Novel chip-based isoelectric focusing device for fractionation of bacteria prior to their mass spectrometry identification

• Published in: Analytica chimica acta Vol. 1192; p. 339333
• Main Authors: Duša, Filip, Šalplachta, Jiří, Horká, Marie, Lunerová, Kamila, Rosenbergová, Kateřina, Kubíček, Oldřich
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2022
• Subjects: Bacteria; Chemical Fractionation; Chip; Hydrogen Peroxide; Intact cell; Isoelectric Focusing; Mass spectrometry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; FFE; IC MALDI-TOF MS/ICMS; DF-IEF; Bacteria; Intact cell; Isoelectric focusing; Chip; Mass spectrometry; SA
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2021.339333

We have developed a planar chip utilizing divergent geometry of separation channel capable of vertical free-flow electrophoresis of particles at flows of lower hundreds of microliters per minute. The divergent flow isoelectric focusing (DF-IEF) chip consists of two sheets of clear polystyrene glass which serve as a base with working channels and a top cover sealing the separation channel. Optimization showed that the chip is capable to form pH gradient within 1 h and separation is completed in 5 or more minutes depending on the sample volume. The vertical position of the chip enabled analysis of sedimenting particles including microorganisms. Four different common bacteria species inactivated with H2O2 vapors were analyzed in a series of experiments. Isoelectric points were determined with capillary isoelectric focusing with following fractionation using DF-IEF with intact cell matrix-assisted laser desorption/ionization mass spectrometry detection. The DF-IEF chip fractionation proved promising for bacterial sample preparation from complex matrices for subsequent identification of whole cells by mass spectrometry. [Display omitted] •Planar divergent-flow isoelectric focusing chip presented and tested.•Vertical flow enables separation/fractionation of sedimenting particles.•Bacterial cultures inactivated using H2O2 vapor isoelectric point analysis.•Isoelectric focusing removed sample matrix and desalted and concentrated bacteria.•Isoelectric fractionation of bacteria greatly increased quality of mass spectra.