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.

Mass Spectrometry–Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping

“…The 2 LC-MS/MS systems, Orbitrap Velos and Orbitrap Fusion, showed higher detectability than the Orbitrap XL. On the basis of our experience, sample load must be adjusted for each instrument to retain high analytical sensitivity and avoid excessive carryover (10,21 ).…”

“…Clinical isolates from January 1, 2011, to April 1, 2015, were obtained from 5 provincial public health laboratories in Canada. All cells were cultured overnight on tryptic soy agar with 5% sheep blood for flagella extraction (10,11 ). Detailed sources and applications of the cells for different analyses are summarized in part 1 of the Supplemental Data File that accompanies the online version of this article at http://www.clinchem.org/content/vol62/issue6.…”

“…coli H antigens were serotyped on the basis of published methods (18 -20 ) and summarized as a standard operating procedure (SOP) (10 ). In brief, motility induction was performed by subculturing a single colony in Craigie tubes containing 0.25% agar and then 0.3% agar to induce and observe flagella development.…”

“…We then performed LC-MS/MS-based sequence analysis (10,11 ) or MALDI-TOF-based peptide mass fingerprinting (PMF) (12 ) through a curated E. coli flagella protein sequence database search (13 ). MS-H was able to identify and differentiate any E. coli H type within 5 min after sample loading by MALDI-TOF (12 ), or within 4 hours by LC-MS/MS after flagella extraction (10,11 ). Currently, overnight bacteria culture is typically needed to extract flagella, but motility induction is not required for the majority of isolates, especially when using LC-MS/MS (10 -12 ).…”

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

“…Recently, we reported an MS-based E. coli H antigen identification and classification method at the flagellar protein sequence level using a liquid chromatography-tandem MS (LC-MS/MS) platform (17,18). The method applied a unique flagellar extraction and on-filter trypsin digestion method together with a curated E. coli flagellar protein sequence database for targeted and specific identification (19).…”