Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins

Mark P. Molloy1

Nikhil D. Phadke2

Janine R. Maddock2

Philip C. Andrews1

1Department of BiologicalChemistry, University ofMichigan Medical School,Ann Arbor, MI, USA

2Department of Biology,University of Michigan,Ann Arbor, MI, USA

Two-dimensional electrophoresis and peptide massfingerprinting of bacterial outer membrane proteins

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional(2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis(2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs,followed by improved solubilization conditions for array by 2-DE using immobilizedpH gradients. In this report, we expanded our study, examining protein componentsfrom the outer membranes of two enteric bacteria, Salmonella typhimurium and Kleb-siella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated,free-living �-proteobacteria Caulobacter crescentus. Patterns of OMPs expressionappeared remarkably conserved between members of the Enterobacteriaceae, whileC. crescentuswas unique, displaying a greater number of clusters of higher-molecular-weight proteins (�80 kDa). Peptide mass fingerprinting (PMF) was used for proteinidentification, and despite matching across-species boundaries, proved useful forfirst-pass protein assignment of enteric OMPs. In contrast, identification ofC. crescen-tusOMPs was successful only when searching against its recently completed genome.For all three microorganisms examined, the majority of proteins identified on the 2-Dgel appear localized to the outer membrane, a result consistent with our previous find-ing in Escherichia coli. In addition, we discuss some of the benefits and limitations ofPMF in cross-species searching.

Keywords: Mass spectrometry / Membrane proteins / Two-dimensional gel electrophoresis /Bacteria / Peptide mass fingerprinting EL 4384

1 Introduction

Gram-negative bacteria possess an outer membrane(OM) external to the cytoplasmic membrane that forms abarrier between the cell and its surrounds. The OM is richin transmembrane proteins, whose roles include trans-porting nutrients to and from the cell [1], conjugation [2]and controlling cell morphology [3]. Unlike �-helical mem-brane proteins (as found in the cytoplasmic membrane)that utilize stretches of aliphatic amino acids to cross thehydrophobic membrane layer, outer membrane proteins(OMPs) employ a repeating chain of hydrophobic-hydro-philic amino acids to cross the bilayer. The resultant ter-tiary structure forms an antiparallel �-barrel (reviewedin [4]). Hydrophobic amino acid side chains interface withthe lipid bilayer, while hydrophilic amino acid side chainsfold inwards forming a channel, allowing the passage ofsmall hydrophilic molecules. Because they lack the

hydrophobic domains of classical transmembranedomains, OMPs are predictably more soluble, and thusare likely to be more amenable to separation by two-dimensional electrophoresis (2-DE). However, extractionefficiency of membrane proteins with conventional solubi-lization reagents used for 2-DE is often poor [5]. In addi-tion, protein losses are commonly noted with the use ofimmobilized pH gradients (IPGs) due to hydrophobicinteractions between certain protein domains and someof the polymers used to form the pH gradient [6]. Conse-quently, only limited reports have demonstrated high-resolution separation of bacterial OMPs by 2-DE usingthe reproducibility and load capacity offered by IPGs(reviewed in [7, 8]).

Recently, we described some technical improvements for2-DE separation of bacterial OMPs using IPGs. Wedemonstrated these methods by recovering manyEscherichia coli OMPs [9]. Using this approach, the OMwas enriched for membrane proteins by removing mostnonmembrane proteins with an alkaline pH wash, beforereconstituting the membrane fraction for 2-DE usingstrong denaturing conditions. The efficiency of thisapproach was assessed by comparing the proteins iden-tified on the 2-D gel to the theoretical protein compositionof the E. coli OM. 80% of the expected OMPs wereobserved within a separation window of pH 4–7 and Mr

Correspondence: Professor Philip C. Andrews, Department ofBiological Chemistry, University of Michigan Medical School,Ann Arbor, MI, 48109-0606, USAE-mail: [email protected]: +1-734-647-0951

Abbreviations: COGs, clusters of orthologous groups of prote-ins; OM, outer membrane; OMPs, outer membrane proteins;PMF, peptide mass fingerprinting

1686 Electrophoresis 2001, 22, 1686–1696

WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 0173-0835/01/0905–1686 $17.50+.50/0

10–80 kDa. Follow-up studies showed that the approachwas also successful with membranes from Pseudomonasaeruginosa [10].

In the present report we expand our study, examining theOMs of two additional enteric bacteria, Salmonellla typhi-murium and Klebsiella pneumoniae (also known as Kleb-siella aerogenes [11]). In addition, we have begun a preli-minary investigation of OMPs from an unrelated microbe,the free-living �-proteobacteria Caulobacter crescentus.Peptide mass fingerprinting (PMF) is a widely used,robust method of protein identification when analyzingproteins from organisms with sequenced genomes [12,13]. This approach is rapid, combining high-throughputscreening capabilities with low ambiguity in proteinassignment. However, it has not been studied in detailwhether the technique can be successfully applied toidentify proteins from unsequenced or partially sequencedgenomes through cross-species peptide matching, wheresingle amino acid substitutions may inhibit accurateprotein assignment. Nonetheless, the merits of the PMFapproach, and the constraints to high-throughput analysisafforded by alternative MS techniques, makes cross-species PMF an enticing proposition to test. In this report,we demonstrate separation of bacterial OMPs by 2-DE,and evaluate cross-species PMF of bacterial membraneproteins from three microorganisms, each with varyingstatuses in genome sequencing projects (S. typhimurium-initiated, limited ORFs called; K. pneumoniae-3X shotguncomplete, limited ORFs called; C. crescentus-complete,ORFs called).

2 Materials and methods

2.1 Cell culture

Salmonella typhimurium LT2 (MS 1868) was cultured inMOPS minimal media [14] at 37�C with shaking (200 rpm).Using these conditions, iron concentration was 10 �M. Foriron limitation experiments, the iron concentration was100 nM. Cells were collected in late log phase at an opticaldensity (OD)420 of 2.0. Klebsiella pneumoniae W70 (KC2668) (Klebsiella aerogenes) (obtained from R. Bender-University of Michigan) was cultured in Luria broth medium(LB) at 37�C with shaking at 200 rpm and collected in latelog phase at an OD420 of 1.8. Caulobacter crescentusCB15N was inoculated in 1 L cultures of peptone yeastextract [15] at a 1:500 dilution. Cells were grown at 30�Cwith shaking at 250 rpm in 4 L baffled flasks till an OD600 of0.8. Collected cells were washed twice in 50 mM Tris/HCl,pH 7.5.

2.2 Membrane isolation

Membranes were isolated as previously described [9].Briefly, the cells were ruptured in an Aminco French presswith two presses at 14 000 psi and unbroken cellsremoved by centrifugation at 2500�g for 10 min. Thesupernatant was diluted with ice-cold 0.1 M sodium car-bonate (pH 11) to a final volume of 60 mL and stirredslowly on ice for 1 h. The carbonate-treated membraneswere collected by ultracentrifugation in a Beckman 55.2Tirotor at 115 000�gavg for 1 h at 4�C. The supernatant wasdiscarded and the membrane pellet resuspended andwashed in 2 mL of 50 mM Tris/HCl, pH 7.5. The washedmembrane was collected by centrifugation as describedabove.

2.3 2-DE

Membranes were solubilized for IEF using a solution con-sisting of 7 M, urea, 2 M thiourea, 1% w/v ASB 14, 0.5% v/vTriton X-100, 40 mM Tris-base, 30 mM DTT, and 0.5% v/vBiolytes 3–10. The sample was placed in a sonicatingwater bath for 5 min and insoluble material removed bycentrifugation for 5 min in a benchtop centrifuge. Sample(1.5 mg) was loaded on an 18 cm, pH 4–7 or pH 3–10 IPG(Amersham Pharmacia Biotech, Uppsala, Sweden) byrehydration and left under oil overnight. IEF was con-ducted for a total of 50 000 Vh using the Multiphor II (Amer-sham Pharmacia Biotech) or Protean IEF Cell (Bio-RadLaboratories, Hercules, CA, USA) at 20�C. SDS-PAGEwas conducted with 11% T gels previously described [9]and proteins detected using Coomassie Brilliant BlueG-250 (Aldrich Chemical, Milwaukee, WI, USA).

2.4 PMF

A minimum of 30 of the most heavily stained spots (repre-senting the most abundant protein species in the extract)were excised using a 1 mm dermal punch (Sklar, West che-ster, PA, USA) and placed in siliconized Eppendorf tubes(Thomas Scientific, Swedesbor, NJ, USA). Protein spotswere washed at 37�C in 50% v/v MeCN/0.1 M ammomiumbicarbonate, dried anddigested overnightat 37�C with por-cine trypsin (Promega, Madison, WI, USA). Peptides wereextracted by sonication in60%v/vMeCN, 1%v/vTFA. Thissolution was dried and peptides resuspended in 8 �L of 3%v/v TFA. Sample (0.8�L) was placed on a gold MALDI targetand 0.8 �L matrix (10 mg/mL �-cyano-4-hydroxycinnamicacid in 50% v/v MeCN, 0.1% v/v TFA) added. Spectra wereacquired using a PerSeptive Biosystems Voyager DE-STRMALDI operating in delayed extraction reflector mode.Spectra were calibrated using the trypsin autodigestionpeaks at 842.5 Da and 2211.1 Da. Monoisotopic peptideions were called automatically using in-house software (G.Rymar, unpublished).

Electrophoresis 2001, 22, 1686–1696 2-DE of bacterial membrane proteins 1687

Proteomicsand2-DE

Figure 1. Phylogenetic organization of proteobacteria based upon 16S ribosomal RNA genes. Gene align-ments were made using CLUSTALX and tree produced using the bootstrap option (n=100) in PAUP (SinauerAssociates, Sunderland, MA, USA). Note that S. typhimurium and K. pneumoniae are evolutionary neighborsof E. coli. In contrast, C. crescentus shows considerable divergence from R. prowazekii; its closest relativewhose genome is sequenced.

2.5 Protein identification

Peptide masses were queried against entries for microor-ganisms in the NCBI nr database orC. crescentusgenomedatabase (made available prior to publication by The Insti-tute for Genomic Research, Rockville, MD, USA) using theMS-Fit program in Protein Prospector (http://prospector.-ucsf.edu). Searches where conducted using a pI windowof pH 3–10 and a mass window of 10–130 kDa. The masstolerance window was set at 0.15 Da. For a match to beconsidered successful, a minimum of three matching pep-tides was required. Where an assignment appearedambiguous (three peptides, MOWSE �100), tandem MSwas conducted using a Q-TOF instrument (Micromass,Manchester, UK) to confirm protein identity.

2.6 Cluster of orthologous groups of proteins(COG) functional grouping

Putative C. cresentus ORFs were assigned into specificfunctional groups based on the cluster of orthologousgroups of proteins (COG) system of protein classifica-

tion [16]. The COG program is based on position-specificscore matrices of conserved regions of protein sequencesimilarity as assessed on a genome-wide basis. The group-ing is conducted using the COGNITOR tool available athttp://www.ncbi.nlm.nih.gov/COG/cog99nitor.html.

3 Results

3.1 2-DE of membrane proteins

We have previously demonstrated recovery of 21 out of26 predicted E. coli OMPs by carbonate treatment, andsubsequent array of these proteins by 2-DE using strongsolubilizing conditions [9]. For successful 2-DE, standardIEF solubilization solutions should be ameliorated toinclude thiourea and an amidosulfobetaine surfactant [17].In this study, we aimed to examine the OMPs from threemicrobes, including two members of the Enterobacteria-ceae (S. typhimurium, K. pneumoniae) and the �-proteo-bacterium C. crescentus. Figure 1 displays a phyloge-netic relationship of these microorganisms based upongenes for 16S ribosomal RNA.

1688 M. P. Molloy et al. Electrophoresis 2001, 22, 1686–1696

Figure 2. Coomassie blue-stained 2-D gels of OMPs from (A) S. typhimurium, (B) S. typhimurium grown under conditionsof iron limitation, (C) K. pneumoniae, (D) C. crescentus.

OMPs from the above-mentioned microorganisms wereseparated by high-resolution 2-DE as shown in Fig. 2A-D.These gels were prepared with preparative protein load-ings (1.5 mg) and display excellent resolution with onlyminimal streaking. Using Coomassie blue staining weobserved that several abundant protein groups domi-nated the gels, although many other minor componentssuitable for MS analysis were also detected. Overall, the

2-D gel patterns of both enteric species display remark-able similarity to each other (Figs. 2A and C), and to thepattern previously established for E. coli OMPs [9]. Sev-eral of the proteins display charge variance, an observa-tion also noted with E. coli OMPs. Recently, the pattern ofcharge heterogeneity that is often observed with proteinson 2-D gels was attributed to protein deamidation in sam-ples of human plasma [18], however, it remains to be


Table 1a. Protein assignments by PMF from S. typhimurium OMPs shown in Fig. 2A

SpotNo.

Protein description Speciesmatched

NCBlnrAccessionNo.

No. ofpeptidesmatched

Other cross-speciesmatchesb)

MOWSEscore

TheoreticalMr/pI

ExperimentalMr/pI

1 Outer membraneantigen Oma90a)

Sf 4902918 11 Ec 2.41E+06 90 553 / 4.93 90 000 / 5.10

2 Organic solventtolerance proteina)

Ec 2507089 6 5.23E+02 89 671 / 4.94 92 000 / 5.20

3 Ferrichrome ironreceptora)

St 3445382 15 1.54E+12 80 570 / 5.24 78 000 / 5.35

4 DnaK St 2495350 7 Ec 7.62E+04 69 258 / 4.83 78 000 / 5.055 30S ribosomal

subunit protein S1Ec 2507321 9 2.00E+07 61 158 / 4.89 75 000 / 5.05

7 Vitamin B12receptora)

St 584858 15 Cf 1.00E+08 68 495 / 5.31 65 000 / 5.50

8 GroEL St 1345766 6 Ec, Sm, Vp,Ng, Nm, Ye

1.22E+03 57 286 / 4.85 62 000 / 5.10

9 TolCa) Se 2495191 6 Ec 1.48E+04 53 726 / 5.42 55 000 / 5.4010 OmpAa) St 129140 7 Ea, Cf, Sd 3.82E+05 37 589 / 5.60 35 000 / 5.5511 Long chain fatty

acid transportera)Ec 7442894 7 3.98E+05 48 772 / 5.09 48 000 / 4.95

12 OmpFa) St 585617 9 Sty 6.31E+06 39 988 / 4.76 38 000 / 4.7013 OmpFa) St 585617 9 Sty 6.31E+06 39 988 / 4.76 38 000 / 4.7015 TsXa) St 731022 4 3.97E+03 32 778 / 4.96 32 000 / 4.9516 ATPase-beta

subunita)St 6625703 8 Ec, Ea, Se,

Cf, Hi6.32E+05 50 311 / 4.90 52 000 / 5.10

19 OmpAa) St 129140 7 Ea, Cf, Sd 3.82E+05 37 589 / 5.60 35 000 / 4.9523 Phospholipase Aa) St 585637 7 Ec 2.92E+04 33 003 / 6.08 30 000 / 5.7024 Hypothetical 29.4 kDa

lipoproteina)Ec 401469 3 3.88E+02 29 431 / 5.13 28 000 / 5.20

26 Peptidoglycan-asso-ciated lipoproteina)

Ec 129595 4 1.57E+03 18 824 / 6.29 18 000 / 5.65

28 Phosphoglyceratekinase

Ec 129920 3 8.80E+02 41 118 / 5.08 40 000 / 5.50

30 Enolase Ec 1706655 6 1.34E+05 45 655 / 5.32 47 000 / 5.7031 Cell division protein Ec 120578 8 2.75E+06 40 297 / 4.72 42 000 / 4.8532 Hsp90 Ec 123726 5 3.12E+03 71 422 / 5.09 70 000 / 5.4034 ATPase-alpha subunita) St 6625701 5 Ec, Mg 1.04E+04 55 236 / 5.80 58 000 / 6.2037 OMP Esterasea) St 2896133 6 5.77E+03 69 862 / 5.37 68 000 / 5.40

a) Membrane proteins. Protein spots analyzed but not identified: 6, 17, 18, 20, 21, 22, 25, 27, 29, 33, 35, 36b) St, Salmonella typhimurim; Se, Salmonella enterica; Ec, Escherichia coli; Sm, Serratia marcescens; Vp, Vibrio parahae-molyticus; Ng, Neisseria gonorrhoeae; Nm, Neisseria meningitidis; Ye, Yersinia enterocolitica; Ea, Enterobacter aero-genes; Cf, Citrobacter freundii; Sd, Shigella dysenteriae; Hi, Haemophilus influenzae; Mg, Mycoplasma genitalium; Sty,Salmonella typhi; Sf, Shigella flexneri

determined if the same protein modification is responsiblefor the patterns seen with bacterial OMPs. In contrast toFigs. 2A-C, the 2-D gel obtained for the �-proteobacter-ium C. crescentus appears unique (Fig. 2D), with many ofthe abundant proteins migrating in charge clusters ofhigher masses compared to OMPs from the enteric spe-cies. To survey the abundant protein composition of theseextracts, a minimum of 30 heavily stained proteins spotswere excised from each gel and subjected to PMF.

3.2 OMPs

3.2.1 S. typhimurium OMPs

Of the three microorganisms examined, we anticipatedprotein identification by PMF to deliver the best correla-tions for S. typhimurium. Even though only approximately20% (782 genes in SWISS-PROT) of S. typhimuriumgenes (based on a genome similar in size to E. coli) arepresent in genome databases, we expected that the


Table 1b. Protein assignments by PMF from S. typhimurium OMPs from cells cultured under iron limitation (Fig. 2B)

SpotNo.


NCBlnrAccessionNo.



MOWSEscore

TheoreticalMr/pI

ExperimentalMr/pI

1 Colicin receptora) Ec 2507462 4 4.61E+02 73 897 / 5.11 75 000 / 5.602 Colicin receptora) Ec 2507462 4 4.61E+02 73 896 / 5.11 75 000 / 5.603 OmpA dimera) Ec 129140 5 Ea, Cf 1.52E+04 37 589 / 5.60 75 000 / 5.554 Vitamin B12 receptora) St 584858 5 1.05E+03 68 495 / 5.31 65 000 / 5.505 Siderophore receptora) Se 2738252 12 4.23E+08 79 549 / 6.06 80 000 / 6.006c) Siderophore receptora) Se 2738252 3 3.80E+01 82 091 / 5.45 83 000 / 6.207c) Ferrienterochelin

receptora)Ec 145942 3 7.90E+01 82 091 / 5.45 83 000 / 6.20

8 Ferrienterochelinreceptora)

Ec 145942 3 2.26E+02 82 091 / 5.45 83 000 / 6.20

9 Colicin receptora) Ec 2507462 4 4.61E+02 73 896 / 5.11 75 000 / 5.60

a) Membrane proteinb) Abbreviations as in Table 1ac) Identity confirmed by MS/MS

Table 2a. MS-Fit search for spot 11 (Fig. 2A)

Rank MOWSEScore

# Massesmatched

TheoreticalMr/pI

Species AccessionNo.

Proteinidentification

1 3.97E+05 7/12 45 900 / 4.99 Escherichia coli 7442894 Outer membrane protein FLP2 3.50E+02 3/12 54 300 / 6.36 Anabaena variabilis 212653 Hydrogenase large subunit3 3.10E+02 3/12 73 700 / 6.18 Campylobacter jejuni 6968135 Potassium transporting ATPase B4 2.02E+02 3/12 49 700 / 5.71 Bacillus subtilis 40 249 Xylose isomerase5 1.76E+02 3/12 61 000 / 5.36 Bacillus subtilis 2498150 L-Ribulokinase

Clear score differences between first- and second-ranked proteins are indicative of a cross-species match.

Table 2b. MS-Fit search for spot 10 (Fig. 2A)

Rank MOWSEScore

# Massesmatched

TheoreticalMr/pI

Species AccessionNo.

Proteinidentification

1 3.82E+05 7/12 35 500 / 5.30 Salmonella typhimurium 129140 OmpA2 6.84E+02 4/12 25 500 / 4.88 Enterobacter aerogenes 148368 OmpA3 6.81E+02 4/12 25 600 / 4.93 Citrobacter freundii 129134 OmpA4 6.66E+02 4/12 37 700 / 5.57 Shigella dysenteriae 129134 OmpA

Matching to the same protein across-species is an indicator of confident assignment.

close phylogenetic relationship to E. coli (whose genomesequence is complete) would aid cross-species proteinidentification. Indeed, our results indicated this was thecase, with 25 unique proteins identified (33 including iso-forms) by matching a minimum of three peptides to eitherS. typhimurium, or another microorganism (Tables 1a and1b). Of the 45 proteins examined by PMF, only 12 proteinscould not be identified. Seventeen proteins were identi-fied based upon matches to species annotated as S.typhimurium or S. enterica, 15 matched to E. coli, while a

further match was with Shigella flexneri. Correct matcheswere clearly discernible by examining the number ofmatching peptides between the first-and second-rankedcandidate proteins (e.g., Table 2a). This relationship wasalso seen in the output from the MOWSE scoring algo-rithm. It is important to note that in 13 cases, the best-ranked candidate protein scored multiple hits across-species, highlighting the conserved nature of the match-ing peptides and adding strong confidence to the assign-ment (e.g., Table 2b).


Table 3. Protein assignments by PMF from K. pneumoniae OMs shown in Fig. 2C

SpotNo.


NCBlnrAccessionNo.



MOWSEscore

TheoreticalMr/pI

ExperimentalMr/pI

1 Pullulanase precursora) Ka 149300 5 1.22E+03 119 143 / 4.70 120 000 / 4.703 Outer membrane

antigen Oma90a)Sf 2506737 7 Ec 1.58E+04 90 553 / 4.93 85 000 / 5.20

4 Organic solventtolerancea)

Ec 2507089 4 8.74E+02 89 671 / 4.94 80 000 / 5.20

12 DnaK Se 2495350 5 Ec 3.50E+04 69 258 / 4.83 70 000 / 4.9015 ATPase B-subunita) Ea 226371 9 St, Ec, Se,

Cf, Ye1.98E+07 50 199 / 4.86 55 000 / 5.00

17 TolCa) Se 2495191 6 Ec 4.24E+04 53 726 / 5.42 55 000 / 5.4018 Maltoporina) Kp 400158 4 5.09E+02 47 804 / 4.80 47 000 / 4.8020 OmpKa) Kp 3914223 4 1.31E+03 39 663 / 4.47 40 000 / 4.6021 OrfX (Orf3) capsular

formationa)Ec 4512004 6 Kp 1.79E+04 55 715 / 5.52 55 000 / 5.50

22 ATPase A-subunita) Ec 399079 9 St, Mg 3.72E+06 55 222 / 5.80 60 000 / 5.9023 ATPase A-subunita) Ec 399079 9 St, Mg 3.72E+06 55 222 / 5.80 60 000 / 5.9025 OmpAa) Ea 149286 8 Kp, Cf, Ec 2.32E+06 37 061 / 5.73 35 000 / 5.6026 PTS system-mannose

specificEc 41976 5 6.70E+03 35 047 / 5.74 35 000 / 6.20

27 OmpAa) Ea 149286 8 Kp, Cf, Ec 2.32E+06 37 061 / 5.73 35 000 / 5.6030 TsXa) Ea 731021 7 Kp 2.52E+05 33 507 / 5.18 33 000 / 5.0031 TsXa) Ea 731021 7 Kp 2.52E+05 33 507 / 5.18 33 000 / 5.0033 OmpK17 (OmpX)a) Kp 1279830 4 Enc 1.11E+04 18 463 / 8.67 17 000 / 6.00

Protein spots analyzed but not identified: 2, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16, 19, 24, 28, 29, 32, 34, 35, 36a) Membrane proteinsb) Ea, Enterobacter aerogenes; Ka, Klebsiella aerogenes; Kp, Klebsiella pneumoniae; St, Salmonella typhimurium; Ec,Escherichia coli; Se, Salmonella enterica; Cf, Citrobacter freundii; Ye, Yersinia enterocolitica; Mg, Mycoplasma genita-lium; Enc, Enterobacter cloacae; Sf, Shigella flexneri

In addition to a favorable ranking, the protein’s chemicalattributes of Mr and pI were also considered to contri-bute to matching confidence. Theoretical evaluation hasshown that across species boundaries Mr is well con-served, while pI is subject to more variation [19]. Examin-ing these attributes from candidate protein lists is impor-tant to eliminate false positive matches. We found itnecessary to impose a maximum mass window upon oursearches to avoid random matches to very large proteins(�100 kDa). We also tested if relaxing the peptide masstolerance window to account for amino acid substitutionswould improve cross-species matching. When this win-dow was set at �1 Da, only minor changes were seento candidate protein lists, with no additional peptidesdetected across species boundaries. Further relaxing thewindow to �10 Da resulted in many false positive assign-ments, indicating this approach to be ineffective.

We had previously shown that many E. coli OM ironreceptors were not detected when cells were cultured inmedia containing micromolar concentrations of iron [9].

To induce expression of iron receptors in S. typhimurium,we conducted an experiment that grew cells in mediacontaining a 100-fold reduction in the iron concentration(Fig. 2B). In this case, we readily observed changes inprotein expression, and used PMF to identify these pro-teins (Table 1b). PMF analysis indicated each of the newlyexpressed proteins were siderophore receptors requiredfor iron transport. However, in two instances the MOWSEscores obtained for a siderophore receptor (spot #6) andthe ferrienterochelin receptor (spot #7) were too low to beconsidered a confident assignment. To clarify the situa-tion, we conducted tandem MS sequence tagging onboth samples. This analysis confirmed that the assign-ment established by PMF was correct, despite only lim-ited PMF sequence coverage and low MOWSE scores.

Figure 3 lists the classes of identified proteins. The major-ity of identified proteins (76%) were described as mem-brane components, consistent with the isolation andseparation method. Furthermore, each of the most abun-dant protein spots corresponded to OMPs. With the


Figure 3. Putative localization of proteins identified inS. typhimurium and K. pneumoniae.

exception of an OM esterase that appears absent in E.coli, all of the identified S. typhimurium OMPs corre-sponded to protein homologues found in E. coli. A pre-vious report had presented a 2-D gel proteome map ofOMPs from S. typhimurium SL1344, using a traditionalapproach to OMP solubilization [20]. The present workreports the identification of 13 additional membrane pro-teins not documented earlier (OstA, FhuA, Btub, FadL,OmpF, TsX, Pa1, PaL, YaeC, ApeE, IroN, CirA, FepA).One explanation for the additional OMPs is the improve-ment in protein solubilization, separation and MS identifi-cation techniques. However, it should be noted that straindifferences could also account for some variation seenbetween OM preparations.

3.2.2 K. pneumoniae OMPs

Identification of K. pneumoniae OMPs using PMF pre-sented a more difficult case than with S. typhimurium, asonly a limited number (254 genes for K. pneumoniae, K.aerogenes, in SWISS-PROT) of its gene sequencesare available. However, as a member of the Enterobacter-iaceae, we expected conservation of some peptidesequences with E. coli, sufficient to establish a basis forcross-species matching. From 36 protein spots (Fig. 2C)we identified 14 unique proteins (38%) (Table 3), 12 ofthese were annotated as membrane proteins. Eight pro-teins were assigned by matching to either K. pneumoniaeor E. aerogenes (K. aerogenes) gene sequences, a furtherfour matches were against E. coli and a match was scoredagainst a protein from S. typhimurium and another from S.flexneri. In the case of nine assignments, the same proteindescription was found for at least one other species as thenext highest ranked candidate protein, providing soundevidence forconfident assignment. As expected, the iden-

tity of the major protein spots corresponded to OMPshomologues observed inE. coli, andS. typhimurium. Inter-estingly, we identified a pullulanase receptor, a large glyco-syl hydrolase lipoprotein (�120 kDa), not detected ineither E. coli or S. typhimurium.

3.2.3 C. crescentus OMPs

Cross-species PMF identification of proteins from C.crescentus proved most problematic due to the evolu-tionary divergence of protein sequence between this�-proteobacterium and the enteric bacteria of the gammasubdivision. As an example, many of the OMPs commonin Enterobacteriaceae (OmpF, OmpC, OmpX, OmpT,PhoE, TsX, LamB, Pa1) were not detected when a BLASTsearch was conducted against the C. crescentus genome(data not shown). However, a similar search revealed sev-eral other E. coli OMPs with C. crescentus homologues(OmpA, ToIC, BtuB, FadL, FepA).

PMF of 30 C. crescentus protein spots (Fig. 2D) searchedagainst the recently sequenced C. crescentus genomeresulted in the putative assignment of 29 proteins toORFs (Table 4). In many cases, examination of the proteindescription, established by blasting ORFs, offered littleinsight into the nature of the protein. At best, a generalclassification of the protein could be obtained (e.g.,OmpA-like protein or putative OMP) as an indicator ofthe likelihood for OM localization. COG functional analy-sis provided some additional annotation. In order toestablish the likely localization of the proteins, supple-mentary evidence was obtained, which included the pre-sence of an N-terminal signal sequence for translocation,and possession of an aromatic amino acid “anchor” at thecarboxy-terminus to stabilize the protein in the OM [21].We observed these two attributes for 11 protein spots.Interestingly, ten other proteins were predicted with N-terminal signal peptides, yet were missing the C-terminalaromatic amino acid typically seen in OMPs. This raisestwo possibilities that require investigation. Quite likely, C.crescentus is not reliant on using aromatic residues forsecuring some OMPs in the bilayer, as is the case in E.coli for OmpA. Alternatively, the stop position of the ORFmay have been incorrectly determined.

In total, we anticipate that at least 21 of the proteins stu-died (all those possessing signal peptides) are localized inthe OM. We attempted to identify each protein by cross-species PMF against all microorganisms in the NCBI nrdatabase, however in all cases, confident assignmentscould not be made, as either the number of matchingpeptides was generally low (�4), and/or there was noappreciable difference between scores for the first-andsecond-ranked proteins, as was typically noted with posi-tive assignments in S. typhimurium and K. pneumoniae.


Table 4. Protein assignments by PMF from C. crescentus OMs shown in Fig. 2D. The presence of a signal sequence waspredicted using the SignalP tool available at http://www.cbs.dtu.dk.

SpotNo.

Proteindescription

ORF No. COG group function No.peptidesmatched

N/MOWSEscore

TheoreticalMr/pI

ExperimentalMr/pI

Predictedsignalsequencec)

C-terminalamino acid

2 OmpA familyproteina)

ORF02690 Flagellar MotB andrelated proteins,PAL/OmpA family

7 7.86E+03 21 466 / 5.85 8 000 / 5.3 Y T

3 Hypotheticalprotein

ORF02264 No COG 4 5.26E+02 15 777 / 6.95 9 000 / 4.3 Y A

6 Conservedhypotheticalprotein

ORF05589 Uncharacterizedprotein CsgG-formation of curlipolymers

4 1.38E+02 23 861 / 5.90 15 000 / 6.0 Y K


ORF06872 ChromosomesegregationATPase

6 7.98E+03 24 969 / 5.75 15 000 / 6.4 N A


ORF06104 Urea amidohydrolase(urease) alphasubunit

4 4.17E+02 44 156 / 5.42 20 000 / 6.4 Y R

11 Hypotheticalproteina)

ORF02621 No COG 12 1.42E+05 43538 / 6.44 45 000 / 6.4 Y F


ORF02621 No COG 11 5.45E+07 43037 / 5.50 45 000 / 6.1 Y F

13 PutativeOMP P1a)

ORF06344 Long-chain fatty acidtransport protein FadL

15 8.24E+07 43037 / 5.50 45 000 / 5.4 Y F

14 Putative OprF ORF01781 Flagellar MotB andrelated proteins,PAL/OmpA family

7 5.08E+02 47 603 / 8.38 38 000 /5.3 N F


5 8.25E+03 47 603 / 8.38 38 000 / 5.4 N F

17 OprMa) ORF03881 Putative outermembrane protein

12 4.69E+07 47 648 / 5.39 48 000 / 5.3 Y T


ORF04087 No COG 8 5.16E+04 49 070 / 5.71 49 000 / 5.7 Y F

19 Outer membraneproteina)

ORF04313 Putative outermembrane protein

7 4.12E+03 54 142 / 7.01 62 000 / 6.3 Y N

20 Outer membraneproteina)

ORF04313 Putative outer membraneprotein

9 1.32E+05 54 142 / 7.01 62 000 / 6.0 Y N

21 OmpA-relatedproteina)

ORF04119 No COG 21 1.44E+13 108 754 / 5.63 110 000 / 5.6 Y F

22 Conservedhypotheticalproteina)

ORF00898 No COG 21 5.23E+11 116 453 / 5.15 120 000 / 5.2 Y M


ORF02871 No COG 11 8.28E+06 120 690 / 6.06 120 000 / 5.0 N F


ORF0440 Outer membranereceptor proteins,mostly Fe transport

7 5.06E+04 61 207 / 5.07 100 000 / 5.0 N Y



9 2.29E+06 61 207 / 5.07 105 000 / 4.8 N Y

26 TonB-dependentreceptora)


28 5.88E+16 69 813 / 4.92 75 000 / 5.0 Y F



12 5.16E+08 67 230 / 5.89 65 000 / 5.8 Y F


Table 4. Continued

SpotNo.

Proteindescription

ORF No. COG group function No.peptidesmatched

N/MOWSEscore

TheoreticalMr/plb)

ExperimentalMr/pl

Predictedsignalsequencec)

C-terminalamino acid



12 3.33E+08 67 230 / 5.89 65 000 / 5.9 Y F



22 1.05E+14 67 230 / 5.89 65 000 / 6.0 Y F



14 1.83E+08 85 580 / 5.61 88 000 / 5.8 Y F



14 1.83E+08 85 580 / 5.61 88 000 / 5.6 Y F


ORF00675 No COG 6 3.64E+03 21 536 / 5.94 10 000 / 5.5 Y Q


ORF02621 No COG 10 1.46E+06 43 538 / 6.44 45 000 / 5.8 Y F


6 2.85E+02 47 603 / 8.38 48 000 / 5.4 N F

86 TonB-dependentreceptor


22 1.07E+12 80 843 / 5.48 80 000 / 5.3 N W

Protein spot analyzed but not identified: No. 7a) Putative membrane proteinsb) Determined after removal of predicted signal peptidec) Y, yes; N, no

These results provide a useful example documenting thelimitations of cross-species PMF between phylogeneti-cally distant bacteria (i.e., alpha vs. gamma). C. crescen-tus joins Rickettsia prowazekii, the causative agent oftyphus fever as the second �-proteobacteria whose gen-ome is sequenced [22]. Unlike the case for enteric bac-teria where cross-species PMF proved successful, wewere disappointed that we did not identify any C. cres-cehtus protein by cross-species matching to R. prowaze-kii. On reflection, this result probably highlights the wideevolutionary diversity of the �-proteobacteria group,which beside the Caulobacters and Rickettsias, includesthe Acetobacteraceae, Rhizobacteria, Rhodobacters,Rhodospirillaceae and Sphingomonas group (Fig. 1).

4 Discussion

Until recently, the isolation and reproducible 2-DE separa-tion of bacterial OMPs has been a challenging prospect.In this report, we have demonstrated a reliable method for2-DE separation of OMPs from three bacterial species.The patterns of resolved OMPs showed remarkable simi-larities between enteric species, offering high hopes for

protein identification through cross-species matching. Incontrast, OMPs from C. crescentus do not align well withenteric OMPs, being larger and more alkaline.

To assess our extraction technique for OMPs, PMF wasused for protein identification. PMF is widely successfulwhen searching against fully sequenced genomes, how-ever, it was unclear whether this technique would be use-ful when searching across species boundaries. Such arapid, high-throughput approach would certainly beattractive for protein identification in organisms whosegenomes remain coded. The inherent problem withcross-species PMF is that a single amino acid substitu-tion occurring in the same peptide from two separate spe-cies will result in a failure to match against that peptide.When substitutions occur in many positions (evolutionarydivergence), the probability of cross-species protein iden-tification diminishes (Table 5). Furthermore, it should bestressed that this problem is not restricted to cross-spe-cies PMF, but is also evidenced between strains of thesame species [23]. In spite of these known drawbacks,we have readily demonstrated that for numerous OMPsof enteric bacteria, sufficient peptides are conserved toestablish protein identity. In the examples we presented,


unambiguous identifications were obtained when cross-species PMF was conducted within a phylogenetic familythat contained a species whose genome was fullysequenced, and thus could be used as a reference formatching (i.e., Enterobacteriacae) (Table 5).

Table 5. As evolutionary distance from E. coli increased(Fig. 1), protein assignment by cross-speciesPMF decreased.

Evolutionary distancea)

(arbitary units)% Proteins IDby cross-species PMF

8 7610 3865 0

a) From E. coli

Peptides that match across species boundaries representprotein architecture that has become evolutionary suc-cessful, as seen for some enteric OMP peptides [24]. Itwas not unexpected that cross-species PMF failed whenwe attempted to match between bacterial subdivisions(alpha vs. gamma), suggesting structural design differ-ences for these OMPs. However, it was interesting thatmatching within the alpha-branch subdivision (C. cres-centus vs. R. prowazekii) failed to produce unambiguousassignments, indicating some level of amino acid diver-sity within the peptide structures used in assemblingOMPs for these species. These accounts reinforce thedifficulties faced in using the PMF technique to matchoutside species boundaries. Nonetheless, with de novoMS/MS, the only viable alternative for protein identifica-tion in organisms with incomplete genome sequences,we consider that cross-species PMF has a role in firstpass, high-throughput screening. As more genome se-quences are unveiled from organisms at more distantphylogenetic branches, we anticipate that cross-speciesPMF may become a more useful tool for rapid proteinidentification. In addition to demonstrating protein identi-fication by cross-species PMF, this work further demon-strated the validity of the isolation and 2-DE separationtechnique for bacterial OMPs. We now present this as ageneric method for studying the OMs of Gram-negativebacteria.

This work was supported by funding to P.C.A. from theNational Cancer Institute (1RO1CA77078-01) and MerckGenome Research Institute. J.R.M. is supported by grantGM-55133 from the National Institutes of Health. The pre-liminary Caulobacter crescentus gene list was obtainedprior to publication from The Institute for Genomic

Research. We thank Kelli Biederman for conducting thetandem MS analysis and Bob Bender for supplying theKlebsiella strain.

Received September 6, 2000

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Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins

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