Isoelectric Focusing Fractionation Method for Signal Enhancement in Detection of Inactivated Biological Agents Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Timely identification of highly pathogenic bacteria is crucial for efficient mitigation of the connected harmful health effects. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells enables fast identification of the microorganisms based on their mass spectrometry protein fingerprint profiles. However, the MALDI-TOF MS examination must be preceded by a time-demanding cultivation of the native bacteria to isolate representative cell samples to obtain indicative fingerprints. Isoelectric focusing (IEF) is capable of separating bacterial cells according to their isoelectric point while effectively removing other non-focusing compounds from sample matrix. In this work, we present a divergent-flow IEF chip (DF-IEF chip) fractionation as an alternative way for sample clean-up and concentration of bacterial cells to prepare samples usable for following MALDI-TOF MS analysis without the need of time-demanding cultivation. By means of DF-IEF chip method, we processed four species of highly pathogenic bacteria (Bacillus anthracis, Brucella abortus, Burkholderia mallei, and Yersinia pestis) inactivated with H₂O₂ vapors or by heat treatment at 62.5°C for 24 h. The DF-IEF chip method continually separated and concentrated the inactivated bacterial cells for subsequent detection using MALDI-TOF MS. The content of the inactivated bacteria in the DF-IEF chip fractions was evaluated with the MS analysis, where inactivated Y. pestis was found to be the most efficiently focusing species. Sensitivity analysis showed limits as low as 2 × 10⁵ colony forming units per mL for inactivated B. anthracis.