerase III subunit gamma and tau (dnaX) Peptide methionine sulfoxide reductase (pmsR) Enolase (eno) ATP synthase C chain (atpE) ATP synthase B chain (atpF) ATP synthase alpha chain (atpA) ATP synthase beta chain (atpD) MG397 homolog Galactose-6-phosphate isomerase subunit (lacA) MG068 homolog Serine hydroxymethyltransferase (glyA) Heat shock protein GroES Heat shock protein GroEL Nonspecified aminopeptidase GTP-binding protein (obg) Probable NH(3)-dependent NAD(+) synthetase (outB) Arginine deiminase (arcA) Glucose inhibited division protein (gidB) MG377 homolog (put. zinc protease) Ribosomal protein L32 Ribosomal protein L7/L12 (`A' type) Ribosomal protein L10 Acetate kinase (ackA) ATP-dependent protease binding subunit (clpB) MG354 homolog Inorganic pyrophosphatase (ppa) Hypothetical protein (ygl3) homolog MG342 homolog RNA polymerase beta subunit (rpoB) Putative lipoprotein, MG260 homolog ± L-ribulose-5-phosphate 4-epimerase (araD) Hypothetical phosphotransferase protein (yjfU) homolog Hypothetical protein (yjfV) homolog ± Putative lipoprotein, MG338 homolog Hypothetical protein (yibD) homolog Hypothetical protein (HI1366) homolog
Numbera) MPNORFa) Numberb) 362 366 368 370 379 384 386c) 388 393 395 396 404 406 407 410 411 412 413 414 415 417 418 420 421 424 431 433c) 437 438 439 444 445 446 447 448 449 450 451 452 454d) 455 457 458c) 459 461 465 476 477c) 483
MPN478 MPN474 MPN472 MPN470 MPN461 MPN456 MPN454 MPN452 MPN447 MPN445 MPN444 MPN436 MPN434 MPN433 MPN430 MPN429 MPN428 MPN427 MPN426 MPN425 MPN423 MPN422 MPN420 MPN418 MPN415 MPN408 MPN406 MPN402 MPN401 MPN400 MPN395 MPN394 MPN393 MPN392 MPN391 MPN390 MPN389 MPN387 MPN386 MPN384 MPN383 MPN381 MPN380 MPN379 MPN376 MPN372 MPN361 MPN360 MPN354
P01_orf235 P01_orf1033 P01_orf293 P01_orf354 H08_orf231 H09_orf1005 H08_orf193 H08_orf672 H08_orf1018 H08_orf289 H08_orf1325 A05_orf1244 A05_orf595 A05_orf270L A05_orf337 A05_orf409 A05_orf320 A05_orf290 A05_orf982 A05_orf348 A05_orf129 A05_orf370 A05_orf241a A05_orf900 A05L380V F11_orf760 F11_orf84 F11_orf483 F11_orf160 F11_orf582 F11_orf133 F11_orf479 F11_orf358a F11_orf327 F11_orf402 F11_orf457 F11_orf339 F11_orf358b F11_orf229 F11_orf793o A19_orf282 A19_orf292 A19_orf277 A19_orf291 A19_orf1140 A19_orf591 H91_orf359V H91_orf97 H91_orf449
N-term. Sequ.e)
X X X
X
X
X X X X X X
X X
X
Annotationa) Hypothetical protein (HI0315) homolog MG328 homolog Homolog (degV) protein X-Pro dipeptidase (pepX) Hypothetical protein (yzaC) homolog Putative lipoprotein, MG321 homolog MG319 homolog Cytadherence accessory protein (hmw3) Cytadherence accessory protein (hmw1) Triacylglycerol lipase (lip) 3 Putative lipoprotein, MG309 homolog Putative lipoprotein, MG307 homolog Heat shock protein DnaK Abc transport ATP-binding protein (cbiO) Glyceraldehyde-3-phosphate dehydrogenase(gap) Phosphoglcerate kinase (pgk) Phosphotransacetylase (pta) Hypothetical protein (yidA) homolog P115 Protein homolog SMC family Cell division protein (ftsY) MG296 homolog Hypothetical protein (HI0174) Glycerophosphoryl diester phosphodiesterase (glpQ) Alanyl-tRNA synthetase (alaS) High affinity transport system protein P37 Putative lipoprotein, MG260 homolog Acyl carrier protein Putative prolyl-tRNA synthetase (proS) Transcription elongation factor (greA) MG281 homolog Adenine phosphoribosyltransferase (apt) NADH oxidase (nox) Pyruvate dehydrogenase E1-alpha subunit (pdhA) Pyruvate dehydrogenase E1-beta subunit (pdhB) Dihydrolipoamide acetyltransferase component (E2) (pdhC) Dihydrolipoamide dehydrogenase (pdhD) Lipoate protein ligase (lplA) MG269 homolog Hypothetical protein (yaaF) Leucyl-tRNA synthetase (leuS) Hypothetical protein (yidA) Hypothetical protein (yidA) Formamidopyrimidine-DNA glycosylase (fpg) DNA polymerase I (polA,5¢±3¢ exonuclease) homolog ± ± Peptide chain release factor 1 (prf1) Ribosomal protein L31 Glycyl-tRNA synthetase (grs1)
3779
MPN667 MPN665 MPN662 MPN653 MPN638 MPN636 MPN632 MPN631 MPN629 MPN628 MPN625 MPN618 MPN607 MPN606 MPN603 MPN602 MPN600 MPN598 MPN596 MPN595 MPN591 MPN576 MPN574 MPN573 MPN572 MPN563 MPN562 MPN560 MPN558 MPN555 MPN540 MPN539 MPN538 MPN533 MPN531 MPN530 MPN528 MPN521 MPN517 MPN516 MPN506 MPN499 MPN498 MPN494 MPN493 MPN491 MPN489 MPN483 MPN479
a)
2-D proteome map of M. pneumoniae
175 177 180 189 204 206 210 211 213 214 217 224 235 236 239c) 240 242 244 246 247 251 266 268 269 270 279 280 282 284 287 302c) 303 304 309 311 312 314 321 325 326 336 343 344 347 348 350 352 358 361
a)
Electrophoresis 2000, 21, 3765±3780
Table 3. continued
Number
MPNORF Numberb)
H91_orf620 H91_orf281 F10_orf795 F10_orf444 F10_orf294 F10_orf286 F10_orf104 F10_orf100a F10_orf100b F10_orf721 F10_orf153 F10_orf339 F10_orf160 F10_orf328 F10_orf380 F10_orf141b F10_orf357 F10_orf1818 F10_orf405 H10_orf508 H10_orf328 H10_orf208 H10_orf149 H10_orf220L H10_orf309 H10_orf196 A65_orf787o A65_orf794 A65_orf569 A65_orf581 A65_orf489 A65_orf100 A65_orf144 A65_orf259 A65_orf145 A65_orf102 A65_orf711 A65_orf572 A65_orf338 A65_orf223 A65_orf251b K04_orf215L K04_orf239 K04_orf216 K04_orf315 G07_orf478o G07_orf478V G07_orf454 G07_orf473
Table 3. continued N-term. Sequ.e) X
X X
X
X
X
X
Annotation
a)
DNA primase (dnaG) MG246 homolog ATP-dependent protease (Ion) Trigger factor (tig) MG237 homolog Endonuclease IV (nfo) Ribosomal protein L27 Hypothetical protein (ysxB) Ribosomal protein L21 Ribonucleoside-diphosphate reductase (nrdE) MG230 homolog Ribonucleotide reductase 2 (nrdF) Dihydrofolate reductase (dhfr) Thymidylate synthase (thyA) Cell division protein (ftsZ) Hypothetical protein (yabB) homolog ± Cytadherence accessory protein (hmw2) Protein P65 Pyruvate kinase (pyk) 6-phosphofructokinase (pfk) Hypothetical protein (P35155) homolog MG211 homolog ± Hypothetical protein (yceC) homolog MG208 homolog Putative lipoprotein, MG260 homolog Putative lipoprotein, MG260 homolog MG139 homolog GTP-binding membrane protein (lepA) Lysyl-tRNA synthetase (lysS) Hypothetical protein (yaaK) homolog Hypothetical protein (hit1) homolog Hypothetical protein (HI0072) homolog Hypothetical protein (ygl1) homolog Thioredoxin (trx) DNA topoisomerase I (topA) Hypothetical ABC transporter (yjcW) homolog UDP-glucose 4-epimerase (galE) MG117 homolog MG116 homolog D-ribulose-5-phosphate 3 epimerase (cfxE) 5¢guanylate kinase (gmk) Polypeptide deformylase (def) Thioredoxin reductase (trxB) Protein (pet112) homolog Amidase homolog (S47454) Putative lipoprotein, MG095 homolog Replicative DNA helicase (dnaC)
Numbera) MPNORFa) Numberb) 601 603 604 605 612 619 624 632 633 638 639 641 645c) 647 652 654c) 655 657 661 663c) 664c) 667 671 677
MPN231 MPN229 MPN228 MPN227 MPN220 MPN213 MPN208 MPN200 MPN199 MPN194 MPN193 MPN191 MPN187 MPN185 MPN180 MPN178 MPN177 MPN175 MPN171 MPN169 MPN168 MPN165 MPN161 MPN155
G07_orf149 G07_orf166 G07_orf215 G07_orf688 G07_orf226 G07_orf1030 G07_orf294 GT9_orf798 GT9_orf760 GT9_orf303 GT9_orf274 GT9_orf327 GT9_orf78 GT9_orf215 GT9_orf184 GT9_orf61 GT9_orf180b GT9_orf122 VXpSPT7_orf273 VXpSPT7_orf87 VXpSPT7_orf287a VXpSPT7_orf287b VXpSPT7_orf445 VXpSPT7_orf617
N-term. Sequ.e)
X
X
Annotationa) Ribosomal protein L9 Single-stranded DNA binding protein (ssb) Ribosomal protein S6 Elongation factor G (fus) Ribosomal protein L1 Protein P100 Ribosomal protein S2 Putative lipoprotein, MG260 homolog Putative lipoprotein, MG185 homolog Histidine transport ATP-binding protein (hisP) Sulfate transport ATP-binding protein (cysA) RNA polymerase alpha core subunit (rpoA) Iniation factor 1 (infA) Adenylate kinase (adk) Ribosomal protein L6 Ribosomal protein S14 Ribosomal protein L5 Ribosomal protein L14 Ribosomal protein S3 Ribosomal protein S19 Ribosomal protein L2 Ribosomal protein L3 MG148 homolog protein synthesis initiation factor 2 (infB)
new ORFs: 346.1 MPN495 460.1 MPN377 563.1c) MPN272 578.1 MPN254 a) According to Himmelreich et al. [22] b) According to the reannotation by Dandekar et al. [23] c) Protein identified on 1-D gel only d) Protein identified by N-terminal sequencing only e) N-terminal sequences provided by Proft, Bosserhoff and Frank ([41] and unpublished results) f) MG, Mycoplasma genitalium ORF number [59]
Electrophoresis 2000, 21, 3765±3780
MPN353 MPN349 MPN332 MPN331 MPN330 MPN328 MPN327 MPN326 MPN325 MPN324 MPN323 MPN322 MPN321 MPN320 MPN317 MPN314 MPN311 MPN310 MPN309 MPN303 MPN302 MPN301 MPN297 MPN295 MPN292 MPN291 MPN288 MPN284 MPN280 MPN279 MPN277 MPN275 MPN273 MPN267 MPN266 MPN263 MPN261 MPN258 MPN257 MPN256 MPN255 MPN251 MPN246 MPN245 MPN240 MPN238 MPN237 MPN233 MPN232
a)
J. T. Regula et al.
484 488 505 506 507 509 510c) 511 512 513 514 515 516 517 520 523 526 527 528 534 535 536 540 541 544 545 548 552 556 557 559 561 563 567 568 571 573 576 577 578 579 582 587 588 592 594 595 599 600
a)
3780
Table 3. continued
