Source of Article:http://www.spectroscopynow.com/coi/cda/detail.cda?id=19864&type=Feature&chId=10&page=1
One of the most common food-borne
diseases is staphylococcal food poisoning, caused by the bacterium Staphylococcus
aureus. It accounts for hundreds of
thousands of cases each year and vies with Salmonella for top spot
in the food poisoning league table. The individual organisms exist in pairs
and in grape-like clusters, which gave rise to the name: the Greek word staphyle means a bunch of grapes.
Attractive though the nomenclature is, the bug is less
alluring, provoking serious vomiting and diarrhoea
for up to eight hours after ingestion. It has also spawned a form known as methicillin-resistant S. aureus
(MRSA) which has played havoc in hospitals, resulting in the deaths of
vulnerable patients.
The active staphylococcal agents are a set of proteins
called enterotoxins, 19 of which have been
identified to date. However, the shortage of specific diagnostic tools for
many of these toxins means that a significant number of suspected
staphylococcal food poisonings have not been proven. The classical methods
involve ELISA immunological tests for just five of the staphylococcal
agents (A, B, C, D and E). Even then, the tests have serious drawbacks,
which have been summarised by a team of French
researchers.
VirginieBrun, Alain Dupuis and
Jerome Garin, all affiliated to the three Grenobleorganisations of
the Institute of Life Sciences Research and Technologies, INSERM
U880 and the University
of Joseph Fourier,
and Jacques-Antoine Hennekinne from the French
Agency for Food Safety, recognised three
problems. Very few specific antibodies are available due to the close structural
similarity between enterotoxins. This is
compounded by the occurrence of some non-specific reactions from food
matrices and interference from a common immunoglobulin-binding protein that
is found in foods.
The French solution approached the enterotoxin
identification problem from a different angle, with mass spectrometry. They
used a technique which they announced in 2007 called Protein Standard
Absolute Quantification (PSAQ) which determines the absolute amounts of
protein by the isotope dilution technique. However, rather than adding isotopicallylabelled
peptides as other protein quantifications have done, they added isotopicallylabelled
versions of the complete enterotoxins which they synthesised on site. This has the marked advantage that
the endogenous toxins and the structurally identical standards (apart from
the labels) have identical biochemical properties and will react the same
way throughout their treatment.
Their method was illustrated with a semi-hard cheese
prepared from cow's milk that had been inoculated with a strain of S. aureus producing staphylococcal enterotoxin A (SEA). A portion of the cheese was
depleted of the abundant caseins and dialysed
before the addition of an isotopicallylabelled SEA. The extract was enriched on an immunoaffinity column designed to capture SE(A to E) and the eluates
subjected to SDS-PAGE. The SE regions of the gel were cut out and digested
with trypsin for analysis by electrosprayionisation LC/MS.
The selectivity of mass spectrometry permits the identification
of peptides that are specific to one particular endotoxin.
In this case, a decapeptide characteristic of SEA
was found in the cheese sample, pinpointing the presence of endogenous SEA.
The amount of the endotoxin was determined by
comparing the integrated peak of the endogenous and labelled
peptides giving a concentration of 2.5 ng/g. This value was consistent with that
obtained by ELISA at 2.9 ng/g.
Following this proof of concept, the method was applied
to a naturally contaminated Chinese dessert that was the focus of a food
poisoning outbreak in France
in 2006 in which a S. aureus strain
encoding SEA was detected. The sample was spiked with the SEA labelled standard and processed as before. In this
case, two endogenous peptides from SEA were found, the decapeptide
found in the cheese sample as well as a second peptide. They were used to
determine SEA at 1.47 ng/g, the
values obtained from each peptide differing by 0.7%.
Brun demonstrated the potential of the technique by analysing the tryptic digests
of 13 staphylococcal enterotoxins by mass
spectrometry. The resulting panel of peptides revealed specific marker
compounds for all 13 toxins.
The sensitivity of the overall method could be
increased by using multiple reaction monitoring and this might reveal some
of the peptides from the 13 enterotoxins that
were predicted but not observed, possibly due to their low levels. Although
the performance promises to be more accurate than ELISA, the unit cost and
throughput time of an analysis are comparatively unfavourable.
However, the researchers argued that it takes up to a year and around one
million dollars to develop an ELISA method. In addition, one batch of labelled protein would suffice for thousands of
quantitative analyses.
The team are now synthesising
a library of isotopicallylabelled
staphylococcal enterotoxins and hope to instigate
a "community-based effort to develop large PSAQ libraries for protein
biomarkers." In the meantime, they believe that the detection of
staphylococcal food poisoning incidents will be facilitated by the PSAQ
method, identifying cases where ELISA will fail.
Related links:
Proteomics
2008, 8, 4633-4666: "Protein
Standard Absolute Quantification (PSAQ) for improved investigation of
staphylococcal food poisoning outbreaks"
