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Characterization of the outer membrane protein profile of clinical

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					                                                                               Characterization of the outer membrane protein profile of clinical Helicobacter pylori isolates
                                                                                               by subcellular fractionation and nano-LC FT-ICR MS analysis
                                                                                                                                        Elisabet Carlsohn1*, Johanna Nyström1, Hasse Karlsson1,Carol L Nilsson2 and Ann-Mari Svennerholm1
                                                                                                                                          1.Institute of Biomedicine, Goteborg University, Sweden 2. Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Tallahassee, Florida


                                                                                                                                                                                                                                                                                                    Proteins identified by nano-LC FT-ICR MS analysis of a single 1D-GE band (*)
                                                INTRODUCTION                                                                                                                                   RESULTS                                                                                              from the clinical H. pylori isolate Can13
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   CONCLUSION
The human gastric pathogen Helicobacter pylori is known to colonize a majority of                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          We have demonstrated that subcellular fractionation combined
                                                                                                                                                                                                                                                                                                                            Table 1                                                                                                                    Figure 2 (A)
the global population. In most infected individuals the colonization is                                                                     Visualization of the protein pattern of four different                                                                                                                                                                                                                                                                                                                                                                         with nano-LC LTQ-FT ICR MS and MS/MS analysis enable
                                                                                                                                                                                                                                                                                                                            Protein name                                 Gene no.      MW (kDa)   # of matched    Sequence      Mowse       RMS
asymptomatic; however, H. pylori infection is known to cause several gastric                                                                                                                                                                                                                                                                                                                         peptides    coverage (%)    score   error (ppm)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           identification of the majority of the theoretically predicted OMPs
                                                                                                                                            subcellular fractions from H. pylori                                                                                                                                            Iron-regulated outer membrane protein        HP1512          97.5           13             5          632         2
disorders such as duodenal ulcer and gastric cancer.
                                                                                                                                                                                                                                                                                                                        *
                                                                                                                                                                                                                                                                                                                            Iron III-dicitrate transport protein         HP0686          87.9            8            13          522         2
                                                                                                                                                                                                                                                                                                                            Omp 28 (BabA)                                HP1243          80.1           15            15          856         2                                                                                                                            in clinical H. pylori isolates.
In order to establish colonization and maintain infection the bacteria express a                                                            The quality and purity of the SLS-insoluble OM sample was                                                                                                                       Omp 2
                                                                                                                                                                                                                                                                                                                            Omp 25
                                                                                                                                                                                                                                                                                                                                                                         HP0025
                                                                                                                                                                                                                                                                                                                                                                         HP1156
                                                                                                                                                                                                                                                                                                                                                                                         78.0
                                                                                                                                                                                                                                                                                                                                                                                         76.7
                                                                                                                                                                                                                                                                                                                                                                                                         2
                                                                                                                                                                                                                                                                                                                                                                                                         8
                                                                                                                                                                                                                                                                                                                                                                                                                       2
                                                                                                                                                                                                                                                                                                                                                                                                                      11
                                                                                                                                                                                                                                                                                                                                                                                                                                   93
                                                                                                                                                                                                                                                                                                                                                                                                                                  452
                                                                                                                                                                                                                                                                                                                                                                                                                                              2
                                                                                                                                                                                                                                                                                                                                                                                                                                              1
variety of different virulence factors. These include a number of outer membrane                                                            determined by analyzing the protein content of the different
                                                                                                                                                                                                                                                                                                                            Flagellar hook FlgE
                                                                                                                                                                                                                                                                                                                            Omp 5 / Omp 29
                                                                                                                                                                                                                                                                                                                                                                         HP0870
                                                                                                                                                                                                                                                                                                                                                                         HP0227/1342
                                                                                                                                                                                                                                                                                                                                                                                         76.2
                                                                                                                                                                                                                                                                                                                                                                                         76.0
                                                                                                                                                                                                                                                                                                                                                                                                        31
                                                                                                                                                                                                                                                                                                                                                                                                         2
                                                                                                                                                                                                                                                                                                                                                                                                                      40
                                                                                                                                                                                                                                                                                                                                                                                                                       7
                                                                                                                                                                                                                                                                                                                                                                                                                                 1943
                                                                                                                                                                                                                                                                                                                                                                                                                                  220
                                                                                                                                                                                                                                                                                                                                                                                                                                              1
                                                                                                                                                                                                                                                                                                                                                                                                                                              2                                                                                                                            We found a large heterogenicity of the omp profiles and none
                                                                                                                                                                                                                                                                                                                                                                                                                                                             22'                                 Time (min)                                            44'
proteins and low-abundance adhesins (e.g BabA, SabA). Outer membrane                                                                                                                                                                                                                                                        Flagellar hook-associated protein 2 (Hap2)   HP0752          74.1           19            19         1014         2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           of the analyzed isolates did express all members of the major
                                                                                                                                            subcellular fractions from the OM enrichment by small-size                                                                                                                      Putative Omp                                 JHP0649         73.1           26            26         1464         1                                     944.96762+
proteins are therefore potential targets in the search for disease-related                                                                                                                                                                                                                                                  Omp 1 (HopZ)                                 HP0009          73.0            6            11          396         1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1131.5912+                     -0.9 ppm                               H. pylori omp family.
biomarkers and potential vaccine antigens.
                                                                                                                                            SDS-PAGE.                                                                                                                                                                       Omp 27 (HopQ)
                                                                                                                                                                                                                                                                                                                            Omp 17 (SabA)
                                                                                                                                                                                                                                                                                                                                                                         HP1177
                                                                                                                                                                                                                                                                                                                                                                         HP0725
                                                                                                                                                                                                                                                                                                                                                                                         70.0
                                                                                                                                                                                                                                                                                                                                                                                         69.3
                                                                                                                                                                                                                                                                                                                                                                                                        10
                                                                                                                                                                                                                                                                                                                                                                                                         8
                                                                                                                                                                                                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                                                                                                                                                                                                                  434
                                                                                                                                                                                                                                                                                                                                                                                                                                  439
                                                                                                                                                                                                                                                                                                                                                                                                                                              1
                                                                                                                                                                                                                                                                                                                                                                                                                                              1
                                                                                                                                                                                                                                                                                                                            Flagellar hook-associated protein 1 (Hap1)   HP1119          68.3           13            16          748         1
                                                                                                                                                                                                                                                                                                                            Urease                                      HP0072          61.8           16            29          985         1
                                                                                                                                                                                                                                                                                                                                                                                                                                                           (B)
                                                                                                                                                                                                                                                                                                                            60 kDa Chaperone
                                                                                                                                                                                                                                                                                                                            Flagellin B
                                                                                                                                                                                                                                                                                                                                                                         HP0010
                                                                                                                                                                                                                                                                                                                                                                         HP0115
                                                                                                                                                                                                                                                                                                                                                                                         58.2
                                                                                                                                                                                                                                                                                                                                                                                         53.8
                                                                                                                                                                                                                                                                                                                                                                                                        16
                                                                                                                                                                                                                                                                                                                                                                                                        10
                                                                                                                                                                                                                                                                                                                                                                                                                      23
                                                                                                                                                                                                                                                                                                                                                                                                                      17
                                                                                                                                                                                                                                                                                                                                                                                                                                  843
                                                                                                                                                                                                                                                                                                                                                                                                                                  531
                                                                                                                                                                                                                                                                                                                                                                                                                                              2
                                                                                                                                                                                                                                                                                                                                                                                                                                              1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           We identified several potential targets for drug discovery
The aim of this study was to establish a method that enables characterization of                                                               75 kDa                                                                                                                                                                       Flagellin A                                  HP0601          53.2           20            36         1499         1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           including Omp11, Omp26 and Omp6. Analysis of the immune
outer membrane protein (omp) profiles of clinical isolates of H. pylori in the search                                                                                                           Lys; total cell lysate, cyt;                                                                                                                                                                                                                                                                                                                                               response to these proteins in serum samples from infected
for new protein markers for disease-related H. pylori strains and identification of                                                                                                            cytoplasmic fraction, OM;
                                                                                                                                                                                                                                                                                                    Table 1. Seventeen unique proteins were detected in sample (*) of the outer                                                                                          754.73023+                                                                                        individuals are currently being performed.
potential vaccine antigens. This was achieved by combining subcellular                                                                                                                         sarcosine-insoluble outer
                                                                                                                                                                                               membrane fraction, IM;                                                                               membrane fraction from a H. pylori strain (Can 13) isolated from a patient with gastric
fractionation of the bacterial outer membrane (OM)1 with 1D SDS-PAGE analysis                                                                                                                  sarcosine-soluble inner                                                                              cancer. The great majority of the identified proteins are known to be expressed on the                                                                                                                          1130                1135
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           We conclude that this rapid subproteome approach is suitable
and high-sensitivity nano-LC FT-ICR MS analysis of tryptic peptides.                                                                                                                           membrane fraction.
                                                                                                                                                                                                                                                                                                    bacterial surface. Protein identity is significant at the 99.9 % level.                                                                                        400                   m/z                1600                                                           for the characterization of the outer membrane profile of
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           human pathogens in the search for novel protein-markers and
                                                                                                                                                                                                                                                                                                    Figure 2 (A) The nano-LC chromatogram of the tryptic peptides from *
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       y8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             QSIQNANNIELVNSSLNYLK
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           new potential target proteins for vaccine development.
                                               METHODS                                                                                         20 kDa                                                                                                                                               (B) The doubly protonated peptide at m/z 1131.591 [M=2261.167] was automatically                                                                       (C)
                                                                                                                                                                                                                                                                                                    selected for ion trap dissociation.                                                                                                                                                                                           y11
                                                    Supernatant                       Supernatant
                                                                                                                                                           s                M                                                                                                                       (C) CID spectrum of the ion at m/z 1131.591. The assigned amino acid sequence is
                                                                                                                                                         Ly




                                                                                                                                                               t


                                                                                                                                                                                IM
                   Ultra-




                                                                                                                                                             Cy
                                                                                                           Solubilization of pellet
                   sonication
                                   Centrifugation                  Centrifugation
                                                                                            waste                  in SLS                                               O                                                                                                                           unique for the SabA adhesin in H. pylori. Only fragment ions corresponding to the                                                                              y3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    y5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 y7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               y9         y10


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        y14


                  20 mM Tris-HCl
                                                                                                             2% SLS in 20 mM                                                                                                                                                                        amino acids marked in bold are shown.                                                                                                                                y4              y6                                              y12    y13
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              y15   y16

                                     9000xg                         50.000xg                                 Tris-HCl, pH 7.5
                  pH 7.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                              400             600              800          1000           1200          1400         1600          1800
     Harvest of 0.5
     g bacteria
                                                    waste                                                                                        Expression of the major H.pylori Omp family in different                                                                                                                                                                                                                                                                                                           m/z




                                 Supernatant                                                        50 µg omp
                                                                                                                                                 clinical H. pylori isolates
                                         Inner          Solubilization of OM        SDS-PAGE
                Centrifugation
                                         membrane       pellet in SDS               10% Bis-Tris                                                                                               H. pylori strains isolated from       H. pylori strains isolated from       H. pylori strains isolated from
                                           Outer
                  50.000xg                 membrane
                                                                                                                                            Protein
                                                                                                                                            Omp 1 (HopZ)
                                                                                                                                                             Gene no.
                                                                                                                                                             HP0009
                                                                                                                                                                            Mw (kDa)
                                                                                                                                                                                 76.6
                                                                                                                                                                                         pI
                                                                                                                                                                                        8.71
                                                                                                                                                                                               gastric cancer patients
                                                                                                                                                                                                    +   +   +   +   +   +        +
                                                                                                                                                                                                                                     duodenal ulcer patients
                                                                                                                                                                                                                                      +       +   +   +   +   +   +    +
                                                                                                                                                                                                                                                                           asymtomatic carriers
                                                                                                                                                                                                                                                                                +   +   +   +   +    +       +
                                                                                                                                                                                                                                                                                                                                              Identification of potential biomarkers and vaccine antigen candidates
                                                                                                                                            Omp 2            HP0025              78.0   9.16    +       +       +   +   +        +    +   +           +   +       +                     +       +    +   +   +
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                ACKNOWLEDGEMENT
    Entire gel lane excised
                                                                                                                                            Omp 3            HP0078/79           63.7   5.01                                                          +   +                                                                                 In total, over sixty unique membrane or membrane-associated proteins were identified from the                                                                                                                                  The authors acknowledge Thomas Larsson at the Sahlgrenska academy
    into 9 equally sized
    slices                                                                                                                                  Omp 4            HP0127              32.0   9.56            +   +                    +                    +   +   +   +         +   +               +    +       +                              different strains. In addition, we identified several hypothetical proteins and a small number                                                                                                                                 proteomic core facility for help with data evaluation.
                Protein               On-line peptide        MS and MS/MS                                                                   Omp 5/Omp 29     HP0227/1342         76.0   8.51    +       +   +   +   +            +    +               +   +   +                     +   +       +    +   +   +   Omp6
                digestion             separation*            analysis**                              Data              Protein              Omp 6            HP0229              53.2   8.51            +       +   +   +   +    +                                +         +   +           +   +    +   +   +                              (less than 10%) of cytoplasmic proteins (data not shown).
                                                                                                     analysis***       Identification                                                                                                                                                                                                                                                                                                                                                                                                                                      We gratefully acknowledge the financial support provided by the Swedish
                                                                                                     NCBI database
                                                                                                                                            Omp 7 (Hop F)    HP0252              52.2   9.55                                                              +                                     +        +   +
                trypsin               17 cm x 50µm            7T FT-ICR MS
                                      column                                         m/z
                                                                                                                                            Omp 8            HP0253              47.5   9.18        +                            +                    +       +   +                         +                                               Of the 33 theoretical predicted H. pylori OMPs we identified all except Omp18, Omp24 and the                                                                                                                                   Research Council (in medicine; project 06X-09084, 03X-14183, 03BI-
                                                                                                                                            Omp 9            HP0317              81.6   8.67        +       +                    +        +       +   +   +                             +
                                                                                                                                                                                                                                                                                                                                            hypothetical protein HorD.                                                                                                                                                                                                     14930), Swedish Society for Medical Research, the ICR Facility at the
                                                                                                                                            Omp 10           HP0324              28.7   7.85        +       +                         +   +   +   +   +           +         +           +   +        +           Omp11                                                                                                                                                                                                                                                     National High Magnetic Field Laboratory, the National Science
                                                                                                                                            Omp 11           HP0472              21.0   9.16    +   +   +   +   +   +   +   +    +    +   +   +   +   +   +   +   +    +    +   +   +   +   +   +    +   +   +
                                                                                                                                                                                                                                                                                                                                            We found that Omp6 was expressed by a majority of the cancer- (67%) and AS isolates (77%)                                                                                                                                      Foundation (NSF DMR-0084173), the Wilhelm and Martina Lundgren
*The peptides were trapped on a precolumn (4.5 cm x 100 m i.d.) and then separated in a                                                    Omp 12/ Omp22    HP0477/0923         41.7   8.94            +                        +    +       +   +   +           +         +   +           +                +

17 cm x 50 µm column packed with 3 µm RS-C18-AQ. A gradient was run from 0–50%
                                                                                                                                            Omp 13           HP0638              34.1   9.78                    +                +    +   +           +   +   +   +         +   +               +    +                                      but was only expressed by 11% of the DU isolates. This indicates that omp6 might have a                                                                                                                                        Foundation, and the Wenner-gren Foundation
                                                                                                                                            Omp 14           HP0671              30.5   9.17    +   +   +   +                         +               +       +   +    +    +   +       +       +            +
CH3CN, 0.1 % HCOOH (50').                                                                                                                   Omp 15 (HopE)    HP0706              30.2   8.98    +   +   +   +   +   +   +        +    +   +   +   +   +   +   +   +    +    +   +       +   +   +    +   +   +                              protective function against the development of duodenal ulcer.
                                                                                                                                            Omp 16           HP0722              70.6   9.31                                     +        +   +                                 +       +   +                                                                                                                                                                                                                                                                              We are indebted to the Knut and Alice Wallenberg Foundation which
                                                                                                                                            Omp 17 (SabA)    HP0725              69.3   9.12                    +           +    +            +           +                     +       +   +   +    +                                      Our study also demonstrates that Omp26, an outer membrane protein with unknown function,                                                                                                                                       provided funds for the purchase of the FT-ICR mass spectrometer.
**Mass spectrometry analysis was performed in a hybrid linear quadrupole ion trap FT-ICR                                                    Omp 19 (BabB)    HP0896              77.7   8.64
MS (LTQ-FT, Thermo Electron) Survey MS spectra (from m/z 400–1600) with r=100,000                                                           Omp 20 ( AlpA)   HP0912              56.1   9.14                    +       +   +    +                    +   +   +        +    +               +        +       +
                                                                                                                                                                                                                                                                                                                                            was expressed by a higher frequency of the disease-related H. pylori strains (83%) compared
were acquired in the FT-ICR and the three most intense doubly or triply protonated ions in                                                  Omp 21 (AlpB)    HP0913              57.2   9.20                +   +   +   +   +    +    +           +   +   +   +             +   +       +   +   +    +   +   +                              to the isolate from asymptomatic H. pylori carriers (33%). Further studies will evaluate the role
                                                                                                                                            Omp 23           HP1107              25.7   9.12                                          +   +           +   +       +         +           +            +       +
each FT-scan were fragmented and analyzed in the linear ion trap.                                                                           Omp 25           HP1156              76.7   9.12        +       +   +   +                                 +   +   +   +         +           +   +   +    +           Omp26
                                                                                                                                                                                                                                                                                                                                            of Omp26 as a potential marker for disease-related H. pylori strains.                                                                                                                                                                                  REFERENCES
                                                                                                                                            Omp 26           HP1157             132.6   8.86        +   +   +   +   +   +   +    +    +           +   +   +   +   +    +        +       +                +                                                                                                                                                                                                                                                                 1. Baik, S. C.; et al,. J Bacteriol 2004, 186, (4), 949-55.
 ***Proteins were identified by automated database searching (Matrix Science, London,                                                       Omp 27 (HopQ)    HP1177              70.0   9.21    +   +   +   +   +   +   +   +    +    +   +   +   +   +   +       +         +   +   +   +   +   +        +                                  The immunoreactive proteinOmp111 was found to be expressed by all examined strains and is
                                                                                                                                            Omp 28 (BabA)    HP1243              80.1   8.95    +       +   +   +   +   +   +    +    +   +       +   +   +   +   +    +    +   +   +   +   +   +    +   +   +                              therefore a potential candidate for a new vaccine antigen against H. pylori infection. The
United Kingdom) of all tandem mass spectra using the NCBI database. For protein                                                             Omp 30           HP1395              26.7   9.65    +   +   +   +       +                 +   +       +   +   +   +   +    +    +   +   +   +
identification the minimum criteria were, one tryptic peptide matched at or above the 99.9%                                                 Omp 31           HP1469              28.2   9.50        +   +   +   +   +   +             +           +   +   +   +   +    +    +               +   +    +       +                              expression pattern and the properties of Omp11 will now be evaluated in more detail.
level of confidence and additional one peptide match at the 95% level.                                                                      Omp 32 (HopW)    HP1501              43.1   9.51    +   +   +   +           +   +         +   +   +   +   +   +   +   +             +   +   +   +   +    +   +   +