protein toxins are a major etiological agent of severe illness caused by foodborne bacterial pathogens .  
the importance of bacterial protein toxins has resulted in the development of techniques to identify them as well as other virulence factors a microorganism may harbor .  
for example , polymerase chain reaction ( pcr ) may be used to amplify short segments of toxin genes demonstrating the presence of such genes and the potential of the microorganism to express the toxin     [ 24 ] .   alternatively , an enzyme immunoassay ( eia ) may be used to indirectly detect the actual expression of a protein toxin by a microorganism     [ 2 , 5 , 6 ] .  
another approach for toxin detection is to measure its toxic effects on mammalian cells in vitro     .   because of its speed , sensitivity , and high specificity ,
mass spectrometry has been used to unambiguously detect , characterize , and identify chemical compounds including protein toxins .   
one of the most important bacterial protein toxins is shiga toxin ( also referred to as shiga - like toxin or verotoxin )     [ 911 ] .  
shiga toxin ( stx ) is an ab5 toxin possessing an - and -subunit .  
a single -subunit is positioned primarily on one side of the -complex ( a1 ) and the central core formed from the -pentamer ( a2 ) .  
a highly accessible 20-residue loop in the polypeptide chain of the -subunit facilitates cleavage of the -subunit into its a1 and a2 fragments .  
when released by bacterial cell lysis in the human gut , the holotoxin attaches itself to the glycosphingolipid globotriaosylceramide ( gb3 also referred to as cd ) receptors on the surface of intestinal endothelial cells and/or other cells in the human body that have the same surface receptor , for example , kidney cells     [ 12 , 13 ] .  
attachment of the -complex to gb3 occurs on the side opposite to that of the -subunit .     
early endosomes are transported , in a retrograde fashion , to the golgi apparatus and eventually to the endoplasmic reticulum ( er ) .   at some point in the early endosome , the -subunit is proteolytically cleaved by furin , a membrane protein .  
however , even after this cleavage , the a1 and a2 fragments are still bound by a disulfide bond .  
this disulfide bond is subsequently reduced in the lumen of the er , and the catalytically active a1 fragment is then translocated to the cytosol where it disrupts eukaryotic ribosomal protein synthesis eventually resulting in cell death     [ 9 , 14 ] .
previous approaches that use matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ) for bacterial identification typically attempt to detect as many bacterial proteins as possible in order to obtain as unique an ms  fingerprint  as possible     [ 1529 ] .  
alternatively , matrix - assisted laser desorption / ionization time - of - flight time - of - flight tandem mass spectrometry ( maldi - tof - tof - ms / ms ) has been used to fragment intact bacterial proteins by ms / ms and identify them ( and their source microorganism ) by top - down proteomic analysis     [ 3033 ] .  
we have recently reported using maldi - tof - tof - ms / ms to ionize , isolate , and fragment intact protein ions from bacterial cell lysates     [ 31 , 32 ] .   using software developed in - house , the mass - to - charge ( m / z ) of ms / ms fragment ions
are compared to a database of in silico fragment ion m / z derived from bacterial protein sequences having the same molecular weight ( mw ) as that of the protein ion .  
a simple peak matching algorithm     [ 31 , 32 ] and a p - value scoring algorithm ( developed by others ) were used to independently score / rank identifications .  
this approach to bacterial protein identification is particularly attractive as sample preparation is simple and does not require pre - enrichment or chromatographic separation of proteins .  
maldi - tof - tof - ms / ms analysis is quickly accomplished after an equally rapid maldi - tof - ms analysis .  
both maldi - tof - ms and maldi - tof - tof - ms / ms approaches are accomplished by culturing bacterial cells on media that facilitates bacterial growth .  
however , such growth conditions do not necessarily result in expression of important bacterial virulence factors which could be used to characterize the pathogenicity of the microorganism .     in the current study
, we induce expression of a specific bacterial virulence factor most often linked to cases of severe foodborne illness     [ 12 , 13 ] .  
we have applied our top - down maldi - tof - tof - ms / ms technique to the identification of the -subunit of shiga toxin 2 ( -stx2 ) from a genomically sequenced strain of e. coli o157:h7     .  
expression of -stx2 was induced by culturing on a conventional solid agar supplemented with an antibiotic that is a powerful inducer of the bacterial sos - response     [ 3538 ] .
the e. coli o157:h7 strain used in the current study is the genomically sequenced strain edl933 ( atcc # 43895 ) which possesses the gene for shiga toxin 2 ( stx2 ) as well as other virulence factors     .  
expression of stx2 in e. coli o157:h7 was induced by overnight culturing at 37c on luria - bertani agar ( lba ) plates supplemented with 20  ng / ml of ciprofloxacin ( cp ) , a fluoroquinolone antibiotic .  
the use of fluoroquinolones to induce expression of stx has been described previously     [ 3538 ] .  
one  l of bacterial cells was harvested with a sterile 1  l transfer loop and transferred to a sterile 2  ml microcentrifuge tube containing 300  l of a sterile solution of 3  mm molecular grade cacl2 ( sigma - aldrich , st .
louis , mo ) in hplc grade water ( burdick and jackson ) and approximately 100  mg of 0.1  mm zirconia - silica beads ( biospec products , bartlesville , ok ) .  
the tubes were bead - beat for 60 seconds on a reciprocating shaker ( mini - beadbeater ; biospec products , bartlesville , ok ) after which they were centrifuged at 14,000  rpm for 2 minutes .   
one l of furin enzyme ( 2000  units / ml , new england biolabs , ipswich , ma ) was added to 30  l of bacterial cell supernatant in a sterile 500  l thin - walled pcr tube and incubated at 37c for 10 , 30 , or 60 minutes .  
the furin recognition site ( rxxr ) for cleavage of the -stx2 is located in a highly accessible loop of the polypeptide chain between two cysteine residues ( cys and cys ) which form an intramolecular disulfide bond ( figure 1 )     [ 14 , 39 ] .  
after furin digestion , 1.5  l of 1  m molecular grade dithiothreitol ( dtt , sigma - aldrich , st .
louis , mo ) was added to the reaction mixture followed by incubation at 70c for 10 minutes in order to reduce the intramolecular disulfide bond and unlink the two fragments ( a1 and a2 ) of the -subunit .
dtt appeared to inhibit furin enzyme activity ; in consequence , it was added after completion of the furin digestion step .  
approximately , 2  l of bacterial cells were harvested from a cp - supplemented lba solid agar plate after overnight growth and transferred to a microcentrifuge tube with 300  l of extraction solution and processed as previously described .   for one sample , 4  l of furin
were added to 30  l of bacterial cell lysate and incubated at 37c for 3 hours followed by a disulfide reduction step as previously described .  
a 19.5  l aliquot of this sample was combined with 6.5  l of nupage lds sample buffer ( invitrogen , carlsbad , ca ) .   for another sample
, 16.9  l of bacterial cell lysate was added to 6.5  l lds sample buffer and 2.6  l nupage reducing agent ( nupage , invitrogen , carlsbad , ca ) and incubated for 10 minutes at 70c .  
a total of 25  l of each sample was applied to separate lanes of a 1.0  mm nupage novex 412% bis - tris mini - gel ( nupage , invitrogen , carlsbad , ca ) .  
the upper ( cathode ) buffer chamber consisted of 200  ml of mes sds running buffer containing 500  l of nupage antioxidant reagent ( invitrogen , carlsbad , ca ) to maintain proteins in a reduced state during electrophoresis .  
a constant - voltage ( 200  v ) was applied for 40 minutes .  
the gel was stained with a coomasie staining solution ( simplyblue - safestain , invitrogen , carlsbad , ca ) using the supplier 's microwave procedure and imaged ( supplementary materials ) .  
relevant gel bands were excised and in - gel digested with porcine trypsin ( promega , madison , wi ) using standard protocols ( digestpro , intavis , langenfeld , germany ) .
twenty  l of each digest were separated by nano - hplc ( lc packings / dionex , sunnyvale , ca ) and analyzed by ms and ms / ms using a q - star - pulsar i ( applied biosytems , foster city , ca )    
.   peptides ( and their corresponding proteins ) were identified by analysis of ms and ms / ms data using mascot   using ncbinr and swissprot databases .
taxonomy was left unrestricted so as to search against both bacterial and bacteriophage databases .  
the top protein / organism identifications were escherichia coli and/or enterobacteria phage bp-933w which is present in the e. coli o157:h7 ( strain edl933 ) .   peptide and protein identifications are provided in supplementary materials available online at doi : 10.1155/2010/123460 .
mass spectrometry and top - down analysis has been described in detail     [ 31 , 32 ] .  
briefly , 0.5  l of sample was spotted onto a 384-well stainless steel target and allowed to dry at room temperature .  
the dried sample spot was overlayed with 0.5  l aliquot of saturated maldi matrix solution of -cyano-4-hydroxycinnamic acid ( hcca , fluka analytical ) or 3,5-dimethoxy-4-hydroxycinnamic acid ( sinapinic acid , fluka analytical ) in 67% hplc grade water , 33% hplc grade acetonitrile , and 0.2% trifluoroacetic acid .  
ms and ms / ms analysis was performed using a 4800 plus maldi - tof / tof tandem mass spectrometer ( applied biosystems , foster city , ca ) equipped with a 200  hz yag laser (  = 355  nm ) .    ms analysis was performed in linear positive ion mode using default instrument settings .  
the linear mode was externally calibrated with a mixture of cytochrome c ( mw = 12,361.088  da ) , lysozyme ( mw = 14,306.200  da ) , and myoglobin ( mw = 16,952.551  da ) .  
the reflectron mode was externally calibrated using the fragment ions at mass - to - charge ( m / z ) of 175.119 , 684.346 , 813.389 , 1,056.475 , and 1,441.634 from postsource dissociation ( psd ) of the singly charged ( protonated ) precursor ion of glu - fibrino - peptide b ( sigma - aldrich , mw = 1,570.60  da ) .    for intact protein ion fragmentation
, the instrument was operated using the default settings of the operating mode : ms / ms 1  kv positive sensitivity .  
the metastable suppressor was enabled and the timed ion selector ( tis ) was operated at a resolution of 100  da .   in order to maximize protein ion fragmentation efficiency , a higher than normal laser fluence was utilized     [ 3032 ] .  
positive ions were accelerated from the source at 8.0  kv , decelerated to 1  kv in the collision cell , and reaccelerated to 15  kv in the second source .  
raw ms and ms / ms data were processed using the commercial software provided with the instrument ( data explorer v4.9 , applied biosystems , foster city , ca ) .  
ms spectra were processed with a noise filter ( correlation factor = 0.7 ) and then centroided .  
ms / ms spectra were processed , first with an advanced baseline correction ( peak width = 32 , flexibility = 0.5 , degree = 0.1 ) , followed by a noise removal ( standard deviation = 2 ) , followed by a gaussian smooth ( filter width = 31 points ) , and then centroided .  
processed ms and ms / ms data were exported from the instrument software as an ascii file ( m / z versus absolute intensity ) and then uploaded to their respective databases in the usda software .
the usda software and its use for top - down proteomic analysis has been described in detail .  
briefly , the program rapidly compares the m / z of ms / ms fragments to the m / z of in silico fragment ions derived from bacterial protein sequences that have the same molecular weight ( mw ) as that of the protein biomarker ion being analyzed .
the program was written using a java runtime environment with mysql database management and a tomcat / apache web interface .  
bacterial protein sequences were retrieved using the tagident software tool from expasy website ( http://www.expasy.org/ ) from uniprotkb / swiss - prot databases ( versions 18-may-2010 or 15-june-2010 ) .
the downloaded multisequence fasta files were processed using a beta - version gpmaw ( version 8.01 a5 ) and the resulting files were uploaded to the in silico database of the usda software .   the usda software uses a simple peak matching algorithm   and a p - value algorithm ( developed by demirev and coworkers ) to independently score / rank ms / ms - to - in silico comparisons .

 figure 1 shows the full protein sequence of the -stx2 from e. coli o157:h7 strain edl933 ( ave .
however , this protein toxin has a 22-residue n - terminal signal peptide as well as a disulfide bridge between cys and cys ( cys and cys in the posttranslationally modified protein ) .  
furin cleavage of the -stx2 ( followed by disulfide reduction ) should result in formation of two fragments : a1 ( a1-stx2 ) with an average mw of 27,927.5  da and a2 ( a2-stx2 ) with an average mw of 5284.9  da .  
figure 2 shows maldi - tof - ms spectra of furin - digested bacterial cell lysate of e. coli o157:h7 cultured on solid agar supplemented with 20  ng / ml of ciprofloxacin .  
each spectrum includes an insert of the expanded display range at m / z 5300 .  
the most prominent peak in the spectra in figure 2 is at m / z 7814 .  
this protein ion peak corresponds to the -subunit of stx2 ( -stx2 ) as identified by top - down proteomic analysis .  
as each -subunit monomer is part of a pentamer complex in solution to which a single -subunit is associated , it is to be expected that the -subunit is the most prominent protein ion given the 1  :  5 stoichiometric ratio of   :   subunits for the ab5 holotoxin .   a peak at m / z ~ 5286 , corresponding to the a2-stx2 ,
is not observed when furin digestion is not included in the sample preparation ( figure 2(a ) ) .  
a peak observed at   m / z 5251 is approximately one - half the   m / z of a peak at   m / z 10497 .  
ms / ms of the peaks at   m / z 5251 and   m / z 10497 was performed which showed several prominent fragment ions that were common to both ms / ms spectra suggesting that these ions are the + 2 and + 1 charge state of the same protein , respectively ( supplementary materials ) .
unfortunately , we were unable to identify this protein by either top - down or bottom - up proteomic analysis .  
the data acquisition upper mass range was extended up to   m / z 30,000 ; however , no peaks were observed that corresponded to a1-stx2 .    furin digestion of the bacterial cell lysate for 10 minutes resulted in the maldi - tof - ms spectrum ( figure 2(b ) ) which shows the sudden appearance of what is presumably a singly charged ion at   m / z 5286 .  
the neighboring doubly charged ion at m / z ~ 5250 is also present .  
as the furin digestion time increases from 10 to 30 to 60 minutes , the absolute intensity of the peak at   m / z 5286 increases proportionally with a concomitant increase in its signal - to - noise ( s / n ) .   a plot of the absolute intensity of the peak at   m / z 5286 as a function of furin digestion time is shown in figure 2(e ) .  
the strong correlation between peak intensity and furin digestion time suggests that the peak at   m / z 5286 is the a2-stx2 .   in order to definitively identify it ,
this peak was subjected to maldi - tof - tof - ms / ms and top - down analysis .   

figure 3 shows the ms / ms spectrum of the peak at   m / z 5286 .  
fragment ions with a corresponding in silico fragment ion match to the top - ranked protein identification ( a2-stx2 in table 1 ) are identified by their m / z , the in silico fragment ion type / number and the two amino acid residues immediately adjacent to the sequence cleavage site that resulted in the fragment ion .  
some of the more intense ms / ms fragment ions are matched to in silico fragment ions adjacent to aspartic acid ( d ) , glutamic acid ( e ) , and proline ( p ) residues .  
fragmentation of the polypeptide chain of singly charged ( protonated ) protein ions at d , e , or p residues has been noted previously     [ 3033 , 43 ] .  
these residues facilitate fragmentation by either transferring a proton from their side - chain to the polypeptide backbone ( as in the case of d and e residues ) or by creating an internal energy bottleneck in the molecule ( as in the case of p residue that results in a 90 turn in the polypeptide backbone ) .  
as shown in figure 3 , a number of ms / ms fragment ions were also matched to in silico fragment ions that are adjacent to non - d / e / p - residues .  
the relatively small size of this protein ion results in fragmentation throughout the entire polypeptide as shown in the insert of figure 3 ( not just at d , e , or p residues ) .  
the fragmentation efficiency of smaller protein ions is higher compared to larger protein ions due to the fact that larger protein ions have a greater number of vibrational degrees - of - freedom to redistribute their internal energy .   in the current experiment ,
the energy is then rapidly redistributed and the metastable protein ion fragments sometime after exiting the source , that is , postsource dissociation or psd .   as the size of the protein ion being analyzed increases , fragmentation is more likely to occur at dissociation channels that require the least amount of energy , that is , cleavage at d , e , or p residues .  
six prominent fragment ion peaks in figure 3 ( marked by solid circle ) had no corresponding in silico match to the a2-stx2 sequence but were found to be present in the ms / ms spectrum of the doubly charged ion at m / z ~ 5250   ( figure s1 , supplementary materials ) .  
even at the highest tis resolution ( 50  da ) , it was not possible to fully resolve and thus mass isolate the ion at   m / z 5286 from the ion at m / z ~ 5251 .   in consequence
, some fragment ions from the precursor ion at   m / z 5251 are present in the ms / ms spectrum for   m / z 5286 .    

table 1 shows the top ranked identifications of the protein ion at   m / z 5286 analyzed by maldi - tof - tof - ms / ms and top down proteomics .  
ms / ms fragment ions were analyzed using a non - residue - specific in silico comparison to 3833 bacterial protein sequences having the same mw as that of the biomarker ( within 10  da ) .  
the top - ranked identification of both the usda score and the p - value is the a2 fragment of the furin - cleaved -stx2 .  
nearly all of the more prominent ms / ms fragment ions shown in figure 3 were matched to in silico fragment ions of a2-stx2 .  
as can be seen from table 1 , the sequence of a2-stx2 is common to several serotypes and strains of e. coli ( including o157:h7 ) as well as acinetobacter haemolyticus and many bacteriophages .  
bacteriophages play a critical rle in facilitating horizontal gene transfer across bacterial genera , species , subspecies , subtypes , and strains     [ 44 , 45 ] .  
the fact that a. haemolyticus has recently been reported to carry the stx2 gene   suggests that the spread of stx genes ( and other virulence factors ) across bacteria , facilitated by bacteriophages , may be increasing .  
the second ranked identifications in table 1 have p - values that are 10 orders - of - magnitude less significant compared to that of the top - ranked identification .  
the % of matched ms / ms fragment ions ( usda score ) is nearly double that for the top ranked identification compared to second - ranked identifications .  
the usda algorithm is approximately 8 times faster ( 84 seconds ) to score than the more mathematically complex p - value algorithm ( 654 seconds ) due to the number of ms / ms fragment ions being compared ( 118 ) and the number of bacterial protein sequences being compared against ( 3833 ) .   
the a1 fragment of the furin - cleaved -stx2 was not observed by maldi - tof - ms .   in consequence , we analyzed , by 1-d page , the proteins from cp - supplemented and cp - supplemented / furin - digested bacterial cell lysates ( figure s3 , supplementary materials ) .
in the bacterial cell lysate of the cp - supplemented sample , we observe a band at 33  kda which is absent from the cp - supplemented / furin - digested sample .  
this gel band was excised , in - gel digested , and analyzed by bottom - up proteomics .  
the band corresponded to -stx2 and , as shown in figure 4 , the sequence coverage included partial sequences a1 and a2 and no sequence coverage of the signal peptide as one would expect for the mature protein .  
the 33  kda also corresponds closely to the theoretical mw of 33,192.3  da .   in the bacterial cell lysate of the cp - supplemented / furin - digested sample ,
we observe bands at 28  kda and 5 - 6  kda which are absent from the cp - supplemented sample .  
these bands were also analyzed by bottom - up proteomics and identified as -stx2 with partial sequence coverage that was consistent with the a1 and a2 fragments , respectively ( figure 4 ) .  
these results strongly suggest that both a1 and a2 fragments of -stx2 are present in the cp - supplemented / furin - digested sample , but only the a2 fragment is successfully ionized by maldi .
there are a number of possible explanations that may account for the failure to ionize the a1 fragment of -stx2 by maldi .  
second , the primary sequence , secondary , or tertiary structure of a1 may not efficiently ionize ( protonate ) compared with other analytes present in the sample .  
we do not observe any evidence for furin cleavage at other potential recognition sites ( rxxr ) in the primary structure of a1 .  
it is possible these other sites are not readily accessible to furin and may suggest a compact tertiary structure for a1-stx2 that thwarts ionization .  
third , a1 is the catalytically active fragment that binds to eukaryotic ribosomal rna and disrupts protein synthesis .  
it has been previously reported that a1-stx2 also binds to prokaryotic rna or dna     .  
it is possible that the difficulty in detecting a1 could be due to its being bound to bacterial rna or dna whose mass exceeds the upper mass detection limit of our instrument .  
preliminary experiments were conducted to process samples with rnaase and dnaase on the assumption that cleaving the rna or dna into smaller units might facilitate ionization of the a1 fragment .   however ,
we have detected and identified the antibiotic - induced , furin - cleaved -stx2 from e. coli o157:h7 using maldi - tof - tof - ms / ms and top - down proteomics .  
bacterial cell lysates from growth on conventional solid agar supplemented with ciprofloxacin strongly induced expression of stx2 allowing ionization by maldi .  
the human enzyme furin was used to cleave -stx2 at the highly accessible polypeptide loop that joins the a1 and a2 fragments of -stx2 .    a further disulfide - reduction step completely unlinked a1 from a2 .   in the absence of furin digestion and/or disulfide reduction ,
no peak was observed that corresponded to the a2 fragment suggesting that both steps , performed sequentially , are necessary for successful release of the a2-stx2 and its ionization by maldi .  
this sequential two - step process follows the model proposed for eukaryotic intracellular processing of -stx .  
the higher mw a1-stx2 fragment , although detected and identified by 1-d gel electrophoresis and bottom - up proteomics , did not appear to ionize efficiently by maldi as an intact protein .   
top - down proteomics utilizing maldi - tof - tof - ms / ms and psd is a rapid technique for identifying high copy proteins expressed under standard culturing conditions or highly expressed proteins that have been induced to express under specific culturing conditions .  
identification of such proteins can assist in the identification or characterization of bacteria and their potential for causing severe illness due to the virulence factors that they can express , for example , protein toxins .
mention of a brand or firm name does not constitute an endorsement by the u.s .
this article is a us government work and is in the public domain in the usa .
 a method has been developed to identify the -subunit of shiga toxin 2 ( -stx2 ) from escherichia coli o157:h7 using matrix - assisted laser desorption / ionization time - of - flight - time - of - flight tandem mass spectrometry ( maldi - tof - tof - ms / ms ) and top - down proteomics using web - based software developed in - house . 
 expression of stx2 was induced by culturing e. coli o157:h7 on solid agar supplemented with an antibiotic that elicits the bacterial sos - response . 
 bacterial cell lysates were incubated in the presence of furin , a human enzyme , that cleaves -stx2 into a1 ( ~28  kda ) and a2 ( ~5  kda ) protein fragments . 
 a subsequent disulfide reduction step unlinked a1 from a2 .   
 maldi - tof - ms of the furin - digested / disulfide - reduced sample showed a peak at mass - to - charge ( m / z ) 5286 that corresponded to the a2 fragment .   
 no peak was observed that corresponded to the a1 fragment although its presence was confirmed by bottom - up proteomics .   
 the peak at m / z 5286 was definitively identified by maldi - tof - tof - ms / ms and top - down proteomics as the a2 fragment of -stx2 . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusions
 Disclosure