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)

• Published in: PloS one 2013-02, Vol.8 (2), p.e57339-e57339
• Main Authors: Cheng, Keding, Drebot, Mike, McCrea, Joanne, Peterson, Lorea, Lee, David, McCorrister, Stuart, Nickel, Richard, Gerbasi, Alyssia, Sloan, Angela, Janella, Debra, Van Domselaar, Gary, Beniac, Daniel, Booth, Tim, Chui, Linda, Tabor, Helen, Westmacott, Garrett, Gilmour, Matthew, Wang, Gehua
• Format: Article
• Language: English
• Subjects: Antibodies; Antigens; Antigens, Bacterial - classification; Antigens, Bacterial - genetics; Antigens, Bacterial - isolation & purification; Biology; Chromatography; Chromatography, Liquid; Classification; Clinical isolates; E coli; Escherichia coli; Escherichia coli - chemistry; Escherichia coli Infections - diagnosis; Escherichia coli Infections - microbiology; Filtration; Flagella; Flagella - chemistry; H antigen; Humans; Identification; Laboratories; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Medicine; Membrane filtration; Membranes, Artificial; Methods; Motility; Pandemics; Peptides; Polymerase chain reaction; Proteins; Proteomics - methods; Public health; Pure culture; Scientific imaging; Search engines; Sensitivity and Specificity; Serotyping; Serotyping - methods; Spectroscopy; Tandem Mass Spectrometry; Trypsin; Typing
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0057339

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.