%0 Journal Article
%T Proteomic Analysis and Label-Free Quantification of the Large Clostridium difficile Toxins
%A Hercules Moura
%A Rebecca R. Terilli
%A Adrian R. Woolfitt
%A Yulanda M. Williamson
%A Glauber Wagner
%A Thomas A. Blake
%A Maria I. Solano
%A John R. Barr
%J International Journal of Proteomics
%D 2013
%I Hindawi Publishing Corporation
%R 10.1155/2013/293782
%X Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide, due to hypervirulent epidemic strains with the ability to produce increased quantities of the large toxins TcdA and TcdB. Unfortunately, accurate quantification of TcdA and TcdB from different toxinotypes using small samples has not yet been reported. In the present study, we quantify C. difficile toxins in <0.1£żmL of culture filtrate by quantitative label-free mass spectrometry (MS) using data-independent analysis (MSE). In addition, analyses of both purified TcdA and TcdB as well as a standard culture filtrate were performed using gel-based and gel-independent proteomic platforms. Gel-based proteomic analysis was then used to generate basic information on toxin integrity and provided sequence confirmation. Gel-independent in-solution digestion of both toxins using five different proteolytic enzymes with MS analysis generated broad amino acid sequence coverage (91% for TcdA and 95% for TcdB). Proteomic analysis of a culture filtrate identified a total of 101 proteins, among them TcdA, TcdB, and S-layer proteins. 1. Introduction Clostridium difficile is a gram-positive, anaerobic, spore-forming, rod-shaped bacterium that can produce at least three toxins including two Rho GTPase-glucosylating toxins (TcdA and TcdB) and the binary C. difficile transferase (CDT) toxin. The organism can cause C. difficile infection (CDI) in humans and animals. C. difficile is considered an important cause of healthcare-associated infection in humans and is the leading cause of antibiotic-associated diarrhea in hospitals worldwide [1, 2]. CDIs are toxin-mediated illnesses that range from mild diarrhea to fulminant pseudomembranous colitis and toxic megacolon, which may result in death [3]. Laboratorial confirmation of CDI needs to be rapidly performed. The methods include detection of C. difficile through cultivation, detection of Tcd A and Tcd B in stool samples using immunoassay and DNA-based methods [2]. Reflecting its changing epidemiology, the incidence and severity of CDI have increased significantly in the past ten years. Among the possible causes of increasing morbidity is the emergence of strains considered to be more virulent [4]. These strains produce greater amounts of toxin than reference strains and are highly transmissible due to their greater sporulation capacity [4]. One example is the rapid emergence of the highly virulent clone-designated BI/NAP1/027 in multiple countries [5]. Additionally, it is estimated that there are 500,000 cases of CDI per year in US
%U http://www.hindawi.com/journals/ijpro/2013/293782/