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Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification

•We found method for rapid separation of Lactobacillus in tapered FS capillary.•CIEF separation capacity and efficiency was increased.•CIEF was coupled with off-line MALDI-TOF MS identification.•Method was tested on spiked milk. In this study, combination of capillary isoelectric focusing (CIEF) in...

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Published in:Analytica chimica acta 2013-07, Vol.788, p.193-199
Main Authors: Horká, Marie, Karásek, Pavel, Šalplachta, Jiří, Růžička, Filip, Vykydalová, Marie, Kubesová, Anna, Dráb, Vladimír, Roth, Michal, Šlais, Karel
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container_title Analytica chimica acta
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creator Horká, Marie
Karásek, Pavel
Šalplachta, Jiří
Růžička, Filip
Vykydalová, Marie
Kubesová, Anna
Dráb, Vladimír
Roth, Michal
Šlais, Karel
description •We found method for rapid separation of Lactobacillus in tapered FS capillary.•CIEF separation capacity and efficiency was increased.•CIEF was coupled with off-line MALDI-TOF MS identification.•Method was tested on spiked milk. In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×106mL−1. Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.
doi_str_mv 10.1016/j.aca.2013.05.059
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In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×106mL−1. Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>23845500</pmid><doi>10.1016/j.aca.2013.05.059</doi><tpages>7</tpages></addata></record>
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source ScienceDirect Freedom Collection
subjects Animals
Bacteria
Calibration
Capillarity
Capillary isoelectric focusing
Cattle
Electrophoresis, Capillary - methods
Food Analysis - methods
Hydrogen-Ion Concentration
Ionization
Isoelectric focusing
Isoelectric Focusing - instrumentation
Isoelectric Focusing - methods
Lactic acid bacteria
Lactobacillus
Lactobacillus - isolation & purification
Lasers
Mass spectrometry
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Milk
Milk - microbiology
Pasteurization
Probiotics
Probiotics - analysis
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Tapered fused silica capillary
title Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification
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