the human gastric 
 pathogen helicobacter pylori ( h. pylori ) is a microaerophilic , 
 spiral - shaped , gram - negative bacterium responsible for the majority of peptic 
 ulcer diseases in humans .
h. pylori has been 
 shown to be the causative agent of type b gastritis and peptic ulcerations [ 2 , 3 ] .
infection with h. pylori increases the risks of 
 developing gastric carcinoma and mucosa - associated lymphoid tissue ( malt ) 
 lymphoma [ 4 , 5 ] . continuously exposed to 
 acidic ph during the process of colonization in the gastric mucus layer
, h. 
 pylori requires mechanisms to survive acid shocks and to enable growth in 
 such acidic conditions .
this bacterium expresses two nickel - containing enzymes : 
 urease and hydrogenase , both of which is important for its colonization .
the h. pylori urease 
 consists of 12 urea and 12 ureb , and activation of this apoenzyme requires the 
 insertion of 24 nickel ions .
urease hydrolyzes urea into 
 carbon dioxide and ammonia , thereby neutralizing the nearby environment [ 79 ] .
[ ni - fe ] hydrogen - uptake 
 hydrogenase contains a heterobimetallic center in the large subunit with nickel 
 coordinating to four cysteines , and permits respiratory - based energy production 
 for the bacteria in the mucosa [ 10 , 11 ] .
therefore , h. pylori needs 
 significant amounts of nickel to satisfy its requirement for the maturation of 
 the nickel - containing enzymes .
two kinds of high - affinity , nickel - specific 
 uptake systems are found in h. pylori : atp - binding cassette ( abc)-type 
 transporter ( abc abcd ) and nickel - cobalt permease ( nixa ) [ 13 , 14 ] . however , when excess nickel 
 ions accumulate , they exhibit toxic effects and thus inhibit bacterial growth [ 1517 ] .
h. pylori has developed a system to maintain nickel homeostasis , 
 keeping a balance between the import , intracellular storage , and export of 
 nickel ions .
analysis of the h. pylori genome sequence has discovered 
 several putative ion binders and membrane transporters involved in metal 
 homeostasis .
however , the related 
 nickel - interacting proteins have not been fully identified so far .
immobilized - metal affinity chromatography 
 ( imac ) is a separation technique commonly used for fractionating proteins based on their different binding affinities of the 
 surface - exposed amino acids towards immobilized metal ions [ 19 , 20 ] .
ni belongs to the 
 group of intermediate metal ions , preferring coordination to nitrogen , oxygen , 
 and sulfur , and especially favors binding to the proteins with two exposed 
 vicinal histidines .
elution of the target 
 proteins is achieved by lowering the ph or by adding a competing reagent such 
 as imidazole .
two - dimensional gel 
 electrophoresis ( 2-de ) is a common choice for separating cellular proteins first 
 by their isoelectric point ( pi ) and then by molecular weight ( mw ) . 

however , low - abundance proteins may not be detected in 
 2-de due to its limited separation capacity .
imac 
 may compensate this , in some way , by specifically serving to enrich the 
 low - abundance metal - binding proteins and to reduce protein complexity . in this study ,
proteomic technology was employed 
 for the first time to isolate and identify candidate proteins involved in 
 nickel transport , storage , and utilization in h. pylori , by integrating 
 the powerful tools of ni - imac , 2-de , and matrix - assisted laser desorption 
 time - of - flight mass spectrometry ( maldi - tof ms ) . those proteins with surface active coordinating residues for 
 binding nickel ( and maybe other metals with similar coordinating features )
the information obtained from the 
 identification and functional analysis of these nickel - related proteins may 
 improve our understanding of nickel homeostasis in bacteria .
xia ( department of medicine , the university of hong kong ) , and cultured in the 
 basal medium , brucella broth ( difco ) with 5% ( v / v ) fetal bovine serum ( fbs ; 
 gibco / brl life technologies ) , for 72 hours with orbital shaking ( 100 rpm ) at 37c in an anaerobic jar with a 
 microaerobic gas - generating kit ( oxoid ) .
solutions used in this study were 
 prepared with ultra - pure water ( 18.2 m ; millipore ) .
h. pylori 11637 cells grown in 100 ml liquid media to 
 mid - log phase were pelleted at 8000g for 5 minutes at 4c , and washed three times with 10 ml 
 ice - cold phosphate buffered saline ( pbs ) .
cell pellets were resuspended in 10 
 ml of ice - cold buffer a ( 20 mm sodium phosphate buffer , 500 mm nacl , 10 mm imidazole , 1 mm pmsf , 
 ph 7.4 ) .
bacteria were ruptured with sonication in the presence of 1% v / v triton 
 x-100 .
proteins were recovered ( 10,000g , 30 minutes ) , and the 
 supernatant was filtered through a 0.45 m 
 cellulose acetate syringe filter ( iwaki glass , japan ) before being loaded onto 
 a house - made , buffer a - equilibrated ni - nta agarose column ( 0.5 ml , qiagen ) .
after 
 washing with 10 column volumes of buffer a , proteins were eluted with buffer b 
 ( 20 mm sodium phosphate buffer , 500 mm nacl , 500 mm imidazole , ph 7.4 ) .
the 
 fractions were concentrated with centricon ym-3 
 ( millipore ) , and buffer - exchanged into rehydration solutions ( 8 m urea , 4% chaps , 1 mm pmsf , 20 mm dtt , 2% pharmalyte ph 311 ) with plusone 2-d clean - up kit ( amersham biosciences , 
 buckinghamshire , 
 uk ) .
protein concentrations were determined by bca assay ( bio - rad , calif , 
 usa ) using bsa as the standard .
2-de was carried out with an ipgphor ii isoelectric focusing 
 ( ief ) unit and hoefer se 600 ruby electrophoresis unit ( amersham biosciences ) 
 according to the method detailed elsewhere .
briefly , 100 g of h. 
 pylori protein samples were diluted in rehydration solutions containing 
 traces of bromophenol blue .
ief was carried out with precast 13-cm immobiline 
 drystrip ( ipg strips ; amersham biosciences ) to generate a nonlinear ph gradient 
 of 310 .
following ief , strips were immediately used for the second dimensional 
 sds - page ( 10 ) .
proteins 
 were visualized with silver stain , and protein molecular weight ( mw ) was 
 calibrated with prestained sds - page marker ( broad range ; bio - rad ) . to minimize 
 gel to gel variations
the silver - stained gels were scanned ( image scanner ; amersham 
 biosciences ) and analyzed with imagemaster 2d elite software ( amersham 
 biosciences ) .
the normalized intensity 
 ( ni ) for each protein spot was calculated as the ratio of the spot intensity versus 
 the sum of intensities of the spots present in the whole gel .
protein spots 
 were cut out from the silver - stained gels and enzymatically digested overnight 
 with sequence grade porcine trypsin ( promega ) at 37c .
the masses of 
 the digested peptides were determined with a voyager - de str biospectrometry 
 workstation ( applied biosystems , calif , usa ) .
protein identification was 
 performed by searching the ncbinr protein database using protein prospector ( http://prospector.ucsf.edu ) ( 3 ) , with 
 pyroglutamic acid modification of n - terminal glutamine , oxidation of methionine , 
 and protein n - terminal acetylation as permissible modifications .
the criteria 
 for searching were set at 50 ppm or better mass accuracy , at least four 
 matching peptide masses , as well as theoretical mw and isoelectric point ( pi ) 
 matching to the estimated values from gels .
to investigate nickel 
 binding proteins in h. pylori under 
 physiologically relevant conditions , a relatively mild and nondenaturing lysis method was applied prior to nickel - imac 
 ( ni - imac ) loading .
our previous experiments have shown 1-d gels for the total extract of h. pylori 11637 cells and extract fractions 
 eluted from a ni - imac column , displaying many protein bands for both cases .
we have now further 
 separated and identified nickel - interacting proteins in h. pylori using 2-de .
the gel image of whole cell extracts 
 of h. pylori 11637 ( see figure 1(a ) ) has a protein distribution very similar 
 to the standard proteome pattern of h. pylori 26695 .
more than 800 protein spots were separated , 
 with molecular weights ranging from 6 to 200 kda and pi values from 3 to 10 .
figure 1(a ) shows a representative gel 
 image of ni - related proteins in h. pylori 11637 .
the ratio 
 between the number of ni - binding proteins and total extracted proteins of whole 
 cell lysates was 1:57 , indicating that nickel specifically binds to a 
 restricted number of proteins .
qualitative comparison of 2-d gels between the ni - enriched 
 eluates ( see figure 1(a ) ) and the total extracts ( see 
 figure 1(b ) ) revealed remarkably different spot patterns .
ni - binding protein spots were excised , 
 trypsin - digested , and subjected to maldi - tof ms sequencing .
mass spectra of tryptic peptides were used to identify 
 proteins by matching to the spectra of the protein sequence databases , and the 
 identification was further verified by comparing to the standard proteomic map 
 of h. pylori . in total , twenty two proteins were identified as candidate 
 ni - interacting proteins , as 
 marked in figure 1(a ) and summarized in table 1 .
some of 
 the protein spots could not be annotated due to no substantial ms signals or inadequate 
 peptide coverage for confident identification .
interestingly , five of the 
 proteins identified , including 
 urea , hspb , fumarase , pfr , and hypothetical protein jhp0301 , show 
 more than one spot 
 in the 2-de map , indicating the presence of posttranslational modifications . 

protein isoforms typically present themselves as a series of spots that differ 
 slightly in their molecular masses and pi , that is , numerous closely 
 spaced spots in 2-de profiles [ 2628 ] .
five proteins ( hspa , hspb , 
 fumarase , tsaa , and napa ) were also identified in the bi - interacting profile , indicating that there was 
 some correlation between bi and ni interactions .
the ni - interacting 
 proteins can be classified into five general functional categories : 
 cellular processes ( hspa , hspb , tsaa , and napa ) , enzymes ( urease , fumarase , 
 guab , cad , ppase , and dmpi ) , membrane - associated proteins ( om jhp1427 and 
 hpaa ) , iron storage protein ( pfr ) , and hypothetical proteins ( hp0271 , hp 
 jhp0216 , hp jhp0301 , hp0721 , hp0614 , and hp jhp0118 ) .
nickel homeostasis 
 is required for the establishment of h. pylori infection in animals , necessitating a balance in the nickel import , storage , 
 and delivery for the synthesis and activity of nickel - dependent metalloenzymes . 

consequently , 
 an analysis to identify the nickel - interacting proteins in h. pylori may be able to reveal candidate proteins for further 
 characterization and validation to provide drug targets . in order 
 to live in the acidic gastric environment , h. pylori
continuously synthesize urease catalyzing the hydrolysis of urea to 
 ammonia and carbamate to elevate ph .
both subunits of urease were observed 
 in the 2-de gel after ni - nta enrichment , suggesting each of them can bind nickel 
 ions ( see figure 1(a ) ) .
expression of active urease is essential for h. pylori infection in all animal models 
 and for acid survival in vitro .
urease activity is 
 significantly enhanced in the presence of nickel , additional incorporation of 
 ni into the apoenzyme is thus the major regulating event upon 
 higher ni availability .
heat shock proteins are a group of highly 
 conserved , abundantly expressed proteins with protective advantage through 
 their functions as molecular chaperones , assisting proper folding of a 
 number of substrate proteins that are otherwise destined to aggregation [ 30 , 31 ] .
urease activity was increased four folds in e. coli when coexpressed with hspa , 
 suggesting that hspa possibly plays an important role in the activation of 
 urease , probably by means of its ni - binding domain in the c - terminus .
hspa has two distinct ni - binding 
 sites : a high - affinity site ( kd  2.8 m ) and 
 a lower affinity site ( kd  30 m ) . 

the h. pylori groel homologue hspb belongs to the hsp60 family , and has been shown to increase the risk of 
 gastric carcinoma , possibly by directly inducing the hyperproliferation 
 of gastric cells .
hspb also was suggested to 
 be responsible for the protection and regulation of urease activity .
many identified ni - interacting proteins in h. pylori are involved in antioxidant , 
 antitoxic , and antiinflammation machinery .
pylori tsaa is a major 
 component of the thioredoxin - dependent thiol - specific antioxidant ( tsa ) system 
 that catalyzes the reduction of hydroperoxides   and peroxynitrite .
the cleavage of tsaa 
 suppresses the protecting response of h. pylori cells against oxidative 
 stress in two possible ways : directly by the way of nucleophilic attacking the 
 peptide bond through metal - bound hydroxide ions , and/or indirectly by way of stimulating 
 the activities of specific proteases .
it 
 was named because of its ability to mediate neutrophil adhesion to endothelial 
 cells , and to bind to mucin and 
 neutrophil glycosphingolipids .
napa was identified as a 150 kda 
 dna - binding dodecamer that protects cells from dna damage caused by free 
 radicals under oxidative stress [ 39 , 40 ] .
napa was found to be positively 
 regulated by iron , repressed by ferric uptake regulator ( fur ) , and unaffected by copper , nickel , 
 or zinc .
an excess of iron is 
 potentially harmful as it catalyzes the formation of toxic reactive oxygen 
 species ( ros ) via fenton chemistry .
ferritin protein pfr , the major iron 
 storage protein of h. pylori , is also 
 regulated by iron , nickel , zinc , and copper .
the accumulation of this protein 
 under iron - rich conditions allows h. pylori to maximize the iron storage 
 capacity in response to an increase in iron availability .
it is essential for the 
 bacteria to adapt to low - iron conditions [ 4245 ] .
obviously , ni - imac is also able to trap pfr with its cation binding 
 affinity .
immobilized - nickel affinity chromatography and proteomic analysis 
 were integrated to separate and identify the ni - interacting proteins in h. pylori .
the results suggest that 
 ni - interacting proteins are mainly involved in cellular processes , oxidative 
 stress , and metabolism of the bacteria .
this study demonstrated that metalloproteomic 
 technique can be utilized to efficiently identify metal - related proteins that 
 may play crucial roles in metal homeostasis .
these proteins may be potential targets 
 for designing and constructing drugs to suppress the bacterial infection .
 
 helicobacter pylori ( h. pylori ) is a widespread human pathogen causing peptic ulcers and chronic gastritis . 
 maintaining nickel homeostasis is crucial for the establishment of 
 h. pylori infection in humans . 
 we used immobilized - nickel affinity chromatography to isolate ni - related proteins from h. pylori cell extracts . 
 two - dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two ni - interacting proteins in h. pylori . 
 these ni - interacting proteins can be classified into several general functional categories , including cellular processes ( hspa , hspb , tsaa , and napa ) , enzymes ( urease , fumarase , guab , cad , ppase , and dmpi ) , membrane - associated proteins ( om jhp1427 and hpaa ) , iron storage protein ( pfr ) , and hypothetical proteins ( hp0271 , hp jhp0216 , hp jhp0301 , hp0721 , hp0614 , and hp jhp0118 ) . the implication of these proteins in nickel homeostasis is discussed . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS AND DISCUSSION
4. CONCLUSION