title: 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).
abstract: 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.
authors: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. Keding.Cheng@phac-aspc.gc.ca
authors: Keding
authors: K
authors: Cheng
authors: Mike
authors: M
authors: Drebot
authors: Joanne
authors: J
authors: McCrea
authors: Lorea
authors: L
authors: Peterson
authors: David
authors: D
authors: Lee
authors: Stuart
authors: S
authors: McCorrister
authors: Richard
authors: R
authors: Nickel
authors: Alyssia
authors: A
authors: Gerbasi
authors: Angela
authors: A
authors: Sloan
authors: Debra
authors: D
authors: Janella
authors: Gary
authors: G
authors: Van Domselaar
authors: Daniel
authors: D
authors: Beniac
authors: Tim
authors: T
authors: Booth
authors: Linda
authors: L
authors: Chui
authors: Helen
authors: H
authors: Tabor
authors: Garrett
authors: G
authors: Westmacott
authors: Matthew
authors: M
authors: Gilmour
authors: Gehua
authors: G
authors: Wang
mesh_terms: D000942:Antigens, Bacterial / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000737:chemistry*; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000382:microbiology; D005374:Filtration; D005407:Flagella / Q000737:chemistry*; D006801:Humans; D008567:Membranes, Artificial; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000942:Antigens, Bacterial; C022863:H antigen; D008567:Membranes, Artificial