Table of Contents
International Journal of Proteomics
Volume 2013, Article ID 293782, 10 pages
http://dx.doi.org/10.1155/2013/293782
Research Article

Proteomic Analysis and Label-Free Quantification of the Large Clostridium difficile Toxins

1Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention (CDC), MS F-50, 4770 Buford Hwy NE, Atlanta, GA 30341, USA
2Association of Public Health Laboratories, Silver Spring, MD 20910, and Oak Ridge Institute for Scientific Education, Oak Ridge, TN 37380, USA
3Universidade do Oeste de Santa Catarina, 89600 Joacaba, SC, Brazil

Received 12 April 2013; Revised 23 June 2013; Accepted 24 June 2013

Academic Editor: Jen-Fu Chiu

Copyright © 2013 Hercules Moura et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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 mL 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.