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Serotyping is the long-standing gold standard method to determine  E. coli  H antigens; however, this method requires a panel of H-antigen specific antibodies and often culture-based induction of the H-antigen flagellar motility. In this study, a rapid and accurate method to isolate and identify the  Escherichia coli  ( E. coli ) H flagellar antigen was developed using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Flagella were isolated from pure culture, digested with trypsin, and then subjected to LC-MS/MS using one of two systems (Agilent-nano-LC-QSTAR XL or Proxeon-nano-LC-LTQ-Orbitrap XL). The resulting peptide sequence data were searched against a custom  E. coli  flagella/H antigen database. This approach was evaluated using flagella isolated from reference  E. coli  strains representing all 53 known H antigen types and 41 clinical  E. coli  strains. The resulting LC-MS/MS classifications of H antigen types (MS-H) were concordant with the known H serogroup for all 53 reference types, and of 41 clinical isolates tested, 38 (92.7%) were concordant with the known H serogroup. MS-H clearly also identified two clinical isolates (4.9%) that were untypeable by serotyping. Notably, successful detection and classification of flagellar antigens with MS-H did not generally require induction of motility, establishing this proteomic approach as more rapid and cost-effective than traditional methods, while providing equitable specificity for typing  E. coli  H antigens.

plos one

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)