Characterization of the outer membrane protein profile of clinical Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis

1
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
RESULTS
Proteins identified by nano-LC FT-ICR MS analysis
of a single 1D-GE band () from the clinical H.
pylori isolate Can13
INTRODUCTION The human gastric pathogen
Helicobacter pylori is known to colonize a
majority of the global population. In most
infected individuals the colonization is
asymptomatic however, H. pylori infection is
known to cause several gastric disorders such as
duodenal ulcer and gastric cancer. In order to
establish colonization and maintain infection the
bacteria express a variety of different virulence
factors. These include a number of outer membrane
proteins and low-abundance adhesins (e.g BabA,
SabA). Outer membrane proteins are therefore
potential targets in the search for
disease-related biomarkers and potential vaccine
antigens. The aim of this study was to establish
a method that enables characterization of outer
membrane protein (omp) profiles of clinical
isolates of H. pylori in the search for new
protein markers for disease-related H. pylori
strains and identification of potential vaccine
antigens. This was achieved by combining
subcellular fractionation of the bacterial outer
membrane (OM)1 with 1D SDS-PAGE analysis and
high-sensitivity nano-LC FT-ICR MS analysis of
tryptic peptides.
CONCLUSION We have demonstrated that subcellular
fractionation combined with nano-LC LTQ-FT ICR MS
and MS/MS analysis enable identification of the
majority of the theoretically predicted OMPs in
clinical H. pylori isolates. We found a large
heterogenicity of the omp profiles and none of
the analyzed isolates did express all members of
the major H. pylori omp family. We identified
several potential targets for drug discovery
including Omp11, Omp26 and Omp6. Analysis of the
immune response to these proteins in serum
samples from infected individuals are currently
being performed. We conclude that this rapid
subproteome approach is suitable for the
characterization of the outer membrane profile of
human pathogens in the search for novel
protein-markers and new potential target proteins
for vaccine development.
Table 1
Figure 2 (A)
Visualization of the protein pattern of four
different subcellular fractions from H. pylori
The quality and purity of the SLS-insoluble OM
sample was determined by analyzing the protein
content of the different subcellular fractions
from the OM enrichment by small-size SDS-PAGE.

(B)
Lys total cell lysate, cyt cytoplasmic
fraction, OM sarcosine-insoluble outer membrane
fraction, IM sarcosine-soluble inner membrane
fraction.
Table 1. Seventeen unique proteins were detected
in sample () of the outer membrane fraction from
a H. pylori strain (Can 13) isolated from a
patient with gastric cancer. The great majority
of the identified proteins are known to be
expressed on the bacterial surface. Protein
identity is significant at the 99.9 level.
Figure 2 (A) The nano-LC chromatogram of the
tryptic peptides from (B) The doubly protonated
peptide at m/z 1131.591 M2261.167 was
automatically selected for ion trap
dissociation. (C) CID spectrum of the ion at
m/z 1131.591. The assigned amino acid sequence is
unique for the SabA adhesin in H. pylori. Only
fragment ions corresponding to the amino acids
marked in bold are shown.
METHODS
(C)
Expression of the major H.pylori Omp family in
different clinical H. pylori isolates
Identification of potential biomarkers and
vaccine antigen candidates In total, over sixty
unique membrane or membrane-associated proteins
were identified from the different strains. In
addition, we identified several hypothetical
proteins and a small number (less than 10) of
cytoplasmic proteins (data not shown). Of the 33
theoretical predicted H. pylori OMPs we
identified all except Omp18, Omp24 and the
hypothetical protein HorD. We found that Omp6
was expressed by a majority of the cancer- (67)
and AS isolates (77) but was only expressed by
11 of the DU isolates. This indicates that omp6
might have a protective function against the
development of duodenal ulcer. Our study also
demonstrates that Omp26, an outer membrane
protein with unknown function, was expressed by a
higher frequency of the disease-related H. pylori
strains (83) compared to the isolate from
asymptomatic H. pylori carriers (33). Further
studies will evaluate the role of Omp26 as a
potential marker for disease-related H. pylori
strains. The immunoreactive proteinOmp111 was
found to be expressed by all examined strains and
is therefore a potential candidate for a new
vaccine antigen against H. pylori infection. The
expression pattern and the properties of Omp11
will now be evaluated in more detail.
ACKNOWLEDGEMENT The authors acknowledge Thomas
Larsson at the Sahlgrenska academy proteomic core
facility for help with data evaluation. We
gratefully acknowledge the financial support
provided by the Swedish Research Council (in
medicine project 06X-09084, 03X-14183,
03BI-14930), Swedish Society for Medical
Research, the ICR Facility at the National High
Magnetic Field Laboratory, the National Science
Foundation (NSF DMR-0084173), the Wilhelm and
Martina Lundgren Foundation, and the Wenner-gren
Foundation We are indebted to the Knut and Alice
Wallenberg Foundation which provided funds for
the purchase of the FT-ICR mass spectrometer.
Omp6
Omp11
The peptides were trapped on a precolumn (4.5 cm
x 100 ?m i.d.) and then separated in a 17 cm x 50
µm column packed with 3 µm RS-C18-AQ. A gradient
was run from 050 CH3CN, 0.1 HCOOH (50').
Mass spectrometry analysis was performed in a
hybrid linear quadrupole ion trap FT-ICR MS
(LTQ-FT, Thermo Electron) Survey MS spectra (from
m/z 4001600) with r100,000 were acquired in the
FT-ICR and the three most intense doubly or
triply protonated ions in each FT-scan were
fragmented and analyzed in the linear ion trap.
Proteins were identified by automated database
searching (Matrix Science, London, United
Kingdom) of all tandem mass spectra using the
NCBI database. For protein identification the
minimum criteria were, one tryptic peptide
matched at or above the 99.9 level of confidence
and additional one peptide match at the 95
level.
REFERENCES 1. Baik, S. C. et al,. J Bacteriol
2004, 186, (4), 949-55.
Omp26