{"id":2034,"date":"2013-12-27T07:00:28","date_gmt":"2013-12-27T12:00:28","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=2034"},"modified":"2013-12-27T07:00:28","modified_gmt":"2013-12-27T12:00:28","slug":"rapid-e-coli-typing-using-tandem-mass-spectrometry-potential-for-clinical-screening","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/proteomics\/rapid-e-coli-typing-using-tandem-mass-spectrometry-potential-for-clinical-screening\/","title":{"rendered":"Rapid E. coli Typing Using Tandem Mass Spectrometry: Potential for Clinical Screening"},"content":{"rendered":"

\"E.

E. Coli<\/p><\/div>Traditionally, Escherichia coli<\/em> outbreaks are managed diagnostically by laborious serotyping methods that require induction of cellular motility and subsequent identification of flagellar H-antigens. Membrane filtration followed by liquid chromatography–tandem mass spectrometry (LC–MS\/MS) using LTQ Orbitrap XL (Thermo Scientific) offers a faster and more sensitive approach.<\/p>\n

Confirmation of serotype involved in E. coli<\/em> 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.<\/p>\n

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<\/span><\/sup> 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.<\/p>\n

The researchers first prepared a custom proteomics database containing all known E. coli<\/em> H-antigen sequences. Using this custom flagellin database, they were able to identify antigen sequences from the flagella purified from cell culture.<\/p>\n

The method was first validated by analyzing motile and non-motile E. coli<\/em> 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.<\/p>\n

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.<\/p>\n

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.<\/p>\n

In summary, Cheng et al. showed that not only is LC–MS\/MS more accurate in identifying E. coli<\/em> 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.<\/span><\/p>\n

 <\/p>\n

Reference<\/strong><\/p>\n

1. Cheng, K., et al. (2013) “MS-H: A Novel Proteomic Approach to Isolate and Type the E. coli<\/em> H Antigen Using Membrane Filtration and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS\/MS)<\/a>,” PLOS One, available at http:\/\/www.plosone.org\/article\/info:doi\/10.1371\/journal.pone.0057339<\/a>.<\/p>\n

X<\/p>\n

Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer; SAHM and expat trailing spouse.<\/p>\n

A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.<\/i><\/p>\n","protected":false},"excerpt":{"rendered":"

Traditionally, Escherichia coli outbreaks are managed diagnostically by laborious serotyping methods that require induction of cellular motility and subsequent identification of flagellar H-antigens. Membrane filtration followed by liquid chromatography–tandem mass spectrometry (LC–MS\/MS) using LTQ Orbitrap XL (Thermo Scientific) offers a faster and more sensitive approach. Confirmation of serotype involved in E. coli outbreaks usually requires colony<\/p>\n","protected":false},"author":21,"featured_media":2033,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[54],"tags":[165,166],"division":[],"class_list":{"0":"post-2034","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-bacteriology","8":"tag-e-coli","9":"tag-lc-msms-serotyping","10":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"\nRapid E. coli Typing using Tandem Mass Spectrometry - 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