| Aim:MALDI TOF MS is a recently developed analytical chemistry technology. For bacterial detecting and identifying, MALDI TOF MS could provide mass spectra containing different biomarkers specific to various bacteria. The aim of this research is to find out the experimental parameters that affecting the results when analyzing bacteria by MALDI TOF MS, and to set up a general sample preparation protocol for establishing mass database of Bacillus anthracis and Yersinia pestis. It will play an important role in rapid identification of bacteria, and tracing its origin in the scenario of bioterrorism and outbreak of infectious disease for microbial forensic analysis. Methods:As for the universalness of sample preparation protocol for bacterial analysis by MALDI TOF MS, bacteria being investigated included: gram-positive bacteria including B. anthracis (spore-producing) and Staphylococcus aureus (non-spore-producing ); gram-negative bacteria such as Y. pestis, Escherichia coli and Burkholderia cepacia with high extracellular polysaccharides; different species of the same genus ( 7 species of Burkholderia ); different strains of the same species ( 20 strains of S. aureus and 11 strains of E. coli). The present study mainly employs three different sample preparation protocols: bacteria directly analyzing, treating bacteria by solvent (single treatment and combined treatment) and enzyme (lysozyme and trypsin). At the same time, different experimental factors such as matrix, solvent of matrix, solvent system used, bacterial quantities, sample application method, were investigated. The role of different factors in sample preparation was illuminated. By using the above protocols, spectra acquired by MALDI TOF MS with mass information will be selected for evaluating the ability to differentiate bacteria of different origins.The reproducibility of the established protocol was evaluated by repeating analysis of same sample and different batches of culture. In addition, safety andstability of the protocol were tested in order to analyze different virulent strains of B.anthracis and Y. pestis deposited in other institutes outside Beijing.Results:In the present research, bacterial analysis was studied systematically through testing different sample preparation protocols and experimental factors by MALDI TOF MS. A universal sample preparation protocol for MALDI TOF MS was established. The protocol is listed as follows. Bacterial cells (4 mg) were washed by 0.1% TFA and chloroform: methanol (1:1) in turn and the pellet was applied to the plate using improved dried droplet method. Matrix CHCA was prepared in ACN: methanol: water (1:1:1) with 0.1 % formic acid and 0.01 M 18-crown-6.This protocol could be used to differentiate B. anthracis, S. aureus, E. coli, Y. pestis and B. cepacia; different species of Burkholderia and different strains of E. coli and 5. aureus according to the typical peak profiles in mass range from 2 000 to 10 000 Da, respectively.The reproducibility was proved perfect using this protocol. Y. pestis was inactivated entirely using established protocol, while B. anthracis was not killed even using additional methods. The results acquired from samples of B. anthracis and Y. pestis, which placed for 7 d after treatment using the established method, is well consistent with results obtained immediately after treatment. Conclusions:Protocols of bacterial analysis by MALDI TOF MS were described systematically. A novel sample preparation protocol was developed for rapid and accurate analysis of bacteria by MALDI TOF MS. This protocol was simple, rapid and easy to perform with excellent reproducibility. It can product enough peaks to identify both gram-positive bacteria, including spore-producing bacteria (B. anthracis) and non-spore-producing bacteria (S. aureus), and gram-negative bacteria such as Y. pestis, E. coli and B. cepacia with high content of extracellular polysaccharides. This protocol was also applied to analyze different species of the same genus and different strains of the same species. Th...
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