Analysis of Whole Bacterial Cells by Flow Field-Flow Fractionation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Abstract

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.

Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare

“…Drawbacks to this approach include the requirement for a highly concentrated sample (10 5 to 10 7 cells/ml for whole-cell analysis), the need to develop complex spectral fingerprints for every target agent, and the possible lack of specificity in complex matrices or mixtures of targets. Flow fieldflow fractionation is being investigated as one way of increasing specificity by separating particles in a complex matrix prior to analysis by MALDI-TOF/mass spectrometry (173,240).…”

Separation and concentration of water-borne contaminants utilizing insulator-based dielectrophoresis.

“…[50,54,55] FFF is another alternative for bacterial separations, an excellent overview of the biological applications of FFF has been reported by Giddings. [57] FFF has also been used as a method for bacterial sample preparation prior to mass spectrometry analysis [58] and to separate selenium-laden cells from normal ones based on their size. [59] Nevertheless, iDEP has a main advantage over the other methods mentioned; iDEP can achieve very high sample concentration (almost to solid density) [6], since bacterial cells can be retained while the background liquid passes through the iDEP device.…”

MALDI‐TOF Mass Spectrometry of Intact Bacteria

“…Field-flow fractionation mass spectrometry was used to study differences in an organism associated with growth stages and other parameters presumed to produce bacteria having different sizes. 26 Later another group proposed to use a form of field-flow fractionation called hollow-fiber flow field-flow fractionation [HF FlFFF] to fractionate bacteria, in a mixture of organisms, for taxonomic identification. Separated fractions were identified by MALDI-TOF MS analysis.…”