Bacterial biomarkers, identification

While this chapter has described two examples of our efforts in bacterial strain identification, a number of other groups have contributed to this research area. It remains to be seen whether mathematical algorithms such as the modified correlation approach described herein are always more effective for strain assignments than the simple use of distinctive biomarker peaks. Nilsson reported MALDI mass spectra of Helicobacter pylori with three different matrices and solvent conditions.59 She showed that some strains of this bacteria yield rather similar mass spectra while others are quite different. Nevertheless, each strain appears to exhibit some unique peaks that might be used for distinguishing strains. 

BACTERIAL PROTEIN BIOMARKER DISCOVERY A FOCUSED APPROACH TO DEVELOPING MOLECULAR-BASED IDENTIFICATION SYSTEMS... 

Additionally it has been our experience that mass spectrometry as a routine detection/identification technique for bacteria is not well received by microbiologists and clinicians who prefer less expensive, less complicated approaches to bacterial typing and identification, such as methods based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). For that reason we have adapted our MS approach to serve as a means of biomarker discovery that feeds candidate proteins or leads into development as PCR targets or other immunoassay techniques. 

Intact-cell MALDI-TOF analysis offers several attractive features for rapid screening of bacterial collections. Analysis is performed directly on the cells after minimal sample preparation, and data acquisition is complete in only a matter of minutes. Intact biomarkers are introduced into the MALDI-TOF instrument under these conditions. Whether the observed biomarker molecules are desorbed directly from the surface of the cell wall or are extracted from the cells and co-crystallized with the matrix is currently unresolved, but MALDI spectra of intact bacteria generally contain a large number of peaks in the mass range 1-20 kDa [31]. For bacterial cells, proteins are the most often observed biomarkers. While this approach samples only a small percentage of the total proteins produced in the cells, these profiles have been reported by many groups to be suitable for taxonomic identification, down to at least the strain level. The wide availability of the MALDI-TOF instrumentation and its relative ease of use, coupled with relatively simple sample preparation procedures, have been key features in the rapid advancement of this approach. 

This chapter describes a step-by-step protocol for top-down proteomic identification of protein biomarkers of bacterial food-borne pathogens using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and web-based software developed at the Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture (USDA). 

Demirev et al. reported the identification of proteins and their source microorganism Bacillus atrophaeus and Bacillus cereus) using a MALDI-TOF-TOF-MS/ MS and top-down proteomic identification with software developed in-house. The software compares the m/z of MS/MS fragment ions to the m/z of in silico fragment ions (a, b, y, and loss of NH3 or H2O) derived from bacterial protein sequences having the same MW as the protein biomarker. A P-value algorithm was also developed to score/rank identifications based on a number of parameters including the number of MS/MS in silico matches [17],... 

In the current chapter, a protocol is presented for top-down proteomic identification of protein biomarkers of food-borne bacterial microorganisms. The protein biomarkers were detected and analyzed using MALDI-TOF-TOF-MS/MS from bacterial cell lysates. Although the identification of food-borne pathogens is the practical objective of this work, this approach is applicable to other organisms. 

Fagerquist, C.K., Garbus, B.R.,Williams, K.E., Bates,A.H., Boyle, S., Harden, L.A. (2009) Web-based software for rapid top-down proteomic identification of protein biomarkers with implications for bacterial identification. Appl. Environ. Microbiol., 75,4341-4353. 

Fagerquist, C.K., Yee, E., Miller, W.G. (2007) Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. concisus for bacterial identification. Analyst, 132,1010-1023. 

Eagerquist CK. Top-down proteomic identification of bacterial protein biomarkers and toxins using MALDI-TOE-TOE-MS/MS and post-source decay. Rev Anal Chem. 2013 32 127-33. 

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