Confirmation of serotype involved in E. coli outbreaks usually requires colony cultivation from clinical samples and induction of motility so that the flagellar H-antigen is expressed. This approach can take a few days and, in addition, risks phenotypic transformation of the colonies, which may prevent accurate identification. Moreover, immunologically classifying the organism requires serotyping for 53 individual H-antigens, as well as maintaining flagellar expression. Flagellar expression can be lost in prolonged culture and by physical stresses as simple as thin needle-shear when transferring samples, or heat stress above 37°C.
Cheng and co-authors report a multi-antigen LC–MS/MS approach that cuts the analysis time from days to hours and shows potential for efficient automation of a laborious diagnostic test.1 Their proteomics-based approach involves isolating cell flagella from pure culture using membrane filtration, before trypsin digestion and subsequent LC–MS/MS analysis. Their results are similar to traditional typing methods, showing even greater sensitivity in identifying two serologically unknown isolates.
The researchers first prepared a custom proteomics database containing all known E. coli H-antigen sequences. Using this custom flagellin database, they were able to identify antigen sequences from the flagella purified from cell culture.
The method was first validated by analyzing motile and non-motile E. coli reference cultures. As predicted, H-antigen was only demonstrated in motile cells. Moreover, H-antigen was detected in the normally motile stock even when motility was not induced in culture, thus speeding up the preparation stage from days to overnight growth.
Using reference colony stock to all 53 H-antigen types, the researchers confirmed assay specificity and sensitivity for accurate identification of flagella. Induction of motility was not required for successful identification by LC–MS/MS.
Further validation steps included assaying clinical samples in tandem with traditional serotyping methods. The LC–MS/MS approach identified all samples correctly and even typed two strains unclassifiable by immunology. Furthermore, LC–MS/MS coped well with colony transformation and loss of phenotype, factors that normally interfere with serotyping, and correctly identified the pathological strains presented.
In summary, Cheng et al. showed that not only is LC–MS/MS more accurate in identifying E. coli strains, the procedure is faster – four hours compared to three to five days for serotyping – and less sample is required per assay. When the researchers performed assays using the proxeon-nano-LC–LTQ Orbitrap XL (Thermo Scientific) system, they noted a conservation of sample as well as increased time savings through the elimination of the vacuum drying step following sample preparation.
1. Cheng, K., et al. (2013) “MS-H: A Novel Proteomic Approach to Isolate and Type the E. coli H Antigen Using Membrane Filtration and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS),” PLOS One, available at http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057339.
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